Biochem. J. (1988) 249, 779-788 (Printed in Great Britain)

The complete amino acid sequence of human skeletal-musclefructose-bisphosphate aldolasePaul S. FREEMONT,* Bryan DUNBAR and Linda A. FOTHERGILL-GILMOREtDepartment of Biochemistry, University of Aberdeen, Marischal College, Aberdeen AB9 lAS, U.K.

The complete amino acid sequence of human skeletal-muscle fructose-bisphosphate aldolase, comprising363 residues, was determined. The sequence was deduced by automated sequencing of CNBr-cleavage,o-iodosobenzoic acid-cleavage, trypsin-digest and staphylococcal-proteinase-digest fragments. Comparisonof the sequence with other class I aldolase sequences shows that the mammalian muscle isoenzyme is one ofthe most highly conserved enzymes known, with only about 2% of the residues changing per 100 millionyears. Non-mammalian aldolases appear to be evolving at the same rate as other glycolytic enzymes, withabout 4 of the residues changing per 100 million years. Secondary-structure predictions are analysed inan accompanying paper [Sawyer, Fothergill-Gilmore & Freemont (1988) Biochem. J. 249, 789-793].

INTRODUCTION

Aldolases (EC 4.1.2.13) are a group of enzymesthat catalyse reversible aldol cleavage/condensationreactions. The glycolytic aldolase is fructose- 1,6-bis-phosphate aldolase:

Trypanosoma brucei (Clayton, 1985). Partial (but ex-tensive) sequences of the human liver enzyme have alsobeen published (Besmond et al., 1983; Costanzo et al.,1983). Crystallographic studies on aldolase from humanmuscle, rabbit muscle and D. melanogaster have beenundertaken, and low-resolution structures have beenpublished (Millar et al., 1981; Sygusch et al., 1985;

203POCH2 CH20P032

kW4H

HO

2 03po 0 OHI 11 ICH2 C-CH2

+2-03po OH 0

l 11CH2 CHlCH

Aldolases can be classified into two groups that havestrikingly different mechanisms of action. Class Ialdolases form a Schiff-base intermediate between theC-2 carbonyl group of the substrate (dihydroxyacetonephosphate in the case of the glycolytic aldolase) and thee-amino group of a lysine residue (reviewed by Horeckeret al., 1972). Class II enzymes, in contrast, do not forma covalent enzyme-substrate intermediate, and a bivalenttransition-metal ion such as Zn2+ is required. Class IIaldolases occur primarily in bacteria and yeast (Rutter,1964; Warburg & Christian, 1943).

Fructose-bisphosphate adolase isolated from verte-brates, insects and higher plants is a tetrameric enzymewith essentially identical subunits of Mr 40000. In mam-malian tissues aldolase exists in three main forms: Apredominates in skeletal muscle, B in liver and kidney,and C, together with a variety of hybrids with A and Bsubunits, in brain. Complete amino acid sequences areavailable for fructose-bisphosphate aldolase from rabbitmuscle (Tolan et al., 1984), rat liver (Tsutsumi et al.,1983), Drosophila melanogaster (Malek et al., 1985) and

Brenner-Holzach & Smit, 1982). Work is in progress byall these groups to extend the crystallographic studies tohigh resolution.We now report here the determination of the complete

amino acid sequence of human skeletal-muscle aldolase.The availability of this sequence allows the rate ofevolution of the muscle isoenzyme to be compared withthat of the liver isoenzyme, and shows that the muscleisoenzyme is much more strongly conserved. An analysisof the predicted secondary structures of the availablealdolase primary structures is presented in the accom-panying paper (Sawyer et al., 1988). The sequenceinformation will be of importance for the interpretationof the crystallographic structure.

EXPERIMENTALPurification and chemical modification of humanskeletal-muscle aldolaseThe purification and carboxymethylation procedures

are described in detail in Freemont et al. (1984).

* Present address: Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT 06511, U.S.A.t To whom correspondence and reprint requests should be sent, at present address: Department of Biochemistry, University of Edinburgh, George

Square, Edinburgh EH8 9XD, U.K. Details of peptide compositions and sequencing yields may be obtained from the authors.

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Succinylation (2-carboxypropionylation) was done bythe addition of a 200-fold molar excess of succinicanhydride (general-purpose-reagent quality from BDHChemicals) over lysine residues as described by Russellet al. (1986).

Purification of fragments for sequencingThe methods for CNBr and o-iodosobenzoic acid

cleavage, and the results of gel filtration of the fragments,are given in Freemont et al. (1984). The details of theconditions used for digestion of fragment CN2 withstaphylococcal proteinase or with trypsin, and fordigestion of aldolase with trypsin, are given in Figs. 1, 2,3 and 4 respectively. Peptides were purified by h.p.l.c.on Waters Associates C18 #sBondapak columns(0.4 cm x 30 cm), with Waters pumps, gradient controllerand detector. The flow rate was 1 ml/min, and peakswere collected manually. Some peptides were furtherpurified on a Waters phenyl ,uBondapak column(0.4 cm x 30 cm). The separations were done with 0.1 %(v/v) trifluoroacetic acid as solution A, and acetonitrile/propan-2-ol/methanol (1:1:1, by vol.) as solution B.Enzymes were from Worthington Biochemical Corp.,and solvents were from Rathburn Chemicals (Walker-burn, Peeblesshire, Scotland, U.K.).

Amino acid analysis and sequence determinationThe procedures for amino acid analysis are given in

Freemont et al. (1984). Sequencing was done auto-matically with a Beckman 890C liquid-phase sequencerequipped with the Beckman cold-trap accessory asdescribed in Russell et al. (1986).

RESULTSSequencing strategy

Fragments of aldolase were generated by cleavagewith CNBr, o-iodosobenzoic acid and proteolyticenzymes as outlined in Scheme 1. The results of the

purification of the CNBr- and o-iodosobenzoic acid-cleavage fragments and the N-terminal sequence analysesof these fragments are presented in Freemont et al.(1984). In the present paper we report the purificationand sequencing of the proteolytic fragments necessary toprovide overlaps between the chemically derived frag-ments and to complete the sequence determination.

Peptides derived from fragment CN2Carboxymethylated fragment CN2 (600 nmol) was

digested with staphylococcal proteinase under conditionsspecific for cleaving peptide bonds C-terminal to glutamicacid residues (Drapeau et al., 1972) as described in Fig.1, and the resulting peptides were separated on a Cl.uBondapak reverse-phase column (Fig. 1). Eighteenpeptides were successfully isolated by this method, and afurther two peptides were purified after an additionalseparation of the peak 13 material on a phenyl,uBondapak column (Fig. 1 inset). Eight of these peptides(SP19, SP3, SP12, SP9, SP8, SPIl, SPI and SP16) werecompletely sequenced as shown in Fig. 5. The initialyields were generally about 15 nmol (range 5-30 nmol),and the repetitive yields were between 85 and 90 %, withone exception. Peptide SP8 had a low repetitive yield(69 %) because of extensive loss of material from thespinning cup after cycle 8. There was good agreementbetween the amino acid compositions of the peptides andthe corresponding amino acid sequences (Freemont,1984).There was extensive cleavage adjacent to aspartate

residues despite the use of conditions favouring specificcleavage at glutamate residues. Thus peptides SP13F4,SP3, SP9, SPI and SP16 all resulted from cleavage ataspartate residues (Fig. 5). In addition, an unusualcleavage of a Ser-Lys peptide bond at residues 99-100was observed. A similar type of cleavage has beenreported for horse pancreas phospholipase A2 (Evenberget al., 1977).

Carboxymethylated and succinylated fragment CN2

S. proteinase R.p. h.p.l.c.--- SP peptidesCN2-

Succinylation/ - R.p. h.p.l.c.-.. ST peptidesCNBr CN3 trypsinf

CN4- Trypsin R.p. h.p.l.c.--* T peptides

CN5

Wi +W2

IOBA- W3

W4

Trypsin R.p. h.p.l.c. AT peptides

Scheme 1. Fragmentation of aldolase

The cleavage and digestion procedures and the methods of purification are described in the text. Abbreviations: IOBA,o-iodosobenzoic acid; R.p. h.p.l.c., reverse-phase h.p.l.c.; S. proteinase, staphylococcal proteinase.

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Amino acid sequence of human muscle aldolase

0 10 20 30 40Time (min)

Fig. 1. Separation of

0 20 40 60 80 100Time (min)

staphylococcal-proteinase-digest peptides from fragment CN2 by h.p.l.c.

Freeze-dried fragment CN2 (600 nmol) was dissolved in 1 ml of 50 mM-NH4HCO3, pH 7.8, digested with 0.2 mg ofstaphylococcal proteinase for 18 h at 37 °C, freeze-dried and redissolved in 0.8 ml of 0.1% (v/v) trifluoroacetic acid. Thepeptides in a 0.15 ml sample were separated on a C18 ,uBondapak column as described in the text. The inset shows the separationof peptides in peak 13 on a phenyl ,uBondapak column. Fractions corresponding to each major peak were collected.A214;------, concn. of solution B.

0 20 40 60 80 100

at

mc0

4-

0Cl)

Time (min)

Fig. 2. Separation of trypsin-digest peptides from succinylated fragment CN2 by h.p.l.c.

Freeze-dried succinylated fragment CN2 (250 nmol) was dissolved in 0.5 ml of 1% (w/v) NH4HCO3, pH 7.8, digested with0.25 mg of trypsin for 4 h at 37 °C, freeze-dried and redissolved in 0.4 ml of 0.1 % (v/v) trifluoroacetic acid. The peptides in a

0.1 ml sample were separated on a C18 1sBondapak column as described in the- text. Fractions corresponding to each major peakwere collected. , A214; ------, concn. of solution B.

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P. S. Freemont, B. Dunbar and L. A. Fothergill-Gilmore

(250 nmol) was digested with trypsin, and the peptideswere separated on a C18 atBondapak column (Fig. 2).Thirteen peptides were obtained, and three of these(ST13, ST8 and STIl) were subjected to N-terminalsequence analysis (Fig. 5). Peptide ST13 was a longpeptide of 65 residues, and the N-terminal 52 residueswere successfully identified (initial yield 13 nmol, repeti-tive yield 94 0%). A comparison of the amino acidcomposition of this peptide with the sequence shows asubstantial discrepancy for valine: there are eight valineresidues in the sequence, and only 4.8 in the composition.An inspection of the sequence shows that there are twoVal-Val and two Val-Ile sequences. The low values in thecomposition were probably a result of incompletehydrolysis of the peptide bonds between these residues.Peptides ST8 and ST 11 had initial yields of about10 nmol, and repetitive yields of 900 and 800 respec-tively. There was good agreement between the amino acidcompositions of the tryptic peptides (other than ST1 3)and the corresponding amino acid sequences (Freemont,1984).

Peptides derived from fragment CN4Carboxymethylated fragment CN4 was digested with

trypsin, and the peptides were separated by reverse-phaseh.p.l.c. (Fig. 3). Seven peptides were purified, and thesequence of peptide T7 was determined to complete thesequence of fragment CN4 (Fig. 5). The initial yield was20 nmol, and the repetitive yield 83 %. The amino acidcompositions of the other peptides agreed well with thecorresponding sequences (Freemont, 1984), except thatpeptide T4 had low values of valine and isoleucine (1.2residues of valine instead of 2, and 2.8 residues ofisoleucine instead of 4). Inspection of the sequence showsthe peptide has two Ile-Val sequences and one Ile-Leu. It

is likely that this is another example of incompletehydrolysis of resistant peptide bonds.

Trypsin digestion of aldolaseTrypsin-digest peptides were isolated from aldolase

primarily to obtain methionine-containing peptides toestablish overlaps between the CNBr-cleavage fragments.The conditions of digestion and the separation of thepeptides by reverse-phase h.p.l.c. are given in Fig. 4.Three of the 40 peptides (AT36, AT3 1 and AT32)contained methionine, and were thus sequenced (Fig. 5).These results provided unequivocal evidence for theorder of the CNBr-cleavage fragments. Peptide AT27was also sequenced to provide overlap informationbetween peptides SP8 and SP 1. The initial yields werebetween 15 and 50 nmol, and the repetitive yields between88 and 92 o.

Generally there was good agreement between theamino acid compositions of the peptides and theirsequences (Freemont, 1984). There were three exceptions.The most significant of these concerns peptide AT28,which had a low value of tyrosine: 1.5 residues instead of2. This peptide corresponds to the C-terminal 22 residuesof aldolase. It has been known for some time that the C-terminal tyrosine residue of aldolase is essential foractivity (Drechsler et al., 1959), and that the enzyme canbe inactivated by carboxypeptidase digestion (Rutteret al., 1961). The low value of tyrosine in peptide AT28would be consistent with the fact that the aldolase usedin these studies had a lower specific activity than expected(about 5 units/mg instead of about 15 units/mg)(Freemont et al., 1984). It is likely that the final tyrosineresidue was missing from a proportion of our sample ofaldolase, probably because of limited carboxypeptidasedigestion in the muscle before fractionation or during theearly purification steps. Further justification for this

80

60

40m0

:30en

20

0

Time (min)

Fig. 3. Separation of trypsin-digest peptides from fragment CN4 by h.p.l.c.Freeze-dried fragment CN4 (400 nmol) was dissolved in 0.3 ml of 1 % (w/v) NH4HCO3,pH 7.8, digested with 0.03 mg of trypsinfor 4 h at 37 °C, freeze-dried and redissolved in 0.4 ml of 1% (w/v) NH4HCO3, pH 7.5. The peptides in a 0.05 ml sample wereseparated on a C18 IuBondapak column as described in the text, except that solution A was 1% NH4HCO.. Fractionscorresponding to each major peak were collected. , A214;- , concn. of solution B.

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16

-360.4 20 40 60 80 10025

0.4 42023 18

3~~~~~~~~~~~~~0 20 40 60 802100

Time (min)

Fig. 4. Separation of trypsin-digest peptides from aldolase by h.p.1.c.Freeze-dried carboxymethylated aldolase (200 nmol of subunit) was dissolved in 0.8 ml of 1%0 (w/v) NH4HCO3, pH 7.8,digested with 0.08 mg of tosylphenylalanylchloromethane ('TPCK ')-treated trypsin for 2 h at 37 °C, freeze-dried and redissolvedin 0.4 ml of 0.1 % (v/v) trifluoroacetic acid. The peptides in a 0.1 ml sample were separated on a C18 ,uBondapak column asdescribed in the text. Fractions corresponding to each major peak were collected. , A214,;----, concn. of solution B.

suggestion comes from peptide AT22, which is sixresidues shorter than peptide AT28, but otherwiseidentical. This peptide was isolated in relatively lowyield, and probably is derived from carboxypeptidase-degraded aldolase.The other two peptides with discrepancies between

their compositions and sequences were AT3 1 and AT40.The former peptide had a low leucine content, which isconsistent with the presence of a Leu-Leu sequence. Thelatter peptide had low values of serine and threonine.These residues were identified unambiguously bysequencing, and it is not clear why the compositionvalues were low.

Complete sequence of human skeletal-muscle aldolaseThe complete amino acid sequence of the enzyme,

comprising 363 residues, is presented in Fig. 5. Thesubunit Mr value calculated from the sequence is 39 262;this value is close to that estimated by physical methods.The order of the CNBr- and o-iodosobenzoic acid-cleavage fragments was established by the purificationand sequence determination of staphylococcal-pro-teinase- and trypsin-digest peptides. Most of the sequenceof the enzyme was established from two or moreindependently isolated and sequenced fragments. In afew cases only a single sequence was determined, whichwas substantiated by the compositions of additionalpeptides derived from the same region. In all these casesthe phenylthiohydantoin derivatives of the amino acidresidues could be identified unambiguously.

DISCUSSIONThe complete sequence of human skeletal-muscle

fructose-bisphosphate aldolase is summarized in Fig. 6,and is compared with the sequences of aldolase fromrabbit skeletal muscle, Trypanosoma brucei, Drosophilamelanogaster, rat liver and human liver. It is apparent

that the sequences are highly conserved, especially whenthe evolutionary distances between mammals, protozoaand insects are taken into consideration. The overalldiversity among the sequences is given in Table 1. Theobserved sequence differences are corrected to PAMs(accepted point mutations) to take account of super-imposed and back mutations. One of the most strikingfeatures is that the rate of evolution is very low. This isparticularly true of the mammalian muscle (or A)isoenzyme, which is tolerating only about 2 PAMs/100million years. It is thus changing at a much lower ratethan, for example, ribonuclease, which has a rate ofmutation acceptance about 20 times greater, or trypsin,which is changing about 6 times faster. By contrast, theliver (or B) isoenzyme of aldolase is changing about 5times faster than the muscle isoenzyme. The overall rateof evolution of aldolase is about 4-6 PAMs/100 millionyears, and is typical of most of the glycolytic enzymes.This rate is the same as the small, highly specialized,cytochrome c molecule.

It is possible that the muscle isoenzyme is so highlyconserved because it experiences many constraints inaddition to the need to maintain catalytic function. It is,for example, present in high concentration in a tissuethat is itself evolving only very slowly. Muscle aldolase isknown to interact with the highly conserved F-actinmolecule (Arnold & Pette, 1968) and with phospho-fructokinase (Hofer et al., 1987). It is important to notein this context that the different isoenzyme sequencesfrom a single species can be strikingly different. Thus thehuman muscle and human liver aldolase sequences are asdivergent from each other as they are from D. melano-gaster aldolase. This would imply that the geneduplication event giving rise to the different isoenzymeseither took place early in vertebrate evolution, or that amore recent gene duplication event was followed by aperiod of exceptionally rapid change.Comparison of the sequence of D. melanogaster

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10 20 30 40 50P Y Q Y PA L T P E Q K K E L S DI A H R I V A P G K G I L AA D E S T G S I A KR L Q S I G T E N

i **ow1. .00000000*egg@@@@@@@@**@@@-

Il.o-oSP13F5 ..00000041 I.-.--oooooooooSP13F400.II.--l . sP1.5-.I@@@-00@@-001.ST12.-II-o.ooooo*oooool..-@ooooST1Ooe...*ooooooo-oo-ooollI@@oe@@ ST4.9@@@0@

I .oooooooAT24ooo. Io.oAT6ool. ....AT23 ..-AT16.

60 70 80 90 100T E E 14 R R F Y R Q L L L T A D D R V N P C I G G V I L F H E T L Y Q K A D D G R P F P Q V I K S K

I SP19 ii SP3 I SP12 - P

-O00AT16 . I...................AT39 ............................AT25.......I

110 120 130 140 150

G G V V G I KV D KG V V P L A G T N G E T T T Q G L D G L S E R C A Q Y K K D G A D F A K W R C V

.1 -SP9 -lSP8 spiS1 spiSP P16 ON

-0( ST13 0000000000000000000000000Si 43 ST8

I AT27 II AT3 I I.- oAT10.I0

l-@AT40@e

160 170 180 190 200

L K I G E H T P S A L A I M E N A N V L A R Y A S I C Q Q N G I V P I V E P E I L P D G D H D L K R

............9....CN2 ooo. ...i1 CN4 o

61P 1 6-S--- .........S ..4

ST11T..f.T4.-IIt.AeT3*-eil' ............................ T4.I

| .~~~AT36 it..._00000*6960 AT34 -e000 -@--@000 -00000 -000-@--@@l0

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Amino acid sequence of human muscle aldolase

210 220 230 240 250C Q Y V T E KV LAAV Y KA L S O H H I Y L E G T L L KP N M V T P G HA CT Q KF S H E E I AM

_FFFFFFFFFF***@@@@@@@@**CN1 ~~0-0** o o o 0@****** ** 1** i@No< CN4-*, -9--*<<@@ CN5- I

oo..T2o@.e..1| -o -T6 . 1 T7|

I.AT13 *.. 4IooooAT20......14 AT31. ....1 AT32

260 270 280 290 300A T V T A L R R T V P P A V T G I T F L S G G Q S E E E A S I N L N A I N K C P L L K P W A L T F S

A oo...........oo.oo.oAT40-oo.oooooo.ooooooooooolooo... Wo--- AT37 o oooo1oo4 P

310 320 330 340Y G RA L QA SA L KA WGG K KE N L KAA Q E E Y V KRA LA N S L A C QG KY T PS G Q AGA

||1@--4..il... AT1 7 ......If..... 1AT7 . 4|."AT12-i ....1AT2'.... |ii@ATl@***12........ .... * ...AT90000I AT22opI

AT28

360AAS E S L F I S DH A Y

.00so-. CN3 .Fo-@@@@@@@

--4 AT22o * * e

- AT28 aI~~~~~~~~~~

Fig. 5. Amino acid sequence of human skeletal-muscle aldolase

The continuous lines ( -) indicate those residues identified by automated N-terminal sequence analysis, and the dotted lines(. ) the unsequenced regions. Peptide nomenclature is given in Scheme 1, and details of the methods are given in thetext.

aldolase with those of the mammalian isoenzymes showsthat it is more similar to the muscle isoenzyme. Thiswould indicate that the ancestral form correspondedmore closely with the muscle isoenzyme, and that theliver isoenzyme arose by gene duplication. It seems likelythat the rate of aldolase evolution in non-mammaliantissues is 4-6 PAMs/100 million years, and that after thegene duplication event the muscle isoenzyme has evolvedmore slowly and the liver isoenzyme more rapidly. (Thistype of information is not apparent from the T. bruceisequences, as the protozoan is too evolutionarily distant,and the isoenzyme differences are swamped by the largenumber of total differences.)The comparisons that have been considered in Table 1

have involved the average differences between entireamino acid sequences, and are appropriate for estimatingevolutionary divergence. Of course, sequence variationsare not randomly distributed, and certain regions can beexceptionally conserved or exceptionally variable.A number of residues have been implicated from

chemical modification studies on Class I fructose-bisphosphate aldolases as being important for activity,and with one exception these residues are conserved inall the sequences. Lys-229 forms a Schiff-base inter-mediate with the substrate dihydroxyacetone phosphate(Horecker et al., 1961), and is thus clearly essential foractivity. It is present in a strongly conserved region. Arg-55 and Lys-146 are both implicated as binding the C-1

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-10

S K R V E V L L T Q

K R L Q S

K R L Q S

KRFG

KR LQD

NR L Q R

N R

1 10 20 30 40

P Y Q Y P A L T P E Q K K E L S D I A H R I V A P G K G I L A A D E S T G S I A

P H S H P A LT P E Q K K E L S D I A H R I V A P G K G I L A A D E S T G S I A

L P A Y N R K T P EAEL I E A K KMTA P G K G L L A A D E S T G SCS

T T Y F N Y P S K EL Q DEL R E I A QI V A P G KG I L A A D E S G T M G

A H R F JA S E Q K K E L SE I A Q R I V A N G K G I L A A D E S V GT M G

S E I A QSIVANG KG I L A A D E S V G TM G

50 60 70I G T E N T E E N R R F Y R Q L L L T A D D R V N P C

I G T E N T E E N R R F Y R Q L L L T A D R V N P C

I G L SN T E R R Q Y RML L E - C E G F E Q Y

I G V E N T ED R R A Y R Q L L F ST D P K L A E N

I K G E N T E E N R R QFL F S V N S I S Q S

K V E N T E|...

120

80 90I G G V I L F H E T L Y Q K A D D G

I G G V I L F H E T L Y Q K A D D G

ING V I LI3E TEJY Q K Aj jG

I [JG V I L F H E T L Y Q K A D D G

I G G V I L F H E T LTQKDSN

130T N|G E T T T Q G L D G L S E R C A Q Y K K

T N|G E T T T Q G L D G L S E R C A Q Y K K

A KI E TjG L D G Y I KER

SEDIEVT QGLDLILA..RCAQYKK

S D KIE T T ITQ G L D G L SE RCA Q K K

140 150 160 170 180

GAD F A K W R C V L K I G|E H T P S A L A I ME N A N V L A R Y A S I C Q Q N G I V P I V E P E

D GIA|D F A K W R C V L K I G|E HIT P SIALAJME N A N V L A R Y A S I C Q Q N G I V P I V E P E

JG jCR FEIK W RE3V3K IQ N G ITIS E A V V R3NAF L A R Y AI Q V P I V E P E

D GIC|D F A K W R C V L K I GKNK PSMQIVLE N A N V L A R Y A S I C QIS Q RII V P I V E P E

D JVDFGKWRAV LR I D Q C[ jSL IQENANALARVAS I CQQNGLVP I V E P E

190 200 210 220 4230I|L P D G U h D L K|R C Q Y V T E K V L A A V Y K A L S D HH I Y L E G T L L K P N M V T P G H A C

I|L P 0 G D H I) L K R C Q Y V T E K V L A A V Y K A L S D H H I Y L E G T L L K P N M V T P G H A C

VMD G H DIET C Q RVS VI V A LEIH E GE L L K P N M V PG FA S

V L P D G D H D RD Q K VT EUV L A A V Y K A L S D H H V Y L E G T L L K P N M V T AGQ-D LIE C E A A V Y K AL N OH H V LEG L L K P N MV VYAG HA

HAEC240 250 260 2 70 280TQKVS H|E E I A M A T V T A L R R T V P P A V T GVT F L S G G Q S E E E A S I N L N A I N KC

JQ K YS H|E E I A M A T V T A L R R T V P P A V T G V T F L S G G Q S E E E A S|I|N L N A I N|K|C

GL KGHAE Q V A Y T V K L R P P A |P G V T F L S G G S5 E 0A S E L N A |N N C

A K KM E E I A LAT VQA L R R T V P A A V T G V T F L S GG Q S E E E A T V N LEIA I N N

K K Y T P E Q V A M A T V T A L|; R T V P A A V PEI C F L S G GM S E EDA T S N L N A IV R 'C

T K K Y T P E Q V M A T V T A L H R T V P A A V P G I|C F L S G G M|S E E D A T L N L N A I N L C

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Rab A

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Rat B

Hun B

Hun A

Rab A

T. b.

D. m.

Rat 6

Hum B

Hum A

Rab A

T. b.

D. m.

Rat B

Hum A

Rab A

T. b.

D. m.

Rat B

Hum A

Rab A

T. b.

0. m.Rat B

Hum B

Hum A

Rab A

T. b.

D. m.

Rat BHum B

1

Amino acid sequence of human muscle aldolase

290 300 310 320P L L K P W A L T F S Y G R A L Q A S A L K A W G G K K E N L KA

P L L K P WA L T F S Y G R A L Q A S A L KA W G G K K E N L KA

P L PR P W K L T F S YVER A L QLSA 1WKW G G K ES G V E A

IP LHR P W A L T F S Y G R A L Q A S L R A WIG K K E N I AAP L P R P W K L S F S Y G R A L Q A S A L A A W G G K A A N K A

P L P K P W K L S F S Y G R A L Q A S A L A A W G G K A A N K EA

330A Q E E Y V K R AA Q E E Y V K R A

G R R A F MHR A

G E K R A

T Q E A F M K R A

T Q E A F M K R A

L A N S L A C Q

L A N S L A C Q

K M|N S L A QJK AN G D A A QV A N C Q A A Q

M A N C Q A1AK

Hum A

Rab A

T. b.

D. m.

Rat B

Hum B

340 350 360* *G K Y T P S G Q A G A A A S ESLFI S N H A Y

G K Y T P S Q A G A A A S E S L F I S N H A Y

|G K Y N R A D D D --K D S Q S L|Y YA T YIG K Y A|G S A G G ,- S S L V A N H A YGQY V|H TIG SSG A AIS T Q S L F TASYT Y

GQ V H T[GjS[SG ST Q S L FI A CVYT

Fig. 6. Comparison of the amino acid sequences of human, rabbit, rat, Drosophila melanogaster and Trypanosoma brucei aldolases

The sequences are available in the following publications: Rab A, rabbit muscle (Tolan et al., 1984); T. b., T. brucei (Clayton,1985); D. m., D. melanogaster (Malek et al., 1985); Rat B, rat liver (Tsutsumi et al., 1983); Hum B, human liver (partialsequences) (Costanzo et al., 1983; Besmond et al., 1983). The residues are numbered in accordance with the human musclesequence. The symbol - indicates that a gap has been introduced to maximize homology, and the symbol * shows the residuesimplicated as being important for activity. Residues that are identical in three or more sequences are boxed in.

Table 1. Evolution of aldolase

The number of residues compared relates only to known matched sequences; deletions and compositions of unsequencedpeptides are not included. The sequences are available in the references given in the legend to Fig. 6. Values for accepted pointmutations (PAM) and estimates of times since divergence are from Dayhoff (1978). Entries in parentheses are particularlyspeculative estimates, but nevertheless give an indication that they are consistent with an overall rate of evolution of4-6 PAMs/100 million years.

Differences Time since PAM/Sequences No. of residues divergence 100 x 106compared compared (no.) (%) PAM (106 years) years

Hum A-Rab AHum B-Rat BD. m.-Hum AD. m.-Hum BD. m.-Rat BT. b.-Hum AT. b.-Hum BT. b.-Rat BT. b.-D. m.

Hum A-Hum BHum A-Hum B

363164360161360360162360357164363

5 1.4 1.413 7.9 8.0

107 29 3758 36 50129 36 50185 51 8683 51 86192 53 92189 53 9259 35 48117 32 43

phospho group of both fructose bisphosphate anddihydroxyacetone phosphate (Lubini & Christen, 1979;Pathy et al., 1979), and both amino acid residues areconserved in all species. As already discussed, Tyr-363 isessential for activity, and it too is invariant in all species.Photo-oxidation studies have indicated that histidine isimportant in the catalytic mechanism (Hoffee et al.,1967; Davis et al., 1970), and chemical modification withN-bromoacetylet-hanolamine phosphate has enabled a

Vol. -249

labelled peptide containing alklylated His-361 to beisolated (Hartman & Welch, 1974). Horecker et al. (1972)have proposed that His-361 catalyses the removal andaddition of a proton from and to C-3 of the enzyme-bound dihydroxyacetone phosphate complex. However,this residue is not conserved in the human and rat liverisoenzyme sequences, nor in the T. brucei enzyme, and itis therefore unlikely that it plays a crucial role in thecatalytic mechanism.

Hum A

Rab A

T. b.

D. m.

Rat B

Hum B

7575

900900900

(1500)(1500)(1500)(1500)

1.9114.35.65.6(5.7)(5.7)(6.1)(6.1)

787

788 P. S. Freemont, B. Dunbar and L. A. Fothergill-Gilmore

The N-terminal region of aldolase is exceptionallyvariable, and, for example, three of the five differencesbetween human and rabbit muscle aldolase involveresidues 2-4. The T. brucei sequence has a ten-residue N-terminal extension, which may be important for ensuringthat the enzyme is correctly addressed for localizationwithin the glycosome organelle found in Trypanosoma(Clayton, 1985). Why should the terminal regions be sovariable? A possible explanation may relate to the factthat terminal regions are usually not buried in theprotein structure. Thus these portions of an enzyme mayvary both in amino acid sequence and in length withoutdisrupting the conserved tertiary structure that is requiredfor activity. In the case of the T. brucei enzyme, variationof the terminal region can be correlated with the'acquisition' of additional properties.

It is relevant to note that chicken muscle aldolaseisolated in the presence of proteolytic inhibitors had anadditional residue, a-N-blocked methionine, at the N-terminus (Lebherz et al., 1984). Aldolase from Drosophilaalso has been shown to have a blocked N-terminus, inthis case an a-N-acetylated threonine (Malek et al.,1985). It would seem likely that samples of aldolasepurified from muscle by traditional methods suffer theproteolytic removal of the blocked N-terminal residue,which may account for the unblocked proline N-terminusobserved in these studies. The presence or absence of ablocked N-terminus appears to have little functionalsignificance, as the specific catalytic activities andthermostabilities of the two forms of chicken musclealdolase are similar (Lebherz et al., 1984).A number of other portions of aldolase are

exceptionally variable: for example, residues 39-48,63-72, 91-99, 108-120, 154-164 and 343-358. All oftheseregions appear to correspond to portions of the poly-peptide chain lying between elements of predicted regularsecondary structure, and may represent surface loops.Residues 91-99 are of particular interest because they arestrongly predicted as a-helix in the D. melanogaster andrat liver sequences but not in the others. The results ofsecondary-structure predictions on class I fructose-bisphosphate aldolases are presented and discussed inthe accompanying paper (Sawyer et al., 1988).

We thank Mrs. Jean Bathgate and Mr. Ian Davidson forexcellent technical assistance. We are grateful to the Scienceand Engineering Research Council for a project grant and forsupport of the Scottish Sequencer Facility. L. A. F.-G. is therecipient of a Wellcome Trust University Award.

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Received 26 March 1987/17 July 1987; accepted 28 September 1987

1988