carbohydrate microorganisms bethesda, maryland · biosynthetic precursors. hydrate metabolism vary...

PATHWAYS OF CARBOHYDRATE METABOLISM IN MICROORGANISMS

I. C. GUNSALUS

Department of Bacteriology, University of Illinois, Urbana, Illinois

B. L. HORECKERThe National Institute of Arthritis and Metabolic Diseases, The National Institutes of Health,

Bethesda, MarylandAND

W. A. WOODDepartment of Dairy Science, University of Illinois, Urbana, Illinois

CONTENTSIntroductory Statement ..................................................... 80I.!Fermentation via Hexosediphosphate Reactions and Pathways ... 81

1. Aldose and Ketose Reactions..................................................... 81a. Kinases ........................................... 81b. Mutases ........................................... 84c. Isomerases ......................................... 84d. Aldolases and ketolases ................................... 85

2. Triosephosphate Reactions..................................................... 87a. Dehydrogenases ....................................... 87b. Mutase...................................................... 88c. Enolase ........................................... 88d. Transphosphorylases..................................................... 88

3. Evidence of an Embden-Meyerhof Pathway in Bacteria ... 89II. Hexosemonophosphate Reactions and Pathways............................................ 90

1. Oxidations..................................................... 90a. Glucose-6-phosphate dehydrogenase................................................... 90b. 6-Phosphogluconate dehydrogenase................................................... 91

2. Isomerization, Phosphorylation and Dehydration........................................ 93a. Phosphopentose isomerase..................................................... 93b. Phosphoribulokinase..................................................... 93c. 6-Phosphogluconate dehydrase..................................................... 93

3. Cleavage and Transfer..................................................... 93a. Transketolase..................................................... 93b. Transaldolase..................................................... 95c. Aldolases..................................................... 96

4. Hexosemonophosphate Oxidation Pathways.............................................. 96a. Interconversions of 5, 6 and 7 carbon phosphate esters ... 96b. Routes from hexosemonophosphate to pyruvate....................................... 98

(1) Pentose phosphate cleavage..................................................... 98(2) 2-Keto-3-deoxy-6-phosphogluconate cleavage...................................... 98

5. Hexosemonophosphate Fermentation Pathways.......................................... 98a. Heterolactic (ethanol-lactic)..................................................... 98b. Bacterial ethanol..................................................... 99c. Induced aldonic acid..................................................... 99d. Pentose..................................................... 99

6. Nonphosphorylated Hexose Oxidation................................................... 101a. Glucose and gluconate oxidation.................................................... 101b. Gluconokinase and 2-ketogluconokinase............................................... 102c. Routes from gluconate to pyruvate................................................... 102

III. Routes to Known Pathways..................................................... 1021. Monosaccharides..................................................... 102

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80 I. C. GUNSALUS, B. L. HORECKER, AND W. A. WOOD [VOL. 19

a. Mannose.................................... 102b. Fructose .................................... 103c. Galactose.................................... 103

2. Disaccharides and Polysaccharides ............................... 104a. Phosphorylases.................................... 104b. Hydrolases................................... 104c. Polyhydric alcohols................................... 104

IV. Pyruvate Reactions ............................. 1051. Reductions .................................... 105

a. Lactic dehydrogenases.................................... 105b. Racemase and amination reactions.................................... 106

2. Decarboxylases and Aldehyde Transfer ............................. 106a. Direct decarboxylases................................... 106b. Acyloin condensations.................................... 107c. Acetate generation.................................... 108d. Acetyl generation.................................... 109

3. Clastic Reactions .................................... 110a. Acetyl plus formate................................... 110b. Acetyl plus C02 and H2.................................... 110

4. Carboxylations .................................... 1105. Acyl Condensations and Reductions ............................... 111

a. Acid activations.................................... 111b. Reductions.................................... 111c. Condensations.................................... 111

Bibliography ........................... 112

Carbohydrates serve as a major source of cording the data and concepts now current, help-carbon and energy for the growth of many micro- ful in planning future experimentation, timeorganisms. Thus, in understanding the chemical saving for instruction, and a helpful guide tobasis of biological processes, a knowledge of car- information on microbial carbohydrate metab-bohydrates, their chemical properties and trans- olism, we shall consider the effort expended informations, and the pathways of carbon and assembling, considering and recording the statusenergy liberation assumes importance, as does of knowledge in this area well spent.control of the reaction routes and formation of The methods and obectives of studying carbo-biosynthetic precursors. hydrate metabolism vary continuously. In the

Several deterrents face the seeker of such economic sense, (a) the quantitative transforma-knowledge, stemming principally from the tion of cheap, often by-product carbohydrate tocomplexity of organisms and their processes, useful products, by fermentation or partiallimitation in imagination of the investigator, oxidation with growing cultures for industrial orparticularly as to the questions asked, in his epicurean purposes, has, at least for the former,ability to generalize fruitfully from the existing given way largely to synthetic industrial prac-data and to recognize its limitations in complete- tice and is replaced by (b) the formation, inness and accuracy. Attempts to collect and to more limited amounts, of more complex mole-present these data in unified form for use by cules of medicinal and nutritional importance.students and other investigators suffer in addition In addition, microbial cells enjoy increased usefrom limitations of the observer and his judgment as (c) food or feed adjuncts for the minor (vita-in selecting pertinent data and of clearly con- min) components, and as tools for (d) the studyceiving and presenting their implications and of the formation of the intermediates in theconsequences. Moreover, these concepts are sub- glycolytic and oxidative pathways (i.e., phos-ject to continuous obsolescence as new data and phate esters) and for the preparation of the co-their interpretation reorient understanding and factor forms of the vitamins as medical curiosrelate seemingly random observations into a and'more fruitfully as tools for the furtherconceptual if not complete scheme. The present elucidation by the chemist and the microbiologist,effort, like many before it, is subject to these of the many chemical processes of the living cellshortcomings. Should it prove accurate in re- still unclassified or undocumented.


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1955] PATHWAYS OF CARBOHYDRATE METABOLISM IN MICROORGANISMS 81

In the search for understanding of living glycolytic scheme and for both the fermentativeprocesses, the objectives have shifted principally and oxidative hexosemonophosphate pathways.from the quantitative description of the conver- Thus in the present discussion, the reactions ofsion of substrate to product and control of product glucose serve as the first example and mainyields, through identification of fermentation theme. The analogous reactions of aldoses andintermediates, description of the reactions they ketoses of 3 to 7 carbon atoms are discussed withundergo and the catalysts responsible for these the documentation of glucose reactions. Theirreactions, to a consideration of the energy, equi- import in carbohydrate pathways is discussed inlibria and mechanisms of these transformations subsequent sections. An attempt to verify theand now principally to their relationship to cell routes followed by carbohydrates in fermenta-formation. tion and oxidation, along with the necessaryMany problems of fermentations studied abun- assignment of sequence to the enzymatic steps,

dantly at the substrate to product level with is postponed until after discussion of the specificgrowing cultures and with cell suspensions have reaction types.not been subjected to the enzymatic and isotopicanalysis essential to an understanding of the 1. Aldose and Ketose Reactionsroutes involved. Particularly outstanding among a Kinases. Kinases which transfer phosphatethese is the propionic fermentation in which from adenosine triphosphate (ATP) to carbonisotopelabeling ampldeme nonstrathesothatthe skeletons, were recognized first for their role inEmbden-Meyerhof scheme is not the sole path- gluos disiiato. Laewt hesprtoway, but enzymatic clarification of the steps i glucs iain -trihtespStoof kinases from several sources, their substratelacking.Altckogh for most microorganisms degrading specificity and role in fermentation of other car-

caltoughyforat most microayorgpanisms radgo bohydrates were clarified. The variety of carbo-carbohydrates,te thayfor pathways of hydrates fermented by microorganisms has in

several instances been traced to ability to formclear, this may prove to be only the phase pre- kinases, either constitutively or in response toceding recognition of stllr further divergences. As added inducers, thereby initiating their dissimila-examples, the recent observations on the function via phosphate esters. With increasing study,tion of ribulose diphosphate in 002 incorpora- kinases have been shown to possess specificitytion, the route of formation of rhamnose and for both the position of phosphorylation and theother cell wall components may be cited.

Also, still unstudied are many problems of configuration of the acceptor. The known carbo-induced enzyme formation, the transport of hydrate in thesccompounds including carbohydrates from the s edoinabl 1.Hezolcinase, which phosphorylates glucose,cellular environment to enzymatic sites, the fsteps in formation of many amino acid and other

futs n ansa eaiertsostesifomaionofan amno ci an oter100:140:30 (24), has been crystallized fromcarbon skeletons and their transport to the sites yeast (13,24),2 an meenc anima tisuofsynthesis. ~~~~~~yeast (13, 24, 230) and measured in animal tissue

such as brain, muscle, heart, liver and kidneyI. FERMENTATION via HEXOSEDIPHOSPHATE REAC- (38, 71, 72, 83, 347, 416).

TIONS AND PATHWAYS A glucokinase which phosphorylates glucose,but not fructose or mannose, has been reported in

Glucose, because of its wide distribution and muscle and in liver (72, 77). In microorganisms,importance in animal metabolism, has served as few studies have utilized purified kinases, but thethe initial substrate for most studies of product phosphorylation of glucose and of other carbo-formation and mechanism of carbohydrate me- hydrates entering the Embden-Meyerhof path-tabolism in microorganisms. Furthermore, glu- way has been shown in a wide variety of fer-cose is assigned a major role: (a) in reactions mentative microorganisms. A glucokinase specificleading from polysaccharides, (b) as a substrate for the conversion of glucose to glucose-6-for phosphorylation prior to rearrangement and phosphate (G-6-P) has been reported in Staph-cleavage in fermentation and oxidation, and (c) ylococcus aureus (Micrococcus pyogenes var.as a point of departure for the alternate pathways aureus) and in Escherichia coli (54) and "hexoki-of the hexosediphosphate, or Embden-Meyerhof, nase" activity shown in extracts of Leuconostoc


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TABLE 1Kinases: Carbohydrate Phosphorylation

Enzyme Substrate Product Source

1. Aldokinase (hemiacetal bond)Galactokinase Galactose GA-1-P Bacteria (54, 331)

Yeast (221, 380)Animal (219)

2. Aldokinases (ester bond)Hexokinase Glucose G-6-P Bacteria (125, 331, 350)

Fructose F-6-P Yeast (24, 230)Mannose MA-6-P Animal (72, 347)Glucosamine GAM-6-P

Glucokinase Glucose G-6-P Bacteria (54)Animal (72)

Ribokinase Ribose R-5-P Yeast (333)Bacteria (69, 180)

Phosphoglucokinase G-1-P G-1,6-P Yeast (311)Muscle (311)Plant (55)

3. Ketokinases (ester bond)Fructokinase Fructose F-1-P Animal (80, 163, 244)

6-Phosphofructokinase F-6-P F-1,6-P Yeast (72)Bacteria (393)Animal (319, 321)Plants (9)

1-Phosphofructokinase F-1-P F-1,6-P Muscle (347)

5-Phosphoribulokinase Ru-5-P Ru-1,5-P Plants (418)

4. Aldonic kinases (ester bond)Gluconokinase Gluconate 6-PG Bacteria (67, 350)

Yeast (332)

2-Ketogluconokinase 2-Ketogluconate 2-KPG Bacteria (88, 289)

Abbreviations: GA-1-P = galactose-i-phosphate; G-1-P - glucose-l-phosphate; G-6-P = glucose-6-phosphate; F-6-P = fructose-6-phosphate; MA-6-P = mannose-6-phosphate; GAM-6-P = glucosa-mine-6-phosphate; R-5-P = ribose-5-phosphate; G-1,6-P = glucose-1,6-diphosphate; F-1-P =fructose-i-phosphate; F-1,6-P - fructose-1,6-diphosphate; 6-PG = 6-phosphogluconate; 2-KPG =2-keto-6-phosphogluconate; Ru-5-P = ribulose-5-phosphate; Ru-1,5-P = ribulose-1,5-phosphate.

mesenteroides (95), Streptococcus faecalis (350), to form ca-galactose-l-phosphate (GA-1-P), hasLactobaciUus bulgaricus (331), and Clostridium been studied extensively in yeast (221, 380) andbutyricum (125), as well as in the aerobic (non- to a lesser extent in animal tissue (219) and inglycolyzing), Pseudomonas aeruginosa (65) and galactose fermenting microorganisms, i.e., E.Pseudomonas putrefaciens (206, 207). coli (54) and L. bulgaricus (331). Galactose-1-

Galactokinase, unique among the aldokinases phosphate is converted to glucose-l-phosphatein that the number one carbon is phosphorylated (G-1-P) by a "galactowaldenase", also found in


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galactose fermenting microorgani(331). "Ga- glucose plus ATP (238). The mechanism waslactowaldenase" requiring uridine diphosphohex- studied with a partially purified preparationose as a coensyme functions in the epimerization and postulated to involve a primary phosphoryl-of GA-1-P to G-1-P (242). This reaction will be ation followed by a transphosphorylation fromdescribed in detail in section III, but is mentioned one glucose-l-phosphate molecule to anotherhere as an example of the route from other carbo- as follows (238):hydrates to glucose phosphates which aremetabolized by a classical pathway bypassing 2 glucose-i-phosphatethekinasesforglucose. -k~ glucose-i,6-diphosphate + glucosethe Idnases for glucose.Fructokinase, which requires magnesium and Pentokinases, phosphorylating the pentoses at

potassium ions for activity (163, 359), has been the fifth carbon atom, have been studied in bac-demonstrated in animal tissues (80, 244). The teria and yeast but with the exception of ribo-phosphorylated product, which accumulated in kinase, purified little if any. The substrate spec-the presence of fluoride, was identified as fruc- ificity and the identity of the first product oftose-1-phosphate (F-1-P). Fructose phosphoryla- phosphorylation in many cases remain to be clar-tion has been observed in Pseuomonas putre- ified. Ribokinase, which phosphorylates ribosefaciens, but purified enzymes have not been to ribose-5-phosphate (R-5-P), has been foundprepared and the product of phosphorylation in yeast (333) and from purified ribose grownhas not been identified. The high fructose con- Aerobacter aerogenes cells (180). Inducible ribo-centration required suggests, however, that the and arabinokinases have been demonstratedreaction is catalyzed by a hexokinase (207). in E. coli (69).

Phosphofructokinase, an enzyme peculiar to A phosphoribulokinase, which converts ribu-the Embden-Meyerhof pathway, resembles lose-5-phosphate to ribulose-1, 5-diphosphate,fructokinase in phosphorylating fructose-6-phos- has been purified from green leaves (418).phate (F-6-P) at carbon 1 to yield fructose-1, 6- Ribulose diphosphate has been found in extractsdiphosphate (F-1, 6-P) (319, 376). Although of algae and of green leaves (22, 23), but itsATP was initially considered to be the only occurrence in nonphotosynthetic microorgan-phosphate donor, uridine triphosphate and isms has not been reported. The enzymatic car-inosine triphosphate have recently been shown boxylation of ribulose diphosphate yields 3-also to serve as phosphate donors (248). A phosphoglycerate (3-PGA) (318). Data obtainedspecific phosphofructokinase has been reported with crude extracts of lactic acid bacteria sug-in plants (9), in brain (321, 389) and purified gest the existence of keto kinases for the keto-from rabbit muscle (319, 376). Phosphofructo- pentoses, xylulose and ribulose (233), but enzy-kinase is presumed to function in those organ- matic reactions have not been reported.isms which accumulate fructose-1,6-diphosphate Gluconokinase, which phosphorylates gluco-during hexose fermentation, but little evidence nate to 6-phosphogluconate (6-PG), was firsthas been published as to its occurrence in micro- demonstrated in gluconate grown E. coli cellsorganisms. An exception is the data of VanDe- and shown to be specific for gluconate as themark and Wood (393) who demonstrated an phosphate acceptor (67). Gluconokinase has beenATP-dependent conversion of fructose-6-phos- demonstrated in gluconate adapted Strepto-phate to triose phosphates in extracts of Micro- coccus faecalis, a fermentative organism (350),bacterium lacticum. A second phosphofructokinase baker's yeast (332), and Aerobacter cloacae (88)which phosphorylates fructose-i-phosphate to as well as in aerobic bacteria such as Pseudo-fructose-1,6-diphosphate has been purified from monas fluorescens (289) and Pseudomonas aerugi-rabbit muscle (347) but so far has not been nosa (65). The presence of gluconokinase is nowstudied in bacteria. considered to be evidence for a nonglycolytic

Phosphoglucokinase, which forms glucose- pathway of hexose utilization.1,6-diphosphate (G-1,6-P) from glucose-i- 2-Ketogluconokinase, which phosphorylates 2-phosphate, has been found in muscle and yeast ketogluconate presumably to 2-keto-6-phospho-(311). An apparently different kinase present in gluconate (2-KPG), has been found in Aerobac-E. coli, Klebsiella pneumoniae and Aerobacter ter cloacae and Pseudomonas fluorescens. Thisaerogenes forms glucose-1,6-diphosphate from kinase, like gluconokinase, is induced by growth


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84 I. C. GUNSALUS, B. L. HORECKER, AND W. A. WOOD [VoL. 19

on the specific substrate. The presence of gluco- and ribose-5-phosphates (147, 208). The enzymenokinase and 2-ketogluconokinase in the pseudo- can be activated by the corresponding diphos-monads, in which the oxidation of glucose to phates or by glucose-1, 6-diphosphate. A mutasegluconate and to 2-ketogluconate, without phos- specific for phosphoribose has been demonstratedphorylation, has been known for some time, in smooth muscle and separated from phospho-indicates that these oxidative processes supply glucomutase (147).the inducing substrates, which activate kinase Phosphoglucomutase has not been isolatedformation and further substrate utilization by or studied in detail in bacterial preparations, butone of the hexosemonophosphate pathways. its activity has been demonstrated in Pseudo-2-Ketogluconate was previously considered to monasfluorescen (431), Lactobacillu8 bulgaricusbe the endproduct of an incomplete oxidation (331), and its presence is assumed in extractspathway. These reactions are discussed in detail which ferment or oxidize glucose-i-phosphate.in section II, 6. Phowphoglyceromutase interconverts 3-phos-

b. Mutases. Mutases transfer phosphate from phoglyceric acid and 2-phosphoglyceric acidone carbon to another, frequently between hemi- (271, 302). A 2,3-diphosphoglycerate isolatedacetal and primary hydroxyl, or between pri- from blood by Greenwald (146) as early as 1925mary and secondary hydroxyls. Such transfer was shown by Sutherland et al. (373) to functionreactions occur with hexose- and pentosephos- by a mechanism analogous to that of glucose-1, 6-phates and with phosphoglyceric acids. diphosphate in phosphoglucomutase. Thus, the

Phosphoglucomutase, which effects the equilib- conversion of 2,3-diphosphoglycerate to pyru-rium between glucose-i-phosphate and glucose-6- vate shown by Lennerstrand (243) was explained.phosphate, has been demonstrated in many This mutase has not been studied in bacteria.tissues and cells including yeast and muscle (75, c. Isomerases. Isomerases interconvert aldo76) and has been crystallized from rabbit muscle and keto groups of carbohydrates. Enzymes(287). A coenzyme, glucose-1, 6-diphosphate, specific for hexose-, pentose-, and triosephos-which mediates in this mutase reaction was iso- phates and for nonphosphorylated pentoseslated from impure fructose-1, 6-diphosphate by have been observed. The isomerization of phos-Leloir and his coworkers (52, 237) and was phate esters, long recognized as important inshown by Capputo et al. (49), Sutherland et al. glycolysis, is now recognized as important also(374), Pullman and Najjar (288, 317), and others in pentose metabolism. The xylose-xylulose(186) to function by a mechanism best outlined (166, 167, 280) and ribose-ribulose (70) isom-as follows: erases have been studied. Many more isom-

a-glucose-1-phosphate + enzyme-phosphate =± glucose-i,6-diphosphate + enzymeenzyme + glucose-i,6-diphosphate = glucose-6-phosphate + enzyme-phosphate

95%a-glucose-l-phosphate - glucose-6-phosphate5%

The mechanism indicated is based on recent erases active on nonphosphorylated sugars seemexperiments of Najjar and Pullman (288) with certain to be discovered.substrate amounts of crystalline phosphogluco- Phosphohexose isomerase which catalyzes themutase in which incubation of the enzyme with reaction:glucose-1- or glucose-6-phosphate formed glucose- 30%1, 6-diphosphate and "dephosphorylated" en- glucose-7phosphate. fructose--phosphatezyme. Upon reincubation with glucose-1, 6- %diphosphate, the enzyme was rephosphorylated. was recognized as a key enzyme in the glycolytic

Phosphoglucomutase is considered to function pathway soon after the identification of glucose-in oligosaccharide degradation by converting 6-phosphate (258). This isomerase was purifiedthe glucose-l-phosphate formed by phosphorlytic from animal tissues (348), yeast (258), and plantscleavage to glucose-6-phosphate. Phosphoglu- (351) and demonstrated in several bacteria in-comutase will also interconvert mannose-1- cluding Pseudomons fluorescens (431) and Micro-and mannose-6-phosphates (239) and ribose-1- bacterium lacticum (393).


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Phosphomannose isomerase (346, 348) which Utter and Werkman (384) obtained similarinterconverts mannose-6- and fructose-6-phos- equilibrium data with E. coli extracts as a sourcephates has been partially separated from the of "zymohexase".isomerase interconverting glucose-6- and fruc- d. Aldolases and ketolases. Aldolases, of whichtose-6-phosphates (Lohmann's isomerase). Since several are now known, catalyze a reversiblefructose-6-phosphate is a product common to cleavage of carbon chains adjacent to secondaryboth mannose-6- and glucose-6-phosphate isom- hydroxyl groups as indicated by the generalerization, the term "phosphoglucoisomerase" reaction:has been suggested as more appropriate for theLohmann isomerase previously termed phospho- OH 0 0 H 0hexose isomerase (258). R-C,-C--C-R- R-C + H-C-C-R

Pentose phosphate isomerase, discovered in H &6H) H &6H)yeast by Horecker et al. (170), catalyzes the reac- The specific aldolases described and assignedtion: functions in carbohydrate metabolism are listed

75% in table 2.ribulose-5-phosphate I ribose-5-phosphate Fructose-i ,6-diphosphate aldolase (table 2,

25% reaction 1), first recognized by Meyerhof (268,The enzyme was demonstrated in animal tissue 269, 272), was reported to show absolute spec-(173) and highly purified from plant sources ificity for dihydroxyacetone phosphate but not(11). Its presence in a wide variety of bacteria for D-glyceraldehyde phosphate, which is re-including Pseudmonas flume=5 (431), Aceto placeable with many aldehydes, including acetal-bacer uboxydan (161), Azotobacter vinelandii dehyde, to form sugar phosphates of various(281) and Microbacterium lacticum (393) has configurations (272, 273). More recent studiesbeen inferred from the accumulation of heptu- with crystalline aldolase confirmed the substratelose phosphate with ribose-5-phosphate as sub- specificity patterns suggested by Meyerhof (272).strate, i.e., transketolase action on ribose- and Among the reactions catalyzed is the condensa-ribulose-5-phosphates-see section II, 3. tion of dihydroxyacetone phosphate with D-

Triosephosphate isomerase interconverts dihy_ and with a-glyceraldehyde forming, respectively,droxyacetone phosphate and D-glyceraldehyde-3- fructose-i- and sorbose-1-phosphates (382).phosphate as follows: A condensation with glycolaldehydephosphate

forms xylulose-1, 5-diphosphate, and with glycol-5% aldehyde forms xylulose-1-phosphate (42).

dihydroxyacetone phosphate9

All of the condensations reported form trans-hydroxyls. One cleavage of a cis-hydroxyl con-

D-glyceraldehyde-3-phosphate taining ester, tagatose-1, 6-diphosphate, wasreported (382).

Muscle (278) and yeast (21) are rich sources of Aldolase has been separated from triosephos-this enzyme which has been purified by Meyerhof phate isomerase (162) and the equilibriumand Beck (278) and cry2stallized from muscle by shown to be 89% toward hexosediphosphateMeyer-Erendtetal. (267a). Beforefrutose-i,6- (270). Aldolase has been purified from yeastdiphosphate aldolase and triosephosphate iso- (415) and crystallized from rabbit muscle (375,merase were separated, Meyerhof (268, 270) 414). In contrast to muscle aldolase, the enzymeshad applied the term "zymohexase" to prepara- from yeast (415), Aspergius niger (187) andtions catalyzing both reactions with the follow- Closridium perfringens (17) show divalent metaling equilibria (277): activation-the yeast and clostridial enzymes are

fructose-i, 6-diphosphate in addition cysteine activated. Among the di-89% valent metals, Zn++, Co++ and Fe++ activate

the yeast and AspergiUus aldolases; but only-glyceraldehyde-3-phosphate = Fe++ activates the clostridial enzyme. Simplified

0.5% aldolase assays, especially the colorimetric pro-

dihydroxyacetone phosphate cedure of Sibley and Lehninger (344), have been10.5% used to demonstrate aldolase in microorganisms


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TABLE 2Aldolases for phosphate esters

Enzyme Substrate Product Source

1. F-1,6-P aldolase DHA-P + G-3-P F-i ,6-P Yeast (415)Plant (371)Animal (414, 375)Bacteria (17, 261)

2. F-1-P aldolase DHA-P + glyceral F-1-P Liver (244, 245)

3. Phosphoketotetrose aldolase DHA-P + HCHO E-1-P Liver (58)

4. Transaldolase G-3-P + 8-7-P E4-P + F-6-P Animal (176)Yeast (182)Plant (10)

5. DR aldolase G-3-P + AcAl DR-5-P Bacteria (322)

6. KDPG aldolase KDPG Pyruvate + G-3-P Bacteria (223, 263)

Abbreviations: DHA-P = dihydroxyacetone phosphate; E-1-P = erythrulose-i-phosphate; G-3-Pglyceraldehyde-3-phosphate; 8-7-P = sedoheptulose-7-phosphate; KDPG = 2-keto-3-deoxy-6-phos-phogluconate; DR-5-P = deoxyribose-5-phosphate; glyceral = glyceraldehyde.

including Clostridium perfringens (17), Peni- by an enzyme distinct from fructose-1, 6-diphos-cilium notatum (261), Aspergillus sp. (6, 187), phate aldolase (58).Bacilus subtilis (124), Escherichia coli (361) Transaldolase, (table 2, reaction 4) a new typeand Lactobacillus bifidus (228). Low activity of aldolase active in the transfer of a dihydroxy-was detected in Pseudomonas fluorescens (431) acetone group from sedoheptulose-7-phosphatewhereas Leuconostoc mesenteroides was devoid to glyceraldehyde-3-phosphate forming fructose-of demonstrable aldolase and isomerase activity 6-phosphate and erythrose4-phosphate (182,(93). 183), was first found in yeast (176, 182). This

Fructose-1-phosphate aldolase, (table 2, reac- enzyme is present in plant and animal tissuestion 2) which cleaves fructose-i-phosphate to (10, 176) and in aerobic bacteria including Pseu-dihydroxyacetone phosphate and D-glyceralde- domonas fluorescens (431), Microbacterium lacti-hyde, was obtained from liver (164,244,245). This cum (393) and Acetobacter suboxydans (161).enzyme is inactive toward fructose-1, 6-diphos- The specificity for dihydroxyacetone is similarphateand is thus distinct from the Meyerhof-Loh- to that of fructose-1, 6-diphosphate aldolase formann, fructose-1, 6-diphosphate aldolase (164). dihydroxyacetone phosphate (182, 183). Trans-The conversion of fructose-i- to fructose-6-phos- aldolase, however, transfers but does not liberatephate as originally observed (80) has been shown dihydroxyacetone.to occur by the combined action of fructose-l- Deoxyribose-5-phosphate aldolase (table 2,phosphate aldolase, triosephosphate isomerase, reaction 5) catalyzes the reversible cleavage offructose-i, 6-diphosphate aldolase and fructose- deoxyribose-5-phosphate to acetaldehyde and1, 6-diphosphate phosphatase (164, 245). Evi- D-glyceraldehyde-3-phosphate (322). The en-dence for an additional pathway of fructose zyme is found in Corynebacterium sp. and wasdegradation via fructose-i-phosphate in muscle purified from E. coli extracts by Racker (322).has been presented (80). The equilibrium favors deoxyribose-5-phosphate

Phosphoketotetrose aldolase. Liver preparations formation; no cofactors have been demonstratedcatalyze an aldolase-type of condensation of for the purified enzyme.dihydroxyacetone phosphate and formaldehyde 2 - Keto - 3 - deoxy - 6 - phosphogluconate aldo-to erythrulose-i-phosphate (table 2, reaction 3) lase (table 2, reaction 6) cleaves 2-keto-3-deoxy-


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19551 PATHWAYS OF CARBOHYDRATE METABOLISM IN MICROORGANISMS 87

6-phosphogluconate (KDPG) to pyruvate and broadened the concepts of their importance inD-glyceraldehyde-3-phosphate. This enzyme was the diverse pathways of carbohydrate metabo-discovered in Pseudmonas saccharophila (262, lism. Especially noteworthy are the acceptor and263) and has been purified from this organism transfer reactions of transaldolase and transketo-and from Pieudomonas fluore8cens extracts (223). lase for 4, 5, 6, and 7 carbon aldose and ketoseThe enzyme converts the KDPG, formed from mono- or diphosphates, and the new aldolase6-phosphogluconate by 6-phosphogluconate de- cleavages of 2-keto-3-deoxy-6-phosphogluconatehydrase, to pyruvate and D-glyceraldehyde-3- forming glyceraldehyde-3-phosphate. These find-phosphate (223, 263). The KDPG aldolase does ings assign to the triosephosphates a role com-not cleave fructose-i,6-diphosphate nor deoxy- mon to several pathways of sugar metabolismribose-5-phosphate (223). The function of this re- (figure 1) similar to the position long enjoyedaction in carbohydrate metabolism will be dealt by pyruvate. Those reactions that are commonwith in greater detail in section II, 3. to triose- and hexosephosphates, i.e., isomerases

Trarwketolase transfers glycolaldehyde among and the enzymes forming the triosephosphatesa number of aldose phosphates (175, 323). by cleavage and transfer, have been discussedLike transaldolase, this is a transfer and not a in the foregoing section dealing with Aldose andcleavage enzyme, i.e., does not form free glycol- Ketose Reactions.aldehyde. Glycolaldehyde donors among the a. Dehydrogenases. The role of the triosephos-ketose phosphates include ribulose-5-phosphate, phates in oxidation and reduction reactions tofructose-6-phosphate and sedoheptulose-7-phos- form 1,3-diphosphoglyceric acid from D-gly-phate; the acceptors include ribose-5- and deoxy- ceraldehyde-3-phosphate and a-glycerol phos-ribose - 5 - phosphates, glyceraldehyde - 3 - phosphate from dihydroxyacetone phosphate wasphate, glyceraldehyde and glycolaldehyde and recognized in the earliest studies of fermentationform the corresponding ketoses, as will be shown mechanisms.in figure 5 (section II, 3). Hydroxypyruvate also Glyceraldehyde-3-phosphate dehydrogenase cata-serves as glycolaldehyde donor producing CO. lyzes the DPN mediated oxidation of D-gly-Diphosphothiamine and a divalent metal serve ceraldehyde-3-phosphate with incorporation ofas cofactors presumably as a carbanion acceptor inorganic phosphate (292, 411) as follows:and donor (175, 323). Transketolase has been D-glyceraldehyde-3-phosphatehighly purified from spinach leaves and liver + DPN+ + Pi - 1,3-diphosphoglyceric acid(178) and crystallized from yeast (87). The + DPNH + H+importance of these transfer reactions in hexose The crystalline dehydrogenase catalyzes themetabolism is discussed in section II, 3. analogous oxidation of acetaldehyde in the

presence of phosphate to acetyl phosphate (158).The crystalline dehydrogenase from yeast (411)

Until recently, the triosephosphates, gly- and rabbit muscle (78) has been used in ex-ceraldehyde-3-phosphate and dihydroxyacetone tensive studies of the reaction mechanism (37,phosphate were considered only as obligatory in- 79, 225, 308, 342, 395). The enzyme contains 2termediates in glycolysis (Embden-Meyerhof fer- molecules of bound DPN and is sensitive tomentation pathway) in which they arise by the sulfhydryl reagents. An enzyme bound -SHreversible cleavage of fructose-i, 6-diphosphate group is implicated in the acyl generation mecha-by its specific aldolase, and undergo further nism. The complete reaction requires inorganicreaction by interconversion catalyzed by triosephosphate which can be replaced with arsenatephosphate isomerase and by oxidation of the yielding 3-phosphoglycerate (291, 411)-pre-glyceraldehyde-3-phosphate to 1 ,3-diphospho- sumably via 3-phosphoglyceryl-1-arsenate, whichglyceric acid by its specific dehydrogenase. A hydrolyzes spontaneously. Glyceraldehyde-3-side reaction of dihydroxyacetone phosphate phosphate dehydrogenase has been shown inleading to glycerol via a-glycerol phosphate is fermentative bacteria, Leuconostoc mesenteroidescatalyzed by its specific dehydrogenase. (93), Clostridium butyricum (125) and manyThe many reports of new enzymatic reactions aerobic bacteria, Microbacterium lacticum (393),

of both triosephosphates have, however, greatly Bacillus subtilis (124), Pseudomonas fluorescens


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IFRUCTOSE-6-PO41 IDESOXYRIBOSE- 5-PO4

DIHYDROY- ACEAA WEHYDRIBULOSE-5-PO4 ACETONE-E D2-KETO-3-DESOXY-

X6-PO4GLUCOATEGLYCOLALDEHYDE- PYRUVATE

DPT-ENZIGLYCERALDEHYDE-3-P 41IDIHYDROXYACETONE- P04 1

FRUCTOSE-1g6-P41 +2H |PYRUVATE!D 2 0-GLYCERALDEHYDE

D-c- GLYCEROL-PO4L-e-GLYCEROL-PO4 IFRUCTOSE-1-PO41

Figure 1. Reactions of triose phosphate

(431), Escherichia coli (361) and Penicillium glyceric acids in TCA extracts of fermentingnotatum (261). cells. These constitute suggestive but not con-

a-Glycerol phosphate dehydrogenase (2 types) clusive enzymatic data.present in muscle oxidize L-a-glycerol phosphate c. Enolase reversibly dehydrates 2-phospho-to dihydroxyacetone phosphate. The soluble glycerate to 2-phosphoenolpyruvate (259). Thetype dehydrogenase is DPN mediated (1) enzyme, crystallized as the mercury salt fromwhereas a particulate type is thought to be yeast by Warburg and Christian (412, 413), iscytochrome linked (142). The soluble glycerol fluoride sensitive in the presence of phosphate,phosphate dehydrogenase was crystallized from presumably through formation of an insolublemuscle (16), and the equilibrium shown to fluoro-phosphate with the enzyme bound magne-favor glycerol phosphate formation. The cyto- sium cofactor (259). In the mid 1930's, phospho-chrome linked enzyme is less well understood glycerate accumulation before a fluoride blockbut appears to function only in glycerol phos- was considered to be a specific criterion of anphate oxidation. The washed particles do not Embden-Meyerhof pathway in bacteria includ-reduce cytochrome b, cytochrome c, flavine ing lactic and propionic acid bacteria, entero-adenine dinucleotide, nor ferricyanide (381). For bacteria, bacilli, clostridia and several Azoto-discussion of the oxidation of glycerol by bac- bacter species (364).teria, see section III, c. d. Transphosphorylases. The energy liberateP

b. Mutase. Phosphoglyceromutase equilibrates during glycolysis and accumulated in the energy-3- and 2-phosphoglycerates via a 2,3-diphospho- rich, mixed anhydride linkages of 1 ,3-diphospho-glycerate coenzyme, as indicated in the Aldose glyceric acid and phosphoenolpyruvate is trans-and Ketose Reactions section. This enzyme has ferred by specific transphosphorylases or kinasesbeen studied in bacteria only by detection of (72) to adenosine diphosphate (ADP) to formphosphoglycerate before a fluoride block (see only slightly less energy-rich phosphate anhy-section I, 3) and by demonstration of phospho- dride bonds of adenosine triphosphate.


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1955J PATHWAYS OF CARBOHYDRATE METABOLISM IN MICROORGANISMS 89

Phosphoglyceric acid transphosphorylase was living cells of Escherichia coli (74), Propionibac-crystallized from yeast by Bicher (43) and shown terium arabinosum (426) and Clostridium aceto-to transfer phosphate to ADP but not to adeno- butylicum (84). Phosphate ester accumulationsine monophosphate, forming ATP. This enzyme (hexosemonophosphates and hexosediphosphate)with triosephosphate dehydrogenase catalyzes was observed with Streptococcus faecalis (309),the incorporation of inorganic phosphate into Lactobacillus casei (401), Staphylococcus aureusan anhydride (pyrophosphate) linkage as fol- (122), Brucella suis (330) and propionic acidlows (275): bacteria (422).

D-glyceraldehyde-3-phosphate jD-3-phosphoglycerate+ DPN+ + ADP + Pi l+ DPNH + H+ + ATP

Phosphopyruvate tranmphosphorylase transfers Phosphoglycerate accumulation in growingphosphate from phosphoenolpyruvate to ADP cells was shown by addition of M/50 sodium fluo-to form ATP. The crystalline enzyme has been ride to the culture medium. Colon-aerogenes,prepared from muscle (227), and the reac- lactic acid, propionic acid bacteria, among othertion shown to be reversible with magnesium organisms (362), accumulated significant(35, 36) and potassium ions (235). These trans- amounts of phosphoglycerate. The amountphosphorylases have not been studied directly accumulated varied greatly with the organismin microbial extracts but are presumed to func- and conditions of culture, i.e., with the pro-tion in the generation of ATP during growth pionic acid bacteria, the formation of a fluorideand fermentation. resistant strain during growth was indicated

(420). The accumulation of phosphoglycerate,3. Evidence of an Embden-Meyerhof Pathway snasidctdb brumpeptbeognc

Bacteria ~~as indicated by barium precipitable, organicBacteria phosphate stable to hydrolysis and by opticalGlycolysis by the Embden-Meyerhof pathway, rotation of the molybdate complex (399), was

as found in yeast and muscle (276), was long widely interpreted as evidence of an Embden-considered a main pathway of carbohydrate Meyerhof glycolysis pattern.fermentation in microorganisms with the diver- Conversion of phosphate esters such as hexose-sity of products attributed to the many pyruvate diphosphate, phosphoglycerate and glycerolreactions observed in microbial extracts, cell phosphate to fermentation products, or to otherpreparations, or in growing cells (115). Evidence glycolytic intermediates, at a rate compatiblefor an Embden-Meyerhof pathway from glucose with the over-all fermentation rate was alsoto pyruvate in microorganisms was based on: considered evidence for this glycolytic scheme in(a) production and utilization of postulated microorganisms. Some limitation to the ap-intermediates; (b) the presence of enzymes cata- proach resides in reduced reaction rates in ex-lyzing reactions of the Embden-Meyerhof path- tracts due to limiting levels of hydrogen or phos-way; (c) sensitivity of these enzymes to inphate donors (or acceptors) and of coenzymes. Ahibitors effective with muscle and yeast enzymes, fluoride sensitive system of E. coli (116) and ofi.e., iodoacetate, m/1,000, and NaF, M/50; propionic acid bacteria (420) was shown in theand (d) fermentation of C"I labeled glucose to presence of acetaldehyde as hydrogen acceptor,products labeled as predicted by Embden- to degrade hexosephosphate esters to pyruvate,Meyerhof pattern, i.e., glucose-3,4-C'4 to car- inorganic phosphate, reducing the aldehyde toboxyl labeled lactate or to labeled CO2 in the ethanol. The phosphoglycerates, which are at theyeast ethanol fermentation (figure 8). The data oxidation level of pyruvate rather than glucose,accumulated with microorganisms were frag- form pyruvate and orthophosphate with Myco-mentary, however, since complete evidence of bacterium phlei (114) and with extracts ofthe glycolytic process was not accumulated for Lactobacillus sp. (200) and Leuconostoc sp. (200).any one organism. Glycolytic enzymes which serve in the Emb-

Pyruvate as an intermediate in fermentation den-Meyerhof pathway have been demonstratedwas partially validated by demonstration of its in numerous bacteria; and their presence isaccumulation in the presence of bisulfite with cited as evidence for this system. Such evidence,


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discussed also in the foregoing portion of this hydrate breakdown have accumulated until it ismanuscript, is not proof of the scheme or of the clear that this is not the sole pattern of carbohy-order in which the enzymes serve since diverse drate degradation in either fermentative or oxi-routes to triosephosphates and pyruvate are now dative microorganisms. The early experiments ofamply documented, as outlined in the section on Warburg and Christian (405, 406, 407, 408), ofhexosemonophosphate pathways (see, for ex- Dickens (98, 99, 100) and of Lipmann (249)ample, figure 1). Thus, only phosphohexokinase, demonstrated the terminal oxidation of glucose-aldolase specific for fructose-1, 6-diphosphate, 6-phosphate as far as pentose phosphate. Atand triosephosphate isomerase, enzymes that that time, an oxidative route, termed a hexoseconvert fructose-6-phosphate via fructose diphos- monophosphate pathway or "shunt", was visu-phate to an equilibrium mixture of glyceralde- alized as successive terminal oxidations and re-hyde-3-phosphate and dihydroxyacetone phos- moval of single carbon atoms bypassing thephate, are unique to the Embden-Meyerhof glycolytic pathway, at least until the triosepathway. All other enzymes of this pathway are stage. Recently, it has become clear that thisalso common to other known pathways of both type of stepwise oxidation and decarboxylationaerobic and anaerobic carbohydrate metabolism. is not the main route of aerobic carbohydrateThe fermentation of glucose-C14 by a variety of degradation, but rather, after the initial oxida-

lactic acid bacteria (127, 316), clostridia (18, tion to aldonic or ketoaldonic acids, cleavage by310, 428), and bacilli (293) has furnished data specific aldolases occurs with further oxidationcompatible with the postulates of the Embden- of the 3, or 2 and 3 carbon fragments. The fol-Meyerhof pathway and with the data obtained lowing discussion deals with the specific enzymesby its fermentation with muscle enzymes. Thus, of hexosemonophosphate pathways.glucose-l-C14 yielded methyl labeled products.Glucose-3,4-C14 yielded carboxyl labeled lactate 1. Oxidationsin Lactobacillus casei (127), but with Clostridium a. Glucose-6-phosphate dehydrogenase. One ofthermoaceticum the methyl of acetate was more the earliest deviations from the Embden-Meyer-heavily labeled although both carbons of acetate hof pathway was reported by Warburg and Chris-became labeled (18, 428) in keeping with their tian (405, 406, 407, 408) who found that bothequilibrium with C1402 in this organism. yeast preparations and hemolysates of erythro-

Other data inconsistent with an Embden- cytes oxidize glucose-6-phosphate to 6-phospho-Meyerhof pattern were obtained by fermenta- gluconate. The enzyme, glucose-6-phosphatetion of glucose-l-C14 with cells of Leuconostoc dehydrogenase or "Zwischenferment", was sub-mesenteroides (150), Propionibacterium pento- sequently purified from red cells (408) and fromsaceum (236, 328) and Pseudomonas saccharophila yeast (218, 292a) and shown to reduce specifically(118). These data and the consequent enzyme a new coenzyme, triphosphopyridine nucleotideexperiments clarifying the pathways in Leuco- (TPN) (408). Thus from its discovery, TPN wasnostoc (95, 97) and P. saccharophila (118, 263) associated with oxidative pathways as a counter-(figure 8) have amply repaid the labor invested part of diphosphopyridine nucleotide (DPN)in checking what at first glance appeared to be in the fermentative systems. The product ofadequate data. Similar data for E. coli (68, 246) glucose-6-phosphate oxidation, 6-phosphoglu-and Streptomyces sp. (66) have more recently conate, also was isolated by Warburg andbecome available and are included in section II Christian (408).on Hexosemonophosphate Reactions and Path- More recently, Cori and Lipmann (81) haveways. In summary, then, Embden-Meyerhof shown the enzymatic oxidation of glucose-6-glycolysis occurs in microorganisms as a means, phosphate, in common with the chemical oxida-but not the sole means, of energy liberation and tion, to proceed in two steps: (a) oxidation ofcarbon transformation. the pyranose ring of glucose-6-phosphate to 6-

phospho-5-gluconolactone, and (b) hydrolysisII. HEXOSEMONOPHOSPHATE REACTIONS of the lactone ring (figure 2). The hydrolysis,AND PATHWAYS which occurs slowly at neutral pH, is catalyzed

In recent years, exceptions to the Embden- by a specific delactonizing enzyme. Brodie andMeyerhof scheme as "the" pathway of carbo- Lipmann (40, 41) have shown 6-gluconolactone


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OH 0 0- 9-0

S

H-C C C-O

I~_IIH-C-OH TPN H-C-OH + OH _H-C-OHHO-C-H l TPNH - HO-C-H -OH HO-C-HH-C-OH IH-C,-OH |H-C-OHI I~~~~~~~~~~II

H-C | H-C- | H-C-OHHfC-OPO12 H-C-OPOh H(COPd

GLUCOSE - 6-PO4 6-PO4-6-GLUCONO- 6-PO4-GLUCONATELACTONE

Figure B. Oxidation of glucose-6-phosphate

to be hydrolyzed by a similar delactonizing en- product (409, 410). Lipmann (249) characterizedzyme in extracts of Azotobacter vinelandii and of the reaction as an oxidative decarboxylationyeast. The equilibrium for the over-all reaction and suggested that arabinose-5-phosphate wouldfor both glucose and glucose-6-phosphate oxida- be formed. With a partially purified yeast prepa-tion is far to the right, but the oxidative steps ration, Dickens (99, 100) obtained evidence(reaction 1) are readily reversible in the presence for the accumulation of pentose phosphate, whichof reduced TPN (TPN-H) and phosphoglucono- he believed to be ribose-5-phosphate rather thanlactone (177) or DPN -H and gluconolactone, arabinose-5-phosphate, since the former wasrespectively, (367). rapidly metabolized by yeast extracts whereas

Glucose-6-phosphate dehydrogenase has been the latter was not. Thus, ribose-5-phosphate wasfound in a variety of animal tissues (101, 136, assumed to be an intermediate in the oxidation137, 138) and in numerous microorganisms in- of glucose-6-phosphate by yeast.cluding Escheichia coli (338), Pseudomonas More than ten years later, Scott and Cohenfluorescens (431), P. saccharophila (118), P. (337) confirmed the formation of ribose-5-phos-lindneri (94), Azotobacter vinelandii (281), phate as a product of phosphogluconate oxidationBacilus subtilis (85), Bacius megaterium (85), by yeast preparations, and, subsequently, Ho-Leuconostoc mesenteroides (96), and Streptococcus recker and Smyrniotis (168) isolated ribose-5-faecalis (350). The E. coli enzyme has been pu- phosphate as a product of 6-phosphogluconaterified and shown to require magnesium or cal- oxidation with a purified enzyme preparationcium ions for activity (338). from yeast. The apparent anomaly in the con-The high affinity of glucose-6-phosphate dehy- figuration of the three hydroxyl group of glucose

drogenase for glucose-6-phosphate has permitted which corresponds to arabinose rather than theits use for quantitative determination of hexose- ribose was assumed to result from epimerizationmonophosphates, TPN, ATP, and also of several of carbon 3 of glucose by a hydrogen shift viaenzymes including hexokinase, phosphohexose enol or enediol intermediates (101, 337). Horec-isomerase, and phosphoglucomutase. Although ker, Smyrniotis and Seegmiller (170), however,TPN is an obligatory coenzyme for the dehydro- isolated and identified the product of 6-phospho-genase from animal sources (408), in crude prepa- gluconate oxidation by purified 6-phosphoglu-rations of Leuconowtoc mesenteroide (96) and conic dehydrogenase (169) as a ketopentose,Pseudomonas fluorescens (431), DPN also serves ribulose-5-phosphate. Ribulose-5-phosphate ac-as a hydrogen acceptor. The enzyme from L. cumulated first in the reaction mixture and wasmesenteroides reacts with both nucleotides (96) converted, subsequently, to ribose-5-phosphatewhereas that from P. fluorescens reduces DPN (figure 3). The latter reaction is catalyzed byvia a TPN-specific dehydrogenase coupled with traces of a pentose phosphate isomerase alsopyridine nucleotide transhydrogenase (73). present in the preparation. At equilibrium, ribose-

b. 6-Phosphogluconate dehydrogenase. The fur- 5-phosphate and ribulose-5-phosphate werether degradation of 6-phosphogluconate was first present in a three to one ratio. The earlier ap-demonstrated in yeast by Warburg and Christian pearance of ribulose-5-phosphate was attributed(406, 407, 408) who found TPN to be the coen- to the oxidation of 6-phosphogluconate at thezyme (410) and obtained evidence for a C5 third carbon atom (168), but the suggested


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CO2O= ,C-O + HH- ,C-OH H2-C-OH HO-C

I IC=O C=O H-C-OHI ~ ~ ~ ~ ~~~~~~II/-~-O H-C-OH H-C-OH-

_CC H-C-OH H-C-0P0HHO=C-0- L C-H C-H PYUVT

I=C- H27C-OPOJ H2-C OP03H2H2C-OHHCOP3H2H-C-OH

IO¢- RIBULOSE-5-PO4 RIBOSE-5-PO4H- C-OH

I -~~~~~2KEO3DEOY

H- ,-OH O`=C, O

H24-CPOP2 0 - O=C,-0 C=O

6-PO4-GLUCONATE | 1IH|3_6-P-GLCN , G YRALE

Fu 8H-CR-Oei H-6-OH +H- ,-OH H-C-OH H-C=O

LH2-C-OPO4rH Hi-C-OPOA2 H-C-OH

2-KETO-3-DESOXY- -aCO0H-6-PO4-GLUCONATE GLYCERALDEHYDE-

3_P04Figure S. Reactions of 6-phosphogluconate

intermediate, 3-keto-6-phosphogluconate, has terial extracts, but the details of the mechanismnot been detected. The lack of evidence of 3-keto- have not been studied. Extracts of Escherichia6-phosphogluconate has led to a suggestion that coli (337) oxidize 6-phosphogluconate, but anthe enzyme possesses two functions, dehydro- analysis of the endproducts produced by purifiedgenation and decarboxylation, similar to that preparations was not reported. Phosphogluconicpostulated by Ochoa (306) for the malic enzyme dehydrogenase has been demonstrated in Bacil-mechanism. lus subtilit, B. megaterium (85), Azotobacter

Although the equilibrium of 6-phosphoglu- vinelandii (281), Leuconostoc mesenkeroides (97),conate oxidation favors oxidative decarboxyla- and Pseudomonas fluorescens (431).tion, the reaction is reversible as demonstrated The oxidation of phosphogluconate to formby the enzymatic fixation of C1402 into carbon keto-6-phosphogluconates has been postulatedatom one of 6-phosphogluconate and by the repeatedly (98, 168, 249, 337, 383), and thereductive carboxylation of ribulose-5-phosphate production of 2-ketogluconate and 5-ketogluco-in the presence of TPNH and C02 (172). The nate from glucose by oxidative organisms lendsequilibrium of lactonization (figure 2) is far to- credence to this possibility. Thus, Uehara (383)ward hydrolysis, and hence would appear to proposed an alternate mechanism of phospho-limit the rate of 6-phosphogluconolactone forma- gluconate oxidation which does not involve directtion to a level incompatible with net hexose oxidative decarboxylation as follows: (a) oxi-regeneration via this route. Recent evidence dation of 6-phosphogluconate to 2-keto-6-demonstrates that photosynthetic carbon diox- phosphogluconate; (b) cleavage of 2-keto-6-ide incorporation into hexose occurs via ribulose- phosphogluconate to hydroxypyruvate and1, 5-diphosphate, which is the primary carbon glyceraldehyde-3-phosphate; (c) transketolasedioxide acceptor in a carboxylation and cleavage transfer between hydroxypyruvate and glycer-reaction which forms phosphoglycerate (318, aldehyde-3-phosphate to yield ribulose-5-phos-418, 419). phate. The proposed mechanism appears unlikely

Pentose phosphate formation occurs in bac- since the yeast preparations which form ribulose-


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5-phosphate from 6-phosphogluconate do not gluconate dehydrase. This enzyme has thus farutilize hydroxypyruvate and glyceraldehyde-3- been reported only in the Acetobacter (223),phosphate (181). Pseudonwnas (223) and Azotobacter species (281).A new pathway of phosphate ester degrada- 3 Cl and Tranfer

tion involving phosphorylated 2-ketogluconate,presumably 2-keto-6-phosphogluconate, has been Ribose-5-phosphate degradation, both aerobicfound in Pseudomonas fluorescene by Narrod and anaerobic, has been observed in red celland Wood (289) and an Aerobacter cloacae by hemolysates (103), in extracts of liver and yeastDeLey (88, 89). The intermediate is further (100, 135, 333) and in microorganisms (25, 26,degraded with the accumulation of phospho- 266, 320, 403). Although Dickens (99) reportedglycerate with the A. cloacae preparation (89) an oxidation of pentose phosphate to phospho-or to two moles of pyruvate with the extract pentonic acid and Sable (333) demonstrated aof P. fluorescens (290). The evidence for these TPN requirement for ribose-5-phosphate oxi-reactions is detailed in section II, 6c. dation, a pentose phosphate dehydrogenase has

not been found. Thus, experiments of Sable (333)2. Isron,Phoyphrlaon and and of Glock (135) suggested an oxidation after

Dehydration a primary attack upon ribose-5-phosphate.a. Phosphopentose isomerase. Pentose phos- Red cell hemolysates (103) and liver extracts

phate isomerase catalyzes an equilibrium be- were shown to convert adenosine and ribose-5-tween ribose-5-phosphate and ribulose-5-phos- phosphate to hexosemonophosphate and hexose-phate, thus clarifying the earlier problem of diphosphate (103, 135, 333). These data wereribose-5-phosphate formation from phosphoglu- interpreted as indicating a cycle in hexose-conate by crude extracts (169). This isomerase is monophosphate oxidation. However, with thesefound in plants (175), animals (173), and yeast preparations glucose-6-phosphate was not formed(168); Axelrod and Jang (11) have purified the from fructose-1,6-phosphate (102). Seegmillerenzyme from alfalfa and studied its characteris- and Horecker (341) with liver and bone marrowtics. The alfalfa enzyme does not isomerize extracts demonstrated the degradation of 6-glucose-6-phosphate or triosephosphates. phosphogluconic acid with a transient accumula-

b. Phosphoribuolcinase. This enzyme has tion of pentose-presumably a mixture of ribu-been purified from spinach extracts and shown lose- and ribose-5-phosphates-followed by theto catalyze the phosphorylation of ribulose-5- formation of glucose-6-phosphate. Later it wasphosphate with the formation of a diphosphate shown that with these preparations, fructose-i, 6-ester of ribulose (418). Ribulose diphosphate diphosphate did not yield glucose-6-phosphate,has been found in extracts of algae and green thereby, eliminating as a mechanism an aldo-leaves (22). Its occurrence in nonphotosynthetic lase condensation of triosephosphates to fruc-microorganisms has not been reported. tose-1,6-diphosphate and its conversion to

c. 6-Phoaphoguconatee dehydrate. 6-Phospho- glucose-6-phosphate by the combined action of agluconate degradation to pyruvate and triose- 1-phosphofructophosphatase and phosphohexosephosphate by enzymes from Pweudomonas isomerase.saccharophila (118) was shown by MacGee Pentose phosphate cleavage to triosephos-and Doudoroff (262, 263) to occur by way of an phate was shown in red cells by Dische (102,intermediate which was isolated and charac- 103) and in bacteria by Marmur and Schlenkterized as 2-keto-3-deoxy-6-phosphogluconate (266). A more detailed study of a similar reaction(figure 3). The enzyme has been purified from with the extracts of yeast and of Escherichia coliextracts of Pseudmonas fluorescens by Kova- by De la Haba and Racker (86) and with yeastchevich and Wood (222) and shown to require and brain tissue by Sable (333) indicated thatglutathione and ferrous ions for activity. The several enzyme steps participated in this path-data are consistent with a mechanism in- way. Efforts to isolate and identify the remainingvolving dehydration between carbons 2 and 3 two-carbon fragments at the oxidation level offollowed by a tautomeric shift of hydrogen as glycolaldehyde were unsuccessful.postulated by Entner and Doudoroff (118); a. Transketolase. The pentose phosphate cleav-hence, the enzyme has been termed 6-phospho- ing enzyme is found in plant (10, 175), animal


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C-OI H2- -OH H-C-OC=O IIH-C-OH + DPTENZ ]-C= + H-C,-OHI I H-C=OH-C4-OH [NDPFENZ-C-OJH-C0-pC-OPO3-H2-C-OPO03 a ACTIVE GLYCERALDEHYDE-

RIBULOSE-5-PO4 GLYCOLALDEHYDE 3-PO4

H H?-v-OHHOC [ ]HO-C-Y

I ~~ ~H-C-OHIH C+ H-C-ODPFENZ + H-C-OH 0H-C---]C[ OHH2-C-OPO DPI-ENZ I

RIBOSE-5-PO4 GLYCOLALIDEHYDE H-C-OPOSEDOHEPTULOSE-

7- P04Figure 4. Sedoheptulose-7-phosphate formation (Transketolase)

(174), and bacterial cells (87, 281a, 393, 431) and phate (87), ribose-5-phosphate (173) and deoxy-has been crystallized from yeast by Racker, De ribose-5-phosphate (325) (figure 5). In linela Haba and Leder (323) and highly purified with these observations both Horecker et al.from spinach and from liver by Horecker, Smyr- (180) and Racker and coworkers (324) haveniotis and Klenow (178). This enzyme contains obtained evidence that transketolase transfersdiphosphothiamin (DPT) as a tightly bound two carbon units between fructose-6-phosphateprosthetic group (175, 323). The DPT was re- and glyceraldehyde-3-phosphate, forming ribu-moved by dialysis against ethylenediamine lose-5-phosphate and a tetrose tentatively identi-tetraacetic acid (versene) or by precipitating fied as D-erythrose4-phosphate. Purified trans-the enzyme at an acid pH. Ribulose-5-phosphate ketolase lacks a high degree of stereospecificityrather than ribose-5-phosphate is the substrate in that carbohydrates containing both cis andcleaved, the reaction occurring only if an ap- trams configurations at carbons 3 and 4 arepropriate acceptor for the two-carbon fragment split and formed (325).also is present (323) (see figure 4); thus the Akabori, Uehara and Muramatsu (3) reporttransketolase is a transferase. The spinach and the formation of pentose phosphate from triose-liver preparations (178) contain pentose phos- phosphate and dihydroxymaleic acid by a mecha-phate isomerase which interconverts ribulose- and nism believed to involve the formation of hy-ribose phosphates, with the latter serving as droxypyruvate and its decarboxylation to "active"active glycolaldehyde" acceptor to yield sedo- glycolaldehyde".heptulose-7-phosphate and glyceraldehyde-3- Free sedoheptulose accumulates in plantsphosphate. Sedoheptulose phosphate has been (230a) and is found as sedoheptulose-7-phosphateisolated and identified by chemical means (173, in photosynthesizing algae (23). Heptulose178). Among the compounds which serve as phosphate formation from ribose-5-phosphate,precursors for "active glycolaldehyde" are hy- presumably by transketolase, also has beendroxypyruvate (87), L-erythrulose (178), fruc- observed in extracts of Rhodospirillum rubrumtose-6-phosphate (324), sedoheptulose-7-phos- (23), Pseudomonas fluorescens (431), Microbac-phate (178) and D-xylulose-5-phosphate (372). terium lacticum (393), and Azotobacter vinelandiiLikewise, several aldehydes can serve as "active (281a). Fractionation of growing Azotobacterglycolaldehyde" acceptors including glycolalde- vinelandii (281a) cells also yielded heptulosehyde, glyceraldehyde, glyceraldehyde-3-phos- phosphate, further strengthening the supposition


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ISEDOHEPTULOSE-7-PO.

| FRUCTOSE-6-P041 I D-RIBULOSE-5-PO41X ~~~~RIBOSE-5-P04

ERYTHROSE-4-P04 O-GLYCERALDEHYOD-3-P04

H27C-OH/

GLYCOLALDEHYOE.L[DPTENZI _" ~~~ACTIVE 0s-G CE LDH EGLYCOLALDGLYCELALDEHYDe CRADH E

IL-ERYTHRUL SE GLYCOLALDEHYDE |D-RIBULOSE I

CO2 -GLYCERALDEHYDE-3-P04

IHYDROXYPYRUVATEI L-XYULOSE-5-P041Figure 5. Reactions of transketolase

that it plays an important role in carbohydrate dilution of the C14 furnished by the top 3 car-metabolism and is not an artifact of the experi- bons of the sedoheptulose-7-phosphate (182).mental conditions. Transaldolase has been purified about 300-

b. Trarsaldolase. Pentose phosphate metab- fold from yeast (179, 182). Evidence for theolism by crude liver (171), spinach extracts presence of a coenzyme or prosthetic group or for(10) and pea (131) preparations yields sedo- the type of linkage between dihydroxyacetoneheptulose-7-phosphate which in turn is trans- and the enzyme has not been obtained. Noformed into glucose- and fructose-6-phosphates substrates thus far tested other than sedoheptu-(figure 6). Fructose-6-phosphate (135) is formed lose-7-phosphate or fructose-6-phosphate havefirst, and glucose-6-phosphate appears only if been found to act as a dihydroxyacetone donor,hexose phosphate isomerase is also present (176, and only glyceraldehyde-3-phosphate and eryth-179). The liver and yeast preparations convert rose-4-phosphate will act as acceptors (182).sedoheptulose-7-phosphate to hexosephosphates The second product of sedoheptulose-7-phos-only if triose phosphate is also added (179). phate cleavage by transaldolase is a tetrose-Evidence for transaldolase is based on: (a) the phosphate, presumably D-erythrose4-phosphateformation of glucose-6-phosphate labeled in (179, 183). The evidence for this structure is: (a)carbon atoms 4, 5, and 6 with uniformly labeled a condensation of this product with dihydroxy-fructose-1,6-diphosphate (source of triosephos- acetone phosphate, catalyzed by muscle aldo-phate) and unlabeled sedoheptulose phosphate as lase, forms sedoheptulose-1 ,7-diphosphate, andthe substrates (182); and (b) free dihydroxy- (b) a reaction with "active glycolaldehyde"acetone added with labeled sedoheptulose- (transketolase) formed fructose-6-phosphate.7-phosphate does not enter the hexosemono- Sedoheptulose-1,7-diphosphate serves as anphosphate fraction (fructose-6-phosphate + efficient precursor of shikimic acid in E. coliglucose-6-phosphate) as indicated by lack of extracts (195).


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96 I. C. GUNSALUS, B. L. HORECKER, AND W. A. WOOD (VOL. 19H -C-OHHO-CHO--H-C-O H H-C=OH-V-OH I + ENZ Z=+ H8H-C-OH .7 -O-H H- -OHH i ENZ H2-C-OP03H2

Hi-C-? PH2 ERYTHROSE-4-PO4

SEDOHEPTULOSE-7-PO4

Ha-V-OH

I-E =I HO- -H~

H-?-OH + [HI-C-OENZ + H m

H2-C-0P03H2.GLYCERALDEHYDE-3- H2-C-OPO3H2

P04 FRUCTOSE-6-PO4Figure 6. Fructose-6-phosphate formation (transaldolase)

c. Aldolases. In addition to fructose diphos- whether or not a particular intermediate accumu-phate aldolase which also splits sedoheptulose- lates depends upon the number and amount of1, 7-diphosphate (183), specific aldolases which enzymes present since these in turn dictate thecleave 2-keto-3-deoxy-6-phosphogluconate and amount of donors and receptors which are presentdeoxyribose-5-phosphate are found in bacteria. for transfer reactions.The former, postulated by Entner and Doudoroff Evidence as to whether one or two dehydro-(118) as part of the mechanism of pyruvate and genations occur at carbon one of glucose is fur-glyceraldehyde-3-phosphate formation, has been nished by measuring the rate of carbon dioxidepurified from extracts of P. fluorescens by Kova- release from glucose--C1 as compared to that ofchevich and Wood (223) and has been found in glucose labeled in other positions. The preferen-several Pseudomonas and Acetobacter species and tial release of carbon atom one by tissue slicesin Escherichia coli (223). No cofactor requirement (2, 32, 33, 68) and by E. coli (68), Streptomyces sp.has been found. Although accurate determina- (66), Aspergiusniger (5), and yeast (20,246) hastions have not been made, at equilibrium the been demonstrated.ratio of pyruvate to 2-keto-3-deoxy-6-phospho- a. Interconversione of 6, 6 and 7 carbon phosphategluconate appears to be about 3:1. esters. Complete oxidation of phosphate esters to

Deoxyribose-5-phosphate aldolase has been puri- carbon dioxide via a hexosemonophosphate path-fied from E. coli extracts by Racker (322) and way can be visualized as shown in figure 7. Theshown to catalyze the reversible formation of cyclic process catalyzed by the combined actiondeoxyribose-5-phosphate from acetaldehyde and of glucose-6-phosphate dehydrogenase, 6-phos-glyceraldehyde-3-phosphate. Crude extracts of phogluconate dehydrogenase, pentose phosphateE. coli form deoxyribose-5-phosphate from ribose- isomerase, transketolase, transaldolase, and hex-5-phosphate when acetaldehyde is present. osephosphate isomerase could yield one mole of

carbon dioxide and two moles of reduced pyridine4. Hexosemonophosphate Oxidation Pathways nucleotide, the latter in turn being reoxidized byThe distribution and importance of the several one mole of oxygen (R.Q. = 1). Only the first two

pathways for carbohydrate oxidation are not steps of such a cycle are oxidative, the second ofcompletely clear. It is evident, however, that these being accompanied by decarboxylation tomore than one pathway exists and that several yield pentose phosphate. Succeeding anaerobicmay contribute to the over-all rate of substrate reactions can regenerate fructose-6-phosphateoxidation. Which pathway is involved and and glucose-6-phosphate by three possible means:


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H

H ] __ HCOHHO-C+H O -2H HO6-H

TPN H6-&OH~

D-PO; H2-C-OPO3GLUCOSE- 6- PO4 6- PHOSPHOGLUCONATE

2H TPN-0C2

IHOCH-OH.~ FC-H O _______________ H-C,-OH

3 H-C-OH H-C-OH HC-OH~ 4>HjH-C-OH H-C-OH H-0

',(Hj-C-OPO; H2-C-OPO;kRIBULOSE-5-PO4 RIBOSE-5-PO4j

iH2-C-OH'= vt c-= H

',HO- :H+ + COH-C-OH 42JHH-6P-O ERYTHROSE-4-PO4

(fRUCTOSE-6-PO4 J__

H-00~~~~~~UH~~-C-OPO3H" OH+\ aC-O-

HO-6-H _|GLYCERALDEHYDE- H o,HO-&H 3-PO4 -P

H-&-OH IISEDOHEPTULOSE-HZIfPO 7-PO4H2-C-0P03 H-C-OH

FRUCTOSE -1,6 di P04 &HZ- 0P05

DIHYDROXYACETONE-PO4

Figure 7. Glucose-6-phosphate metabolism: oxidative pathway

(a) the conversion of pentose phosphate to fruc- phatase is converted to fructose-6-phosphate. Thetose-6-phosphate by the transketolase-trans- fructose-6-phosphate may reenter the cycle afteraldolase sequence; (b) condensation of the tet- isomerization to glucose-6-phosphate. It is note-rosephosphate remaining from (a) with "active worthy that glucose-6-phosphate could be com-glycolaldehyde" derived from the cleavage of a pletely oxidized by repetition of this reactionthird molecule of pentose phosphate to form series, without other steps of the Embden-fructose-6-phosphate and yielding an additional Meyerhof pathway or the Krebs tricarboxylictriose phosphate; and (c) condensation of two acid cycle participating.moles of triosephosphate to form fructose-1, 6- Horecker et al. (180) have demonstrated hex-diphosphate, which in the presence of a phos- osemonophosphate formation in rat liver prepara-


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tions by the incorporation and rearrangement of covered the intermediate phosphate ester as alabeled carbon from ribose-5-phosphate-1-C14 and crystalline sodium salt and established a structureribulose-5-phosphate-2,3-C14, into the hexose as that postulated. This pathway has been dem-molecule. The labeling of the glucose-6-phosphate onstrated to be of major importance in Pseudo-isolated after enzyme action indicated two mech- monas fluorescenm (431). The enzymes convertinganisms of fructose-6-phosphate formation; one 6-phosphogluconate to pyruvate and triosephos-involving transaldolase and transketolase with phate have been separated and characterized bysedoheptulose-7-phosphate as a substrate; the Kovachevich and Wood (222, 223). The firstother involving transketolase and erythrose4- enzyme, 6-phosphogluconate dehydrase, formsphosphate with ribulose-5-phosphate as the 2 2-keto-3-deoxy-6-phosphogluconate and requirescarbon donor. glutathione or cysteine and ferrous ions for activ-

b. Routesfrom hexosemonophosphate to pyruvate. ity. The second enzyme, 2-keto-3-deoxy-6-phos-(1) Penrtose phosphate cleavage. Degradation of phogluconate aldolase, cleaves this ester to pyru-ribose-5-phosphate by Microbacterium lacticum vate and D-glyceraldehyde-3-phosphate. Evi-extracts (393) has been studied with the following dence for this alternative pathway of 6-phospho-findings: (a) under anaerobic conditions the suc- gluconate degradation has now been found incessive formation of sedoheptulose-7-phosphate several members of the genus Pseudomonas (223),and fructose-6-phosphate was observed as re- Azotobacter (281a), and in Escherichia coli (223).ported by Horecker et al. (180) and by Axelrod etal. (10); (b) aerobically, ribose-5-phosphate was 5 Hexoseophosphate Fermation Pathwaysconverted largely to pyruvate; hexose and heptu- Evidence of a hexosemonophosphate pathwaylose phosphate esters did not accumulate. It in which intermediates derived from the sub-appears that a pentose phosphate cleavage differ- strate replace oxygen as the hydrogen acceptorent from the known reactions of transketolase has directed attention to the role of this pathwayexists and that glyceraldehyde-3-phosphate de- in anaerobic processes. As presently visualized,hydrogenase competes with transaldolase by the fermentative hexosemonophosphate mech-oxidizing glyceraldehyde-3-phosphate, the poten- anism may involve coupled oxidative reactions totial three carbon acceptor. These data suggest yield 6-phosphogluconate, which is either oxidizedthat the importance of the oxidative cycle is to ribulose-5-phosphate or converted anaerobi-governed by the amount of triosephosphate cally to 2-keto-3-deoxy-6-phosphogluconate. Thedehydrogenase and the enzymes which convert pentose phosphate may be cleaved betweenphosphoglycerate to pyruvate relative to the carbon atoms 2 and 3 as in the aerobic pathwaysamount of transaldolase activity. or the 2-keto-3-deoxy-6-phosphogluconate con-

(2) d-Keto-3-eoxy-6-phosphogluconate cleavage. verted to pyruvate and triosephosphate.Doudoroff and his collaborators (118, 263) in a. Heterolactic (ethanol-lactic). A fermentativestudies with Pseudomonas saccharophila have hexosemonophosphate pathway was postulatedexpanded the concept of diverse pathways of in Leuconostoc mesenteroides by DeMoss, Bardcarbohydrate metabolism by their demonstration and Gunsalus (93) and Gunsalus and Gibbs (150)of a third pathway of hexosemonophosphate upon the following evidence: (a) equimolar quan-utilization by which glucose yields nearly two tities of carbon dioxide, ethanol, and lactate weremoles of pyruvate. The carboxyl group of one produced, and the ratio of these was constantpyruvate is derived from carbon atom one of under varying conditions; (b) aldolase could notglucose. The mechanism was suggested by the be demonstrated, (c) carbon dioxide arises fromfinding that 6-phosphogluconate is converted to carbon 1 of glucose, the lactate from carbons 4, 5pyruvate and triosephosphate under anaerobic and 6 and the methyl carbon of ethanol fromconditions (118). Since glyceric acid was not con- carbon 2 of glucose (figure 8). Although theverted to pyruvate, Entner and Doudoroff postu- mechanism is still obscure, extracts of the organ-lated the formation of a new ester, 2-keto-3- ism were shown to possess glucose-6-phosphatedeoxy-6-phosphogluconic acid, as an intermediate dehydrogenase (TPN- or DPN-linked) (95), andand cleavage by an aldolase-type enzyme to form a DPN dependent 6-phosphogluconate dehydro-pyruvate and glyceraldehyde-3-phosphate (figure genase (97). Ribulose-5-phosphate and carbon3). MacGee and Doudoroff (262, 263) have re- dioxide were among the endproducts. At present,


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co?CHI CH3

HO-C-HHO-C-O CHOH

O:C-OH 1. casel L esntrodeoH-C-OH CH3 HOHO-C-

CH I- HO-C CH3

L(+)LACTATE O='&C-OH HO-C-H D(LACTATE+ETHANOL+CO2

H-¶Z- H-!ZJCH3 CI HO-=COC/H2OH H2g-OP03H2 CH 0

'Cog CH3GLUCOSE C=O - ~~CH2OH*C0O2 0 PYRUVATE GU SD 3 P. H3'CO2 ~~~~~~~~~~~~~~P.Andoieri CH°C02 YEASTS Psaccharophiad HO-!C=O *C°2CHAOH (anaerobic) Afluorescens1 23 (P/indnerl) LC-0 CH2O

H3 CH3ETHANOL + CO2 PYRUVATE ETHANOL+ CO2

Figure 8. The distribution of glucose carbons in products from various fermentations

key problems are the steps in ethanol formation been clarified by the demonstration of a yeast-and the means of forming the three-carbon frag- type carboxylase (326). This is the distribution ofment. If the mechanism involves ribulose-5- carbon expected from a pathway generatingphosphate cleavage to "active glycolaldehyde" pyruvate by the Entner-Doudoroff cleavage ofand glyceraldehyde-3-phosphate, the extracts 6-phosphogluconate (figure 8).could convert the latter to lactate (128). Although c. Induced aldonic acid. Streptococcus faecahsthe route from "active glycolaldehyde" to ethanol can be induced by growth on gluconate or 2-keto-is obscure, whole cells of this organism reduce gluconate to fermentation by the hexosemono-acetate to ethanol. The sedoheptulose phosphate phosphate pathway as indicated by: (a) gluconatecycle cannot be involved because ethanol derived but not glucose grown cells ferment gluconate-from glucose-3,4-C'4 contains C14 only in the 1-C'4 to yield labeled carbon dioxide and labeledcarbinol group (150). lactate. The adapted cells also ferment 2-keto-

b. Bacterial ethanol. Pseudomonas lindneristud- gluconate and yield one mole each of carbon diox-ied by Kluyver and Hoppenbrouwers (209), ide and lactate, (b) hexokinase and gluconokinaseferments glucose to 1.8 moles of ethanol, 1.8 are present as are dehydrogenases for glucose-6-moles of CO2 and traces of lactate (128). The phosphate and 6-phosphogluconate. The fermen-pattern resembles that of Leuconostoc mesenter- tation mechanism is still under study (350).oides in that glucose-i-C'4 yields labeled carbon d. Pentose. Pentose fermentation, adaptive indioxide. In contrast to L. meenteroides, glucose- lactic acid bacteria (232), yields labeling patterns3,4-C14 yields 50% of the C14 of glucose as C02; in the products from pentose-1-C'4 which impli-the specific activity of the C02 was one-half of cate a cleavage of ketopentose between carbons 2that in either carbon 3 or 4 of the glucose (130). and 3 (figure 9). Such a cleavage would be similarFermentation of 2- and 6-labeled glucose has to the transketolase reaction except that thedemonstrated that ethanol arose from carbons 2 2 carbon fragment would be free rather thanand 3 or from 5and 6 with the carbons 2 and 5 con- transferred to an acceptor. Thus, D-ribose-1-C'4tributing the carbinol carbon. The mechanism has and D-xylose-1-C'4 fermentation by LactobaciUus


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dH3-CHOH-CHOH-CH3CeH3 CeH3 2,3 BUTYLENE GLYCOL

IH-C-OH H-C-OHC C-OOH FC--H C H3

LACTATE 1 L 3 C-Hz. ~~~~C=O*C~H3C!-H3 CH ETHANOLC=O C=OC OOH -'-- C-OOH eo? CO2 --H3ItPYRUVATE t H-C-OOH H-C-OOH C-OOH.I.:,.2*.3 e .... -/-ACETATE

.H?-C-OH-:' FHZ-C-OC-0.1".. C=O = H-C-O

IHO-C-H - C UNLABELED-~~~~~~~~~~~~~~-----1- - I-.

HH-C-0H] H-C-OH H-C-OH C-OOH LACTATEII J

.H-C-OHI -D H-C-OH - H-C-OH - C=O - 2,3-BUTYLENE

_-CPOP- - C, H?-C-OPOJPP C-H1 GLYCOLETHANOL

FRUCTOSE-6-PO4 PENTOSE-5-PO4j PYRUVATE FORMATE

INTERMEDIATES IN PENTOSE-I-CLFERMENTAT/ON

Figure 9. Pathways to pentose fermentation

pentou (30, 126, 231) and L-arabinose-1-C4 by Cohen (70), and of -arabinose to L-ribulosefermentation by LactobaciUus pentoaceticus (327) by extracts of L. pentosu by ampen(234). Theyielded methyl-labeled acetate; unlabeled lactate induced formation of a specific D-arabinokinasewas produced by the lactobacilli. D-Xylose-1-C4 and a ribokinase has been reported by Cohen etfermentation by Fusarium lini (132) yielded al. (69).ethanol, acetate and C02 with the label in the Fermentation of pentose-1-C14 by Escherichiamethyl group of acetate. coli (133) and by Aerobacter aerogenee (294)

Although many reactions of pentoses have been indicates the operation of a different pathway inreported (34) including isomerization, before or these organisms. With xylose-1-C04 and D-arabi-after phosphorylation, the pathways of degrada- nose-i-C14, resting cells of E. coli, at an acid pH,tion have not been defined. The phosphorylation formed more than one mole of lactate per mole ofof xylose by extracts of xylose adapted L. pen- pentose fermented, as Carson (56a) had alreadytosus ultimately yielded ribose-5-phosphate; observed with xylose. The methyl groups ofxylose-5-phosphate was not found (233). Ribu- lactate and acetate contained the highest radioac-lose-5-phosphate was postulated as the precursor tivity. C14 also was present in the lactate carboxylof ribose-5-phosphate in this reaction and also as group and in formate, whereas the # carbon ofthe intermediate cleaved to 2 and 3 carbon units. grou and i thcarbonaofXylose also was converted to xylulose by the (133)l With D- andr larabinose-t-Ct the prod-same extracts (280) and by an enzyme from (133 .With atn a abiose- thepo-Peudomonas hydrophila (166, 167). Thus, the uctsof fermentation by A. aerogenes were la-reaction sequence was postulated to be xylose beled as if deved from methyl and carboxylxylulose -_ xylulose-5-phosphate -_ ribulose-5- labeled pyruvate (294). The complete conversionphosphate. This mechanism has been in part of pentose to triosephosphate via sedoheptulose-confirmed by the isolationof xylulose-5-phosphate 7-phosphate and fructose-6-phosphate was pos-(372). Similar isomerizations of D-arabinose to tulated to account for the pattern of labeling inD-ribulose by E. coli enzymes have been reported the products (figures 7, 9).


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6. Nonphowphorylated Hexose Oxidation In further studies of glucose oxidation byMany organisms of the Pudomonas and Ace- Pseudomonas fluorescens, Wood and Schwerdt,

toatr genera oxidize glucose with the accum- ug cell-free extracts, demonstrated in additionulation of gluconate (45,255) and ketogluconates to the oxidation of glucose and gluconate to(28, 29, 210, 229, 370). A similr oxidation of 2-ketogluconate (430) the oxidation at similarlactose and maltose to their bionic acids (211, rates of glucose-l-phosphate, fructose-6-phos-362) and pentoses to the corresponding pentonic phate,glucose-6-phosphate, 6-phosphogluconate,acids (256) has been reported. The further degra- and ribose-5-phosphate; with each phosphatedation of 2-ketogluconate by growing cultures ester carbon dioxide was evolved (431). The

yields a-ketoglutarate (257), pyruvate (213, 404) 2-ketogluconate-forming system was separatedand acetate (47), whereas the oxidation of 5 from phosphate ester-oxidizing system, and inketogluconate yields tartarate, glyoxalate, oxa agreement with Campbell's conclusion (363), thelate, and formate (185). The accumulation of oxidation of glucose to 2-ketogluconate wasthese acids, particularly gluconate and 2-ketoglu- shown to proceed without phosphorylation Aconate, is dependent upon the cultural condition similar and separate pathway for hexosephos-including the iron (213) and nitrogen content of phate oxidation also has been demonstrated in P.the medium (213, 214). The pseudomonads are aeunma (65).reported to accumulate 2-ketogluconate (255), The main contribution of these studies has beenwhereas different acetobacter strains accumulate a clarification of the previous findings that living2-ketogluconate (28, 29), 5-ketogluconate (29, pseudomonad cells oxidize part of the glucose to370), both 2-ketogluconate and 5-ketogluconate carbon dioxide, whereas dried cells and extracts(28, 229), or 2,5-diketogluconate (201). oxidize glucose only to the 2-ketogluconate stage.

a. Glucose and gluconate oxidation. From study- The demonstration of both a hexosemonophos-ing the mechanism of gluconic and ketogluconic phate pathway and a pathway from glucose toacid formation and oxidation by cell suspensions, 2-ketogluconate without phosphorylation hasEntner and Stanier (117) concluded that in widened the concept of carbohydrate oxidation inPeudomonas fluorescens the oxidation of glucose these organisms. The steps in the further oxida-to gluconate is affected by constitutive enzymes, tion of the ketogluconates, for which evidence iswhereas the further oxidation of gluconate to accumulating (89, 289), still require clarification.2-ketogluconate is dependent on an inducible Although the oxidation of glucose and gluco-system formed in response to gluconate. Theyals nate to 2-ketogluconate does not appear to fur-concluded from stoichiometric data that 2-keto- nish useful energy at the substrate level, nor togluconate per se is not an intermediate in glucose generate intermediates for biosynthesis, a cyto-oxidation and suggested that its accumulation chrome system mediates in the hydrogen trans-might result from the dephosphorylation of a port and may well yield hih energy phosphatehypothetical 2-keto-6-phosphogluconate.. via oxidative phosphorylation. Some knowledgeOn the other hand, Stokes and Campbell (363), of this electron transport system has been derived

using dried cells of Pseudomonas aeruginosa, and from studies of cell extracts and particulateClaridge and Werkman (64), with extracts of the preparations of P. fluoress(430). In thissame organism, reported a conversion of both oaism no evidence for a pyridine nucleotideglucose and gluconate to 2-ketogluconate with the linked glucose dehydrogenase such as found in theuptake of two and one atoms of oxygen per mole mammalian lver (367) or for a flavoprotein-typeof these substrates, respectively. There was no glucose oxidase as found in molds (82, 203) hasevidence for the further metabolism of 2-ketoglu- been obtained. So far the complete oxidationconate or for the participation of phosphate esters system, which exists in particles (334, 430)in 2-ketogluconate formation. The conclusion separable by high speed centrifugation or bythat a phosphorylation mechanism was absent aimonium sulfate precipitation, has not beenwas based on the observations that glucose oxida- solubilized or the individual components defined.tion was insensitive to M/50 sodium fluoride, was With such preparations, the formation of reducednot stimulated by ATP (363), and that phosphate bands corresponding in wavelength to the cyto-esters could not be extracted from freshly har- chrome "b" and cytochrome "c" bands appearsvested glucose grown cells (48). on the addition of glucose and disappears on


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102 I. C. GUNSALUS, B. L. HORECKER, AND W. A. WOOD [voL. 19

aeration (430). Since absorption was not observed deoxy-6-phosphogluconate and then to pyruvatein the 600 m;& to 620 mg region and the oxidation and glyceraldehyde-3-phosphate may be in-of glucose and other substrates was not sensitive volved. In the case of 2-ketophosphogluconateto 10-8 M cyanide, no evidence was obtained for a degradation, however, pyruvate and 3-phospho-cytochrome oxidase. These observations demon- glycerate would be formed and the latter con-strate a linkage of glucose oxidation with cyto- verted to pyruvate.chromes probably by means of unidentified Thus, two pathways of glucose oxidation existcarriers and enzymes. Until these enzymes can be in these pseudomonads (figure 10): (a) glucose issolubilized, it is not possible to determine if a new oxidized to gluconate and the latter phospho-pyridine nucleotide component or flavoprotein rylated to form 6-phosphogluconate--the 6-phos-carrier is involved. phogluconate is then converted to pyruvate and

b. Gluconokinase and 2-ketogluconokinase. The triosephosphate by the two enzyme systemrelationship between phosphorylated and non- discovered by Entner and Doudoroff (118) andphosphorylated carbohydrate oxidation pathways found in P. fluorescens (431), or oxidized viahas been clarified recently by the demonstration pentose-5-phosphate, presumably involving sedo-of gluconokinase and 2-ketogluconokinase in heptulose-7-phosphate and fructose-6-phosphateextracts of glucose grown P. fluorescens (289) and as intermediates; (b) glucose is oxidized toan inducible 2-ketogluconokinase in Aerobacter 2-ketogluconate before phosphorylation, phos-cloacae (89). With an ammonium sulfate fraction phorylated by 2-ketogluconokinase and thenof either P. fluorescens (290) or A. cloacae (89), degraded to pyruvate by an unidentified path-the product of 2-ketogluconate phosphorylation way. Glucose is not degraded via Embden-accumulates. This acid stable ester has been iso- Meyerhof glycolytic system as indicated by: (a)lated, purified by cellulose column (290) or paper the inability to isolate phosphate esters fromchromatography (89), and partially characterized glucose grown P. aeruginosa (48); (b) the lack ofas 2-keto-6-phosphogluconate. hexokinase and phosphohexokinase in extracts ofThe formation of gluconokinase and 2-ketoglu- glucose grown P. fluorescens (289); (c) the

conokinase of A. cloacae (88) and P. fluorescens distribution of C14 from position labeled glucose(290) is induced by growth on the specific sub- in the pyruvate and acetate isolated (246, 247)strate. Since growth on 2-ketogluconate does not and the inability of resting cells of P. aeruginosainduce gluconokinase formation, gluconokinase to ferment glucose or glucose-6-phosphate (65).and 2-ketogluconokinase appear to be distinctenzymes. A different enzyme termed "hexono-kinase" is found in A. cloacae following growth on The occurrence in nature of a wide variety ofD-galactonate (400). This kinase also phospho- monosaccharides other than glucose and fructose,rylates gluconate and 2-ketogluconate. as well as oligo- and polysaccharides, implies the

c. Routes from gluconate to pyruvate. In the occurrence of enzymatic pathways for theirpresence of ATP, crude extracts of P. fluorescens formation and use. An array of mono- andanaerobically convert gluconate or 2-ketogluco- oligosaccharides has been used as energy andnate to pyruvate (289), whereas 6-phosphogluco- carbon sources to characterize microorganisms. Atnate and 2-keto-6-phosphogluconate are con- an enzymatic level, oligo- and polysaccharidesverted toapproximately 1 and 2molesof pyruvate, are converted by hydrolysis or phosphorolysis torespectively, in the absence of ATP (290). monosaccharides, and the monosaccharides ofGlyceraldehyde-3-phosphate is not an interme- other configurations are converted to interme-diate in 2-keto-6-phosphogluconate degradation, diates of glucose or ribose metabolism. Thus far,and hydroxypyruvate does not accumulate. evidence has not been found for independentCrude extracts of A. cloacae (89) degrade 2-keto- routes for the metabolism of other carbohydrates.6-phosphogluconate to a mixture of phosphate 1. Monwsacch(&i&8esters (88) containing 3-phosphoglycerate (89).Although steps in the degradation of 2-keto-6- a. Mannose. Hexokinase (table 1) convertsphosphogluconate remain to be elucidated, dehy- mannose to mannose-6-phosphate (230); and adration and cleavage reactions similar to those specific isomerase, mannose-6-phosphate isom-converting 6-phosphogluconate to 2-keto-3- erase, converts this substrate to fructose-6-


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GLUCOSE 1/2 02 GLUCONATE 1/2 2-KETOGLUCONATE

I~~~~~~~~~~~~~~A- ATP ATP ATP

DPNGLUCOSE-6-P04 - P-- 6-PHOSPHOGLUCONATE 2-KETO-6-PHOSPHO-TPN GLUCONATE

AATP Fe GSHV ~1 IFRUCTOSE-1,6-diPO4 2-KETO-3-DEOXY- (INTERMEDIATE)

6-PHOSPHOGLUCONATE

GLYCERALDEHYDE-3-PO4 PYRUVATE C3

DPNFigure 10. Pathways to pyruvate: Pseudomonas fluorescens

phosphate (346, 348)-a known intermediate in Among the unresolved problems in fructoseglucose metabolism. This isomerase, isolated from metabolism are the routes in Pseudomoncaliver by Slein (348), is distinct from phospho- fluorescens and Pseudwmonas 8accharophila by wayglucoisomerase (section I). Phosphoglucomutase of mannose (113) to accumulate D-mannonicinterconverts mannose-1- and mannose-6-phos- acid (313).phates (239). The implication of these reactions c. Galactose. Galactose fermentation was shownand of diverse mannose compounds, for example, by Leloir et al. to occur through galactose-1-guanosine diphosphate mannose (46) is not clear. phosphate (GA-1-P) and its conversion toCertain bacteria ferment mannose at approxi-mately 60% the rate of glucose fermentation glu co enby gaodenaseh(50,their enzymatic patterns have not been studied 51, 53). A new coenzyme, uridine diphosphoglu-(123). cose (UDPG) and uridine diphosphogalactose

b. Fructose. Hexokinase phosphorylates fruc- tJDPGA) was isolated and shown to mediatetose to fructose-6-phosphate, an intermediate in the conversion (51, 53).glucose fermentation (table 1). Fructose-6-phos- A specific galactokinase which converts galacphateiseonvertedinaerobic organismsto glucose- tose to galactose-i-phosphate (table 1) is of wide6-phosphate by phosphoglucoisomerase to enter occurrence in yeast (221, 380), animal tissuesthe hexosemonophosphate pathway. Cleavage by (219) and bacteria (54, 331).transketolase may also occur (180, 324) as The interconversion of galactose and glucosediscussed fully in section II. Fructose is also through hydroxyl inversion of the 4 carbon, asmetabolized by another route; a specific fructo- first postulated by Kosterlitz (220), has beenkinase forms fructose-i-phosphate (80, 244) studied with preparations of yeast (221) and L.which is cleaved by a specific aldolase, 1-phospho- bulgaricus (331) and is best summarized asfructoaldolase (section I) to dihydroxyacetone follows:phosphate and free glyceraldehyde (56, 164, 244,245). The latter is phosphorylated by a specific GA-1-P + UDPG G-1-P + UDPGAkinase (164) to glyceraldehyde-3-phosphate. UDPGA UDPG


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The two reactions have not been separated. P. saccharophila by demonstrating: (a) an ex-Uridine triphosphate activates crude prepara- change of other monosaccharides for the fructosetions (285) but with purified enzymes, UDP moiety of sucrose in the absence of glucose-1-hexose (157, 196) is specifically required. The phosphate (109, 110, 159); (b) P" incorporationequilibrium between glucose-i- and galactose-l- in glucose-l-phosphate in the absence of fructosephosphate is about 3:1 as shown with phospho- (159); and (c) an arsenolysis of glucose-l-phos-glucomutase-free preparations of L. bulgaricus phate (111).(156), or with arsenate present to inhibit the Uridine coenzymes have not been implicatedmutase (240). Excellent reviews of these studies in the sucrose phosphorylase, but sucrose isand of the isolation, chemistry and function of formed by an independent enzyme found inUDPG have been written by Leloir (239, 242) and seeds with UDPG + fructose (241) as substrates.by Kalckar and Klenow (197). The general avail- Maltose phosphorylase differs from sucroseability of the uridine coenzymes should accelerate phosphorylase in that (a) a-glucose-i-phosphateclarification of these reactions. is formed, and (b) the P12 exchange and arsenolysisSome evidence was presented for an alternate reaction with j3-glucose-i-phosphate do not occur,

fermentation route paralleling the Embden-Mey- thus indicating a different reaction mechanismerhof pathway from galactose-6-phosphate via (121).tagatose-6-phosphate and tagatose-i, 6-diphos- b. Hydrolases. Enzymes such as invertase,phate (379). maltase, lactase, amylase and cellulase form

Several lactic acid bacteria, which catalyze a glucose from polysaccharides by hydrolysis ofhomolactic fermentation of glucose, have been the glycosidic linkages. These enzymes have beenreported to form only 1 mole of lactate per reviewed elsewhere (140, 286, 301, 315, 394).galactose (360); the reactions have not been c. Polyhydric alcohols. Many hexitols andinvestigated at the enzyme level. glycerol are fermented or oxidized by microor-

ganisms. Since the hexitols, compared to the2. Disaccharides and Polysaccharides hexoses, possess 1 additional pair of hydrogen

a. Phosphorylases. Phosphorolysis, an enzy- atoms and glycerol an additional pair as com-matic transfer of a terminal glycosyl unit to pared to triose, fermentation of these substratesinorganic phosphate, occurs with sucrose, malt- leads to more of the reduced products or occa-ose, starch and glycogen to form, respectively, sionally to new products. The known enzymaticfructose, glucose and polysaccharides plus glu- steps of metabolism of these substrates arecose-l-phosphate. The sucrose phosphorylase oxidative either with or without phosphoryla-cleaves glycosyl-1,2 bonds, whereas the other tion.three cleave glycosyl-1,4 bonds. In both cases, Mannitol and sorbitol oxidation by P. .fluores-the equilibrium favors the formation of the cens cells occurs via 2 inducible enzymes, oneglycosidic bond, though in the presence of oxidizing mannitol and sorbitol to fructose, andphosphoglucomutase, glucose-6-phosphate ac- the other sorbitol to L-sorbose (340). The data documulates. The sucrose and maltose phosphoryl- not indicate whether or not phosphorylation isases are of bacterial origin and the glycogen and involved. Hexitol phosphate (mannitol phos-starch phosphorylases are of general occurrence phate) is formed by a DPN-linked fructose-6-(160). phosphate reductase from E. coli (425) with anSucrose phosphorylase which catalyzes the equilibrium favoring hexitol phosphate forma-

reaction- tion.Glycerol is oxidized to dihydroxyacetone by

sucrosee + Pi (inorganic phosphate) Acetobactr suboxydans (402), Escherichia coli=a-glucose-i-phosphate + fructose (7) and Aerobacter aerogenes (44). The enzymes

has been purified from Pseudomonas saccharophila from E. coli and A. aerogenes have been studied(107, 108) and is also found in P. putrefaciens in extracts and shown to be DPN-linked. The(112) and L. mesenteroide8 (194, 417). analogous pathway via a-glycerol phosphate hasEarly evidence for an enzyme substrate com- been shown in extracts of Escherichia freundii

pound and for a "transglycosidase" reaction was (279), A. aerogenes (264), Streptococcus faecalisalso obtained with the sucrose phosphorylase of (48) and Mycobacterium sp. (184). The S. fae-


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calis glycerol phosphate dehydrogenase, appar- CO2 and H2 (212), differs from previously knownently linked through flavoproteins to oxygen, DPT-mediated reactions in the apparent directgenerates hydrogen peroxide (106, 148), whereas generation of acyl (as thioester or phosphatethat of A. aerogenes involves cytochromes but not anhydride) without aldehyde as an intermediate.DPN and forms H20. The occurrence of still Microbial fermentation studies revealed inanother mechanism for glycerol oxidation involv- addition to reduction and decarboxylation a thirding neither a-glycerol phosphate nor DPN has pyruvate reaction-carboxylation to 4 carbonbeen suggested by data obtained with Acetobacter dicarboxylic acids. The CO2 fixation occurs bysubozydans (204). Much of the mechanism of two routes-(a) reductively to malate (202, 305),polyhydric alcohol formation and use remains and (b) ATP-mediated to oxalacetate (388, 397).unknown, both at the enzymatic and at the An alternate route of carboxylation at a morefermentation balance level. reduced level, yielding succinate as the C4 dicar-

boxylic acid, has also been implicated in microbialIV. PYRtVATE REACTIONS (90, 91, 421) and animal (307) enzyme studies of

Pyruvate gained favor as a key intermediate propionyl metabolism. A further diversity ofleading to the many products of microbial fermen- products is assured through condensation of thetation in the early 1940's when phosphorylative carbon skeletons at the aldehyde (carbanion)glycolysis was established as a main route of and acyl (carbonium ion) oxidation levels. Onlycarbon and energy transformation during anaero- those pyruvate reactions, arising during carbo-bic carbohydrate breakdown. The formation of hydrate fermentation and studied at an enzy-pyruvate by dephosphorylation of phosphoenol- matic level, will be documented in this section.pyruvate concludes the energy yielding phase offermentation, leaving of interest only problems of 1. Rproduct formation, their economic use, the source a. Lactic dehydrogenases. Four lactic dehydro-of biosynthetic intermediates, and of curiosity as genases have been observed in microorganismsto the reaction mechanisms. The pyruvate reac- (figure 11). The most common DPN-linkedtions best understood at the enzyme level were forming L(+) lactic acid has been crystallizedreduction to L(+) lactic acid by DPN-linked from tumor, pig heart (227, 365) and rat liverlactic dehydrogenases (119, 274, 335) and cleav- (134). This enzyme is found in homofermentativeage to acetaldehyde plus CO2 (260, 295, 398) by lactic acid bacteria and in Escherichia coli (202,diphosphothiamin mediated carboxylases. 216, 217). A second lactic dehydrogenase DPN-As means for growing microorganisms and for linked but yielding D(-) lactic acid is present in

extracting their enzymes developed, evidence for heterofermentative lactic acid bacteria such asDPN-linked lactic dehydrogenases and "yeast Leuconmtoc mesenteroides (93) and in LactobaciUmtype" carboxylases in bacteria accumulated. In arabinosus (202). The latter organism formsaddition, unmistakable evidence also accumu- DL-lactic acid from glucose and, in extracts,lated for other reactions of pyruvate, as well as racemizes lactate by linking two stereospecificfor other types of lactic dehydrogenases and DPN dependent dehydrogenases (202).pyruvate decarboxylases. In fact, more recent A third lactic dehydrogenase, flavin coenzymedata assign to diphosphothiamin (cocarboxylase, linked, is present in Lactobacilus delbreiiDPT) a broader role than decarboxylation co- (155, 250). This lactic dehydrogenase, measuredenzyme, and now implicate this cofactor in the by Krebs dismutation reactions or by oxygentransketolase (175, 323), dicarbonyl, and pyru- utilization, is independent of pyridine nucleotidesvate cleavages (149, 336, 343), as a carbanion (155). Such lactic dehydrogenases may be wide-acceptor and transfer agent. The cleavage func- spread in microorganisms, particularly the lactictions include a variety of a-keto compounds, acid bacteria. A similar lactic dehydrogenase notforming "aldehydes", and an equally wide variety requiring a pyridine nucleotide carrier has alsoof acceptors for these aldehydes, in some cases been observed in Proteus vulgaris (284).with acyl generation (152, 153). An additional A fourth lactic dehydrogenase (12,188), crys-DPT-mediated pyruvate reaction of microbio- tallized from yeast by Appleby and Morton (4),logical importance, the "clastic" reaction (198), contains both flavinmononucleotide and cyto-yielding acetyl plus formate (252, 385), or plus chrome b. A less specific a-hydroxy acid oxidase


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L(--ALANINE LACETALDEHYDE

I~ ACID AEADHD

D (+-ALANINE D ACETOIN

ACID i-co2DH-LACTATE -

OPMPYREDPN ' ACETOLACTATEDPN.#H

PYRUVATE -

DPN [ 1 DIACETYL

L W-LACETATE = PHOPHENL ACEALEHACETYL AET

DPNH L ~~~DPT J

PLAVOPROtI APC22'1N6L (+-LACTATE *---or-I ,6 _-jNDO --ACETATE

CYTCRV if£PATCSJ

PHOSPHOENOL- UO-MALATE- l%,r# PYRUVATE ACETYL-S-UP-SH

DPN-H OPN ±0 O1/ ~~~~~~HCOOHFLAVO- ACETYL-P04+ PROTENOXALACETATEPHOSPHOENOL- rACETYL']OXALACETATE D~PT CA

+ - ACETYL-S-CoAH2 + CO2

Figure 11. Reactions to pyruvate (See text, IV. 2, for detailed description ofthe reactions shown at right)

of apparently similar nature is present in rat Transamination, from the viewpoint of the ketokidney (31). acid aminated, is a reductive reaction-the amino

b. Racemase and amination reactions. Racemi- acids are at the oxidation level of their hydroxyzation of D- or L-lactic acid by lactate forming acids rather than of the keto acids.organisms has been clarified only with respect to Amino acid racemase, specifically alanineLactobaciluo arabino8us through the revelation of racemase (429), unlike the DPN-mediatedtwo stereospecific dehydrogenases (202). Lactate racemization of lactic acid by the coupling of twoforming organisms, especially clostridia, and dehydrogenases seems not to occur throughseveral strains of lactic acid bacteria (63, 205) pyruvate. Known pathways for formation ofpossess soluble DPN independent racemases, other D-amino acids, however, do involve pyru-These enzymes, plus a similar soluble racemase vate: i.e., amination to L-alanine by transami-for mandelate (151, 358) another a-hydroxy acid, nase,racemization toD-alanine, and transferof thepresent an interesting area for study since neither amino group from D-alanine to a keto acid form-their mechanism nor their prosthetic groups have ing the D-amino acid in question with regenera-been clarified. Racemization of the analogous tion of pyruvate (377). Thus pyruvate woulda-amino acid, alanine, is pyridoxal phosphate fulfil the role of a coenzyme in the equilibrationmediated and appears not to involve pyruvate as of D- and L-amino acids other than alanine,an intermediate (429) (see figure 11). which is the real site of shift in configuration.Amino acid formation from pyruvate occurs by

transamination (267) with several potential 2. Decarboxylases and Aldehyde Transferamino donors of which glutamate is considered a. Direct decarboxylases. Either direct or oxida-the most important quantitatively (39, 120). tive cleavage of keto acids, and of other dicar-


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bonyl compounds, occurs as a DPT-mediated dehyde from the oxidative carboxylases-pre-reaction in most, if not all, microorganisms. The sumed to possess only step (a)-by the bacterialdirect and the oxidative decarboxylases differ not yeast-type carboxylase of P. lindneri (326) isin the decarboxylation step but in the subsequent presumptive evidence for a two-step reaction.fate of the aldehyde-the specificity for which Direct pyruvate decarboxylases have been foundresides in the enzyme. These reactions are most in Acetobacr sb dans (326) and Pseudmonasrationally viewed as forming "aldehyde-DPT" or lindneri (326) and their properties compared to"aldehyde-DPT-enzyme" compounds, followed the yeast and wheat germ enzymes (143, 226,by donor reactions for the "aldehyde", as indi- 326, 343). These carboxylases vary slightly incated in figure 11. Although a direct demonstra- dissociation constant for DPT, pH optimum andtion of an "aldehyde-DPT" is lacking, several in the amount of acetoin formed per unit ofevidences favoring this concept can be cited: carboxylase. They are similar in Mg++ activa-(a) all DPT-mediated keto-, and dicarbonyl, tion, sensitivity to sulfhydryl reagents, particu-cleavage systems yield at least traces of free larly p-chloromercuribenzoate, and to heavyaldehyde-interpreted as a measure of the insta- metals. The location of the -SH group, enzymebility of the "aldehyde-DPT" compound (105, or DPT, the chemical mechanism of decarboxyl-152,336); (b) the addition of yeast-type carboxylation, and the structure of the intermediatesase to an E. coli type pyruvate system decreases will require further study.the acyl hydroxamate formed without inhibiting b. Acyloin cndensations. Neuberg and Hirsch'scarbon dioxide release, suggesting a competition (296) concept of acyloin formation by yeastfor free "aldehyde-DPT" (61), i.e., diversion of decarboxylase included; step (a), "carboxylase"the aldehyde from acyl forming to free acetalde- now viualized, as indicated, in figure 11, ashyde forming reactions. Similar evidence arises acetaldehyde-DPT" formation-; step (b),from the release of acetaldehyde by Pseudomonaslindneri carboxylase during acetyl phosphatereduction with dimercaptooctanoic acid in the free aldehyde forming acyloin. The reaction hasreductionwithdimercaptoocbeeniindicated as:presence of E. coli fraction A (61). These experi- been idicated asments are far from complete and should be 0considered indicative rather than proof. Similar 0 _ _attempts to divert the acyloin formation toward [DPT]+ II + C-CH,4acyl generation with A. aerogenes and E. coli LCH,-C:extracts did not yield evidence for such a diver-sion (369)-thus suggesting caution. 0 0 0 OHAmong the DPT-mediated reactions outlined ii II H+ 1I I

in figure 11, as occurring through "aldehyde- CH,-C-0-CH, 4 CH-C-C-CH3DPT", is (a) the yeast type carboxylase, with a H Hproton as acceptor. This reaction may occurslowly in the absence of enzyme, but, if the Acetaldehyde as Acceptor. Reaction 2, upperformulation is correct, it is greatly enhanced in right of figure 11, expresses the Neuberg anddirect decarboxylases yielding free acetaldehyde. Hirsch postulate of acetoin formation by theThe postulated mechanism, like the early formu- yeast enzyme (296). The direct yeast and oxida-lation of Neuberg et al. (296, 297, 298, 299, 300), tive E. coli decarboxylases differ in one respect-assigns two sequential steps to the carboxylase the yeast enzyme liberates acetaldehyde to servereaction: (a) a cleavage of pyruvate forming as acceptor whereas the E. coli oxidative decar-"acetaldehyde-DPT" with a release of C02; (b) boxylase does not form acetoin unless acetalde-transfer of "acetaldehyde" to a proton thereby hyde is added. Acetoin formation relative to CO2forming free acetaldehyde. To date, neither the release increases with aldehyde concentrationyeast nor bacterial yeast-type carboxylases have (192, 193) for both enzymes. The acceptorbeen separated into two protein fractions. As aldehyde is virtually nonspecific though alkylindicated above, however, the release of acetal- aldehydes are more active acceptors than the


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more acidic, aryl, or substituted aryl aldehydes of acetyl acetoin. With pyruvate as substrate,(165, 297, 299, 300, 349). The several yeast acetyl acetoin is formed if diacetyl is present incarboxylases retain during purification a constant sufficient concentration to compete as acceptorratio of C02 release to acetoin formation, but this for aldehyde (192, 336). The pig heart and E. coliratio differs among enzymes-possibly resulting systems also form a-acetolactate (DL racemate)from a partial separation of steps 1 and 2. but lack acetolactate decarboxylase and thus doFurther evidence of a 2 step catalysis in acetoin not form acetoin from pyruvate alone (193).formation was presented by Gibbs and DeMoss The products of these pyruvate reactions-(129) who obtained partial removal of acetoin acetaldehyde, acetoin, acetylacetoin-are reducedforming activity from the P. lindneri decarboxyl- in the bacterial fermentations to etha-ase, and by Chin (61) who removed by dialysis nol, 2,3-butanediol, and acetylbutanediol (190).and adsorption much of the acetoin forming Complete reduction of acetylbutanediol and ofactivity of the E. coli decarboxylase without a-acetolactate to analogous tri- and dihydroxydestroying decarboxylase activity. alcohols has not been achieved. Reduction ofThe optical rotation of the acyloins formed acetoin to 2,3-butanediol is catalyzed by a

depends on the enzyme, e.g., the yeast carboxyl- pyridine nucleotide mediated dehydrogenasease yields primarily (+) acetoin-and is used (92). The optical isomers of butanediol-2for the commercial production of mixed acyloins, asymmetric carbons-formed by various micro-i.e., biologically active l(+) ephedrine (297). organisms have been only partially clarified (339,The wheat germ carboxylase forms racemic 357).acetoin with a slight excess of (+) isomer [72% c. Acetate generation. Acetaldehyde, formed by(+), 28%(-)] (343). yeast type decarboxylase in several species of thePyruvate as Acceptor. The bacterial acetoin genus Acetobacter, is oxidized to acetate by both

forming system from Aerobacter aerogenes (27, DPN- and TPN-specific dehydrogenases (326).189) and Streptococcus faecalis (104) among other This pathway can be considered as an alternategenera (189) forms acetoin through (+)a-aceto- route to acetate formation. Other acetate gener-lactate (189) with pyruvate acting as acceptor ating mechanisms include the oxidation of the(reaction 3, figure 11) (189). A manganese but not "aldehyde-DPT" formed by bacterial oxidativeDPT-activated ,-carboxylase, specific for the pyruvate decarboxylases by: (a) chemical elec-(+) isomer of a-acetolactate, forms only (-) tron acceptors, ferricyanide and 2,6-dichloro-acetoin (104), through optical specificity of both phenol indophenol, and (b) oxygen as acceptorthe "condensing" and "decarboxylating" en- via bacterial particles (282, 283, 329) (figure 11).zymes. The yeast type pyruvate decarboxylases These acceptors may or may not involve furtherform acetoin slowly from acetaldehyde alone, enzymatic steps. A decrease in the rate of decar-thus indicating the partial reversibility of the boxylation during purification of both the E. coli"aldehyde-DPT" + H+ acetaldehyde + DPT (153) and pig heart enzymes (336) measured inreaction (reaction 1, figure 11) (144, 343, 378). the presence of these electron acceptors as com-

Diacetyl as Acceptor. Diacetyl was shown by pared to the dismutation assay and Dolin's (106)Juni and Heym (190, 191) to accept "aldehyde" recent separation of flavoproteins from Strepto-from "aldehyde-DPT" forming acetyl acetoin- coccm faecalU with substrate specificity for thesealso termed diacetyl methyl carbinol (DAMC). dyes indicate that enzymatic steps are involved.Diacetyl as both aldehyde donor and acceptor The oxidation systems are cytochrome mediatedforms acetyl acetoin rapidly in an enzyme system (cyanide sensitive) in both Proteu vulgaris andstudied fi~rst in Aerobacter aerogeres and in asoryned first in e..bacterbyerenrcmnd tw E. coli (329), but the nature of the coupling at

acetoirn as substrate (190, 191). More recently, the "aldehyde-DPT" level is unclear. Both E.this system has been found in other aerobic and colt and P vugar, however, contain pyruvatefacultative bacteria-.e., Pseudomonas fluoes- decarboxylases-in the lipoic acid mediatedcens, Micrococcus lysodeikticus (192, 193). Diace- fraction A-which do not connect to bacterialtyl is also a substrate for the oxidative pyruvate particles (329). The diacetyl cleavage system ofdecarboxylases of pig heart (145, 193) and of Corynebacterium sp. and Aerobacter aerogenes alsoStreptococcus faecalis (105) with some formation fails to link to the bacterial particles.


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The diacetyl cleavage enzymes described by vate decarboxylase by addition of lipoic acidJuni are acetate generating (192): show its function (149).

diacetyl -+ "acetaldehyde-DPT" + acetate (1)

"acetaldehyde-DPT" + diacetyl -+ acetyl acetoin (2)

[acetyl acetoin ° acetoin + acetate] (3)

acetoin -+ diacetyl + 2,3-butanediol (4)or butanediol + acetyl acetoin -+ acetyl butanediol + acetoin (3a)

[acetylbutanediol -20 acetate + butanedioll3 diacetyl 4 acetate + 1 butanediol (Sum via Reactions

1 to4orl,2,3a,4,5)Both the initial cleavage and the subsequent The lipoic acid-mediated systems transfer the

hydrolyses-still tentative-generate acetate and acyl group from acetyl lipoate to coenzyme A byappear to constitute an independent oxidative a lipoic transacetylase (152) and thence tomechanism for generating acetate (192). phosphate by phosphotransacetylase. The pres-

d. Acetyl generation. The pyruvate decarboxyl- ence of lipoic transacetylase has been shown inases of Escherichia coli, Proteus vulgaris and the E. coli, S. faecalis, P. vulgaris and pig heartStreptococcus faecahs have been measured in (152) pyruvate decarboxylase systems and shownextracts by following the Krebs' dismutation to function before coenzyme A by the accumula-reaction (217), tion of stoichiometric amounts of acetyl lipoate

in the absence of CoA (59). Acetyl lipoate is2 pyruvate -~ acetyl + CO2 + lactate arsenolyzed in the presence of CoA, phospho-

Intermediate steps require DPT, presumably transacetylase and lipoic transacetylase. Theforming "aldehyde-DPT" and lipoic acid. The slow ferricyanide oxidation of "aldehyde-DPT"reductive acylation of lipoic acid is suggested to (figure 11) is not lipoic or CoA dependent, but theoccur by a mechanism silar to the acetoin more rapid oxidation by S. faecalis apopyruvatecondensation generating a free mercapto group decarboxylase with ferricyanide as electron ac-and a thioester, rather than a hydroxyl and a ceptor is lipoic dependent presumably by reoxi-carbon-carbon bond (152, 153, 345). The reduc- dation of reduced lipoic through DPN (lipoictive acylation step has been visualized as: dehydrogenase) and a flavoprotein DPNH-

[ 01S-°CH2 H+-S

CH,-CJ [DP1r]+ + H+ >CH2 oLH. CS--CH II

I CH,-C-(C112)4

| RCOOH

If this, or a related formulation is correct, the oxidase (106). In S. faecalis, the ferricyanidenew disulfide cofactor assumes importance in reaction rate equals the dismutation rate, whereasenergy rich bond formation at the substrate in E. coli the ferricyanide reaction is only aboutlevel (152, 153, 345). Lipoic acid, through its 1/1,000 as fast (154).sensitivity in the reduced state to trivalent The Lactobacillus delbrucii pyruvate oxida-arsenicals (314), is implicated in the brain (60), tion system generates acyl phosphate (251) bypig heart (329), E. coli (149) and P. vulgaris still another mechanism requiring neither lipoicoxidative acyl generating pyruvate decarboxyl- acid, CoA, nor presumably DPN (155). Thisases (329). In S. faecalis both trivalent system does require DPT and flavin adeninearsenical sensitivity and activation of apopyru- dinucleotide and is linked to a flavoprotein lactic


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dehydrogenase-see reductive pyruvate reac- requirement, thus indicating a terminal functiontions, section IV, 1. for phosphate in acetyl phosphate formationThe reactions of DPT, indicated at the right (424).

side of figure 11, are only part of the reactions in The enzymes have not been separated, butits role as an aldehyde acceptor-the transketo- fractions have been obtained in which the hydro-lase and dicarboxyl cleavage enzymes, not gen liberating mechanism is lost and decarboxyla-shown, also involve formation of "aldehyde- tion will occur if artificial electron acceptors,DPT" complexes. particularly furacin and neotetrazolium, are

added. These, however, block the CO2 exchange3. Clastic Reactions reaction, presumably by displacing the electron

Lipmann (253) equated three DPT-mediated equilibrium.bacterial pyruvate cleavage reactions throughtheir formation of acetyl phosphate. Two of 4. (arboxylatthese were termed phosphoro- "elastic" through As has been already indicated, three carboxyl-the demonstration with bacterial extracts of ation systems are known in bacteria: (a) theEscherichia coli (198, 386) and Clostridium butyl- malic enzyme first discovered by Ochoa and co-icum (212) that phosphate incorporation con- workers in animal tissue (305) and studied inserves the bond energy as contrasted to hydrolysis Lactobacillus arabinosus as an inducible enzyme(hydro- "clastic") liberating approximately 15 (215); (b) an ATP-activated carboxylation ofkilocalories as heat. Even though knowledge is phosphoenolpyruvate or decarboxylation of phos-still fragmentary, and the terminology transient, phoenoloxalacetate-the Wood-Werkman reac-these two reactions appear to differ from the tion (figure 11); (c) the formation and cleavage ofpyruvate~~~~~~~~~~~~,decarbxfigureiyiedin

(c)"aeoraetyndeeaaP opyruvate decarboxylases n yielding an"acetyl-" C4 dicarboxylic acids via succinate forming or(carbonium ion) rather than an "aldehyde- carboxylating propionyl CoA (307).(carbanion). In addition, rapid equilibration The equilibrium between malate plus DPN,with the carboxy2l cleavage product, formate and pyruvate, C02 plus DPNH favors decarboxyl-(387), or0c2 + H2 (424), differs from the slight ation but can be forced toward synthesis by high002 exchange by the decarboxylases (139, 306). concentrations of both reduced pyridine nucleo-

a. Acetyl plus forma. The colon-typhoid bac- tide and carbon dioxide (305).teria posss the formate producing clastic reac- The pyruvate carboxylation via phosphoenol-tion. In extracts of E. coli, the forward and for- pyruvate to phosphoenoloxalacetate has beenmate exchange reactions require inorganic phos- studied by carbon dioxide exchange reactions inphate, CoA, and DPT (57, 198, 366, 368). The chicken liver and in spinach by Utter et al.acetyl fraction, however, does not equilibrate if (390, 391, 392) and by Bandurski et al. (14, 15).added as acetyl phosphate even though it can be The decarboxylation by the liver enzyme hasdemonstrated as the ultimate product. Attempts been shown to be dependent upon catalyticto separate the enzymes and clarify the mech- amounts of ATP or ITP. The formation ofanism are still in progress (304). phosphoenolpyruvate from pyruvate may occur

b. Acetyl plus C02 and H2. The clostridia pos- through reductive carboxylation by malic en-sess a clastic reaction forming C02 and hydrogen zyme, plus oxidation and phosphorylation towhich provides the source of hydrogen among phosphoenoloxalacetate and decarboxylation tothe fermentation products of this organism. phosphoenolpyruvate (224).Experiments with extracts of Clostridium butyr The third carbon dioxide fixation reactionicum (423) have demonstrated a DPT, CoA, m-organic phosphate, and Fe++ requirement for the inca pyruvate butforward reaction, yielding acetyl phosphate, of propionate. This mechanism involves theC02 and H2. For C402 exchange, DPT, CoA, carboxylation of propionate to succinate andinorganic phosphate and Mn++ are required requires substrate amounts of ATP and cofactor(424). The ferrous iron presumably functions in amounts of CoA (90, 91, 421). Recent enzymethe hydrogen forming mechanism and is, there- experiments by Flavin and Ochoa (307) havefore, not essential for 01402 exchange. Substrate implicated methyl malonate as an intermediateamounts of CoA replace the inorganic phosphate between propionyl CoA and succinyl CoA.


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5. Acyl Condensations and Reductions equilibrates an acyl with a free acid, i.e., acetyl

Free fatty acids arising via fermentation can with propionate, or as shown with acetate-C'4,form fermentation products of greater chain acetyl with acetate as follows:length and more reduced nature, or enter biosyn- acetyl-CoA + propionatethetic pathways (62), after activation by one of acetate + propionyl-CoAthe three mechanisms described below. Suchfatty acids, for example acetate, are formed Delwiche (91) has presented data for a trans-during fermentation by: (a) oxidation of pyru- phorase equilibrating succinyl and propionate invate to acetate via a socalled bypass mechanism Propionibacterium, and Stadtman (356) and oth-(section IV, 2c); (b) hydrolytic action on thio- ers for equilibration of acetyl with acids fromester or acylphosphate formed by acyl forming propionate to caprylate as well as vinylacetate,pyruvate cleavage (section IV, 2d), i.e., CoA- among other acids.deacylase (325a) or acetylphosphatase (253); or Acetyl-CoA generated by these and other reac-

(c) by transferring an energy rich link to other tions can in microbial systems (a) be reduced tosystems, i.e., thioester linkage via CoA-transfer- ethanol or (b) undergo condensations followedases (356), or phosphate anhydride to ATP via by reduction to form longer chain fatty acids andacetokinase. their alcohols.

a. Acid activations. Acetokinase (199, 330a) b. Reductions. Ethanol formation from acetyl-which catalyzes the reaction CoA through DPN linked aldehyde dehydrogen-

ase was described in Clostridium kluyveri (44a).Acetate + ATP kI 10 Ethanol dehydrogenases, both DPN and TPN

linked, are known in a wide variety of bacteriaAcetyiphosphate + ADP + Pi (96a, 326, 335). In the absence of enzymatic data,

has been purified from E. coli and Streptococcus the reductions of acetate-C" to ethanol duringhemolyticu, shown to be activated by Mn++ or glucose fermentation in species of LeuconotocMg++, not to require CoA and is presumed to (150) and Aerobacter indelogenes (344a) and toproceed by the reaction of both acetate and ATP butanol in clostridia (427) are presumed to occurwith the enzyme since the exchange of either by related or similar reactions.acetate-C" or HP32O, requires both, plus ADP. After condensations (see below) acetoacetyl-This enzyme, forming acetylphosphate, is found CoA is reduced via fl-hydroxybutyryl-, crotonyl-only in organisms which contain phosphotrans- orvinylacetyl-CoA to butyryl-CoA (19, 354). Theacetylase and appears to furnish acetyl through latter is deacylated, hydrolytically or throughformation of acetyl-CoA (326). CoA transphorase to yield butyrate (356) or isATP-CoA-Acetate "Transferase" (254, 325a) thought to be reduced to butanol by enzymes

catalyzes the reaction similar to the bacterial acetyl to ethanol system(352, 355, 427).

ATP + CoA + acetate c. Condensations. Longer chain fatty acids are= ADP + acetyl-CoA + PP formed in C. kluyveri under reducing conditions

(PP-pyrophosphate) (excess of ethanol over acetate) (352) and in C.

The enzyme has been studied in yeast and animal 8accharobutyricum under reducing but not oxi-tissue (254) and is found in those aerobic bac- dizing conditions (8). Butyrate formation interia devoid of phosphotransacetylase, i.e., both animal tissue (261a, 265) and bacterialspecies of Acetobacter and Pseudomonas (326). preparations (356a) follows condensation via thio-An analogous enzyme for succinate forms lase catalyzing the reaction

succinyl-CoA and Pi-though by a somewhat 2 acetyl-CoA Acetoacetyl-CoA + CoAdifferent mechanism (325a). Succinyl-CoA en-zymes liberating succinate by deacylase (325a) Among the reactions of acetoacetyl-CoA, theand generating energy rich phosphate have also reductions are considered under (b) above. Itsbeen studied (202a). These may play a role in deacylation forming free acetoacetic acid mayfermentative succinate formation and in the precede acetone formation via a specific decar-propionic acid fermentations (91). boxylase (84a, 341a). This is inferred from the

CoA-transphorase described by Stadtman (356) data of Stadtman and others (356).


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Summary acid biosynthesis. Biochem. J., 54, 508-

The reactions leading to products of carbohy- 7. 5 R. E., AND BRODIE, A. F. 1953 Adrate metabolism by microorganisms via pyru- glycerol dehydrogenase from EscF9erichiavate or other intermediates of fermentation or coli. J. Biol. Chem., 208, 153 159.oxidation pathways have been discussed insofar 8. AUBEL, E., AND PERDIGON, E. 1940 Etudeas enzymatic reactions for their formation are de l'action de l'oxygbne sur la productionknown or may be deduced with reasonable cer- de corps en C4 et en C2 par un anaerobictainty. Often, the complete scheme even for strict. Compt. rend., 211, 439-441.known pathways is not documented for a single 9. AXELROD, B., SALTMAN, P., BANDURSKI, R.known paor even for a single genus. In many more S., AND BAKER, R. S. 1952 Phosphohexo-species , . .In . kinase in higher plants. J. Biol. Chem.,instances, the reaction mechanism is little under- 197, 89-96.stood, and pertinent data are fragmentary or 10. AXELROD, B., BANDURSKI, R. S., GREINER,unavailable. Reactions one or more steps beyond C. M., AND JANG, R. 1953 The metabo-pyruvate, acetaldehyde, triosephosphates or lism of hexose and pentose phosphates inother intermediates of carbohydrate fermenta- higher plants. J. Biol. Chem., 202, 619-tion or oxidation are, for the most part, omitted 634.from the present discussion; for example, CO2 11. AXELROD, B., AND JANG, R. 1954 Purifica-+ H2 generation from formate by the hydrogen- tion and properties of phosphoriboisomer-lyase of the the colon-aerogenes bacteria. Many ase from alfalfa. J. Biol. Chem., 209,further degradation and energy liberating reac- 847-855.tions, and their relationship to biosynthetic 12. BACH, S. J., DIXON, M., AND ZERFAs, L. G.

1946 Yeast lactic dehydrogenase andpathways, remain to be clarified. Although in cytochrome b2. Biochem. J., 40, 229-239.some instances, the reactants may be formed by 13. BAILEY, K., AND WEBB, E. C. 1948 Puri-the pathways of carbohydrate metabolism, and fication of yeast hexokinase and its reac-in still others the mechanisms may be similar to tion with 0,0'-dichlorodiethyl sulphide.known reactions in glycolysis or other pathways, Biochem. J., 42, 60-68.these are beyond the scope of the present effort. 14. BANDURSIU, R. S., GREINER, C. M., AND

BONNER, J. 1953 Enzymatic carboxyla-BIBLIOGRAPHY tion of phosphoenolpyruvate to oxalace-

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78. COrn, G. T., SLEIN, M. W., AND COrn, C. F. chim. et Biophys. Acta, 13, 302.1948 Crystalline D-glyceraldehyde-3-phos- 90. DEIWICHE, E. A., PHARES, E. F., ANDphate dehydrogenase from rabbit muscle. CARSON, S. F. 1953 Succinate decar-J. Biol. Chem., 173, 605-618. boxylation reaction in Propionibacterium.

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80. COrn, G. T., OCHOA, S., SLEIN, M. W., AND 198.Cow, C. F. 1951 The metabolism of 92. DEMoss, R. D., GUNSALUS, I. C., ANDfructose in liver. Isolation of fructose-1- BARD, R. C. 1951 New diphosphopyri-phosphate and inorganic pyrophosphate. dine nucleotide-linked dehydrogenases inBiochim. et Biophys. Acta, 7, 304-317. Leuconostocmesenteroides. Bacteriol. Proc.,

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82. COULTEARD, C. E., MICHAELS, R., SHORT, formation. J. Bacteriol., 62, 499-511.W. F., SYEs, G., SKRnMSHExE, G. E. H., 94. DEMoss, R. D., AND GIBBS, M. 1952STANDFAST, A. F. B., BIEKINSHAW, J. H., Mechanism of ethanol formation byAND RMSTRICK, H. 1945 Notatin: an Psudomonas lindneri. Bacteriol. Proc.,anti-bacterial glucose-aerodehydrogenase 146.from Penicillium notatum Westling and 95. DEMoss, R. D. 1953 Routes of ethanolPenicillium resticulosum sp. nov. Bio- formation in bacteria. J. Cellular Comp.chem. J., 39, 24-36. Physiol., 41, Suppl. 207.

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378. TOMIYASU, Y. 1937 tMber die Verbreitung Unpublished data.der Carboligase. Enzymologia, 3, 263- 394. VEIBEL, S. 1950 Hydrolysis of galacto-265. sides, mannosides and thioglycosides. In

379. TOTTON, E. L., AND LARDY, H. A. 1949 The enzymes, pp. 621-634. Vol. I. EditedPhosphoric esters of biological importance. by J. B. Sumner and K. Myrback. Aca-IV. The synthesis and biological activity demic Press, Inc., New York, N. Y.of D - tagatose - 6 - phosphate. J. Biol. 395. VELICK, S. F., AND HAYES, J. E., JR. 1953Chem., 181, 701-706. Phosphate binding and the glyceralde-

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alcohol fermentation with heavy carbon 430. WOOD, W. A., AND SBCHWDT, R. F. 1953acetic and butyric acids and acetone. Carbohydrate oxidation by PseudomonasArch. Biochem., 6, 243-260. fluorescens. I. The mechanism of glucose

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carbohydrate microorganisms bethesda, maryland · biosynthetic precursors. hydrate metabolism vary...

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