biochemistry: vitamins & cofactors p. 1 of 49 many cofactors are derived from vitamins we...
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Biochemistry: Vitamins & Cofactors p. 1 of 49
Many cofactors are derived from vitamins We justify lumping these two topics together because many cofactors are vitamins or are metabolites of vitamins.
Biochemistry: Vitamins & Cofactors p. 2 of 49
What we’ll discuss
Tightly-bound metal ions as cofactors
Activator ions as cofactors
Cosubstrates Prosthetic groups
Vitamins Water-soluble vitaminsAscorbateCofactors
Fat-soluble vitamins
Biochemistry: Vitamins & Cofactors p. 3 of 49
Family tree of cofactors Cofactors, coenzymes, essential ions,
cosubstrates, prosthetic groups:
Cofactors(apoenzyme + cofactor holoenzyme)
Essential ions Coenzymes
Activator ions(loosely bound)
Ions inmetalloenzymes
Prosthetic groups(tightly bound)
Cosubstrates(loosely bound)
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Metal-activated enzymes
Absolute requirements for mobile ions Often require K+, Ca2+, Mg2+
Example: Kinases: Mg-ATP complex Metalloenzymes: firmly bound metal ions in active site Usually divalent or more Sometimes 1e- redox changes in metal
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Coenzymes Organic moeities that enable enzymes to perform their function: they supply functionalities not available from amino acid side chains
Cosubstrates Enter reaction, get altered, leave Repeated recycling within cell or organelle
Prosthetic groups Remain bound to enzyme throughout Change during one phase of reaction, eventually get restored to starting state
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Major cosubstrates Facilitate group transfers, mostly small groups
Oxidation-reduction participantsCosubstrate Source FunctionATP Transfer P,NucleotideS-adenosylMet Methyl transferUDP-glucose Glycosyl transferNAD,NADP Niacin 2-electron redoxCoenzyme A Pantothenate Acyl transferTetrahydrofolate Folate 1Carbon
transferUbiquinone Lipid-soluble e- carrier
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Major prosthetic groups Transfer of larger groups
One- or two-electron redox changesProsth.gp. Source FunctionFMN, FAD Riboflavin1e- and 2e- redox transfersTPP Thiamine 2-Carbon transfers with C=OPLP PyridoxineAmino acid group transfersBiotin Biotin Carboxylation, COO- transferAdenosyl- Cobalamin Intramolec. rearrangements cobalaminMeCobal. Cobalamin Methyl-group transfersLipoamide Transfer from TPPRetinal Vitamin A VisionVitamin K Vitamin K Carboxylation of glu
residues
Biochemistry: Vitamins & Cofactors p. 8 of 49
Adenosine triphosphate Synthesizable in liver (chapter 18)
Building block for RNA Participates in phosphoryl-group transfer in kinases
Source of other coenzymes
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S-adenosylmethionine Made from methionine and adenosine Sulfonium group is highly reactive: can donate methyl groups
Reaction diagram courtesy of Eric Neeno-Eckwall, Hamline University
Biochemistry: Vitamins & Cofactors p. 10 of 49
UDP-glucose Most common donor of glucose Formed via:Glucose-1P + UTPUDP-glucose + PPi
Reaction driven to right by PPi hydrolysis
Structure courtesy of UIC Pharmacy Program
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NAD+ and NADP+
Net charge isn’t really >0 ;the + is just a reminder that the nicotinamide ring is positively charged
Most important cosubstrates in oxidation-reduction reactions in aerobic organisms
Structure courtesy of Sergio Marchesini, U. Brescia
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Differences between them
The chemical difference is in the phosphorylation of the 2’ phosphate group of the ribose moiety
The functional difference is that NAD is usually associated with catabolic reactions and NADP is usually associated with anabolic reactions
Therefore often NAD+ and NADPH are reactants and NADH and NADP+ are products
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How do we get back to the starting point? NADH is often oxidized back to NAD+ as part of the electron-transport chain
Imbalances can be addressed viaNAD Kinase (S.Kawai et al (2005), J.Biol.Chem. 280:39200) and NADP phosphatase
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iClicker quiz: single question What would you expect to be the phosphate donor in the NAD kinase reaction?
(a) free phosphate (b) pyrophosphate (c) ATP (d) pyridoxal phosphate
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Reduced forms of NAD(P)
Reduction occurs on the nicotinamide ring
Ring is no longer net-positive
Ring is still planar but the two hydrogens on the para carbon are not
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FAD and FMN Flavin group based on riboflavin Alternate participants in redox reactions
Prosthetic groups: tightly but noncovalently bound to their enzymes
That protects against wasteful reoxidation of reduced forms
FADH2 is weaker reducing agent than NADH
These are capable of one-electron oxidations and reductions
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FAD and FMN structures FAD has an AMP attached P to P
Structure courtesyPaisley University
Biochemistry: Vitamins & Cofactors p. 18 of 49
Reaction diagram courtesy of Eric Neeno-Eckwall, Hamline University
FMN/FAD redox forms
Two-electron version: H+ + :H- transferred
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Coenzyme A Reactive portion is free sulfhydryl at one end of the molecule
Can form thioester with acetate, etc.
Pantoate +-alanine = pantothenate
Structure courtesy ofMPB project, George Washington University
(ADP-3’P)
(Pantoate)
-alanine)
2-mercapto-ethylamine)
Biochemistry: Vitamins & Cofactors p. 20 of 49
Thiamine Pyrophosphate
Based on thiamine, vitamin B1 Carboxylases and oxidative decarboxylases use this coenzyme
So do transketolases (move 2 carbons at a time between sugars with keto groups)
Thiazolium ring is reactive center:pKa drops from 15 in H2O to 6 in enzyme
Biochemistry: Vitamins & Cofactors p. 21 of 49
TPP reactions
Diagram courtesy ofOklahoma State U.Biochemistry program
pyrimidine
thiazolium
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Pyridoxal phosphate
PLP is prosthetic group for many amino-acid-related enzymes, particularly transaminations
Carbonyl group of PLP bound as a Schiff base (imine) to -amino group of lysine at active site
First step is always formation of external aldimine; goes through gem-diamine intermediate to internal aldimine
Biochemistry: Vitamins & Cofactors p. 23 of 49
Biotin Rarity: vitamin is the prosthetic group
Used in reactions that transfer carboxyl groups
… and in ATP-dependent carboxylations
Biochemistry: Vitamins & Cofactors p. 24 of 49
Biotin reactivity
Covalently bound to active-site lysines to form species called biocytin
Pyruvate carboxylase is characteristic reaction:
Diagram courtesyUniversity of Virginia Biochemistry
Biochemistry: Vitamins & Cofactors p. 25 of 49
Tetrahydrofolate Primary donor of one-carbon units(formyl, methylene, methyl)
Supplies methyl group for thymidylate
Dihydrofolate reductase (DHFR) is an interesting drug target Methotrexate as cancer chemotherapeutic: cancer needs more thymidylate than healthy cells
Trimethoprim as antibacterial:Bacterial DHFR is somewhat different from eucaryotic DHFR because bacteria derive DHF from other sources; humans get it from folate
Biochemistry: Vitamins & Cofactors p. 26 of 49
THF structure and function
Figure courtesy horticulture program, Purdue
Biochemistry: Vitamins & Cofactors p. 27 of 49
Cobalamin Largest B vitamin Structure related to heme but missing one carbon in ring structure
Cobalt bound in core of ring system
Involved in enzymatic rearrangements Catabolism of odd-chain fatty acids Methylation of homocysteine Reductive dehalogenation
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Adenosyl-Cobalamin
Diagram courtesy of Swiss Food News
“Missing” carbon
ReactiveCo-C bond
Biochemistry: Vitamins & Cofactors p. 29 of 49
Lipoamide Protein-bound form of lipoic acid Contains five-membered disulfide ring
Covalently bound via amide to protein lysine sidechain
Involved in swinging arm between active sites in multienzyme complexes
Disulfides break periodically Example: pyruvate dehydrogenase complex
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Lipoamide 2e- reduction Cf. Scheme 7.6: thioester starting point
Fig. Courtesy Biochem and Biophysics program, Rensselaer
Biochemistry: Vitamins & Cofactors p. 31 of 49
iClicker revisited
Which coenzyme would you expect would be required for the reactionoxaloacetate + glutamate aspartate + -ketoglutarate?(a) ascorbate(b) PLP( c) thiamine pyrophosphate(d) NAD(e) none of the above
Biochemistry: Vitamins & Cofactors p. 32 of 49
Vitamins: necessary micronutrients that cannot be synthesized internally What’s a vitamin for one organism is not for another
Primates and some rodents are the only vertebrates that don’t synthesize ascorbate
E.coli can make almost everything given energy and sources of atoms
Biochemistry: Vitamins & Cofactors p. 33 of 49
Why wouldn’t organisms make everything?
Complex metabolites require energy for synthesis
Control of their synthesis is also metabolically expensive
Cheaper in the long run to derive these nutrients from diet
Biochemistry: Vitamins & Cofactors p. 34 of 49
Vitamins: broad classifications Water-soluble vitamins
Coenzymes or coenzyme precursors Non-coenzymic metabolites
Fat-soluble vitamins Antioxidants Other lipidic vitamins
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