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THIJOURNAL OFBIOLOGICALCHEMISTRYVol.247, No. 11, Issue of June 10, pp. 3618-3621, 1972Printed in U.S .A.

A Chymotrypsin-catalyzed Modification ofRabbit Muscle Aldolase*

(Received for publ icatio n. February 15, 197~)

CHRISTIAN F. MIDELFORT AND ALAN H. MEHLERFrom the Department of Biochemzstry, Medical Collegeof Wisconsin, Milwaukee, Wisconsin 53233

SUMMARYIn the presence of a diphosphate substrate or substrate

analogue, rabbit muscle aldolase undergoes a limited pro-teolys is by chymotrypsin. The sole products are an enzymewith modified cataly tic properties and two hexapeptides re-moved from the COOH terminus of the a! and 0 subunits,respectively. The enzyme product has cataly tic propertiessimilar to those of aldolase modified by carboxypeptidase Ain that it retains full act ivi ty with fructose l-phosphate assubstrate but has lost 95% of the original act ivi ty with fruc-tose 1,6-diphosphate as substrate. The hexapeptides havethe structures Ile-Ser-Asn-His-Ala-Tyr (a! subunit) and Ile-Ser-Asp-His-Ala-Tyr (p subunit) and are produced in ap-proximately equal amounts from aldolase preparations ob-tained from adult animals.

Rabbit muscle aldolase is known to catalyze the aldol cleavageof certain sugar phosphates by an ordered mechanism (1).Ketose phosphate 1 Aldehyde 2 Dihydroxyacetone phosphate

+ e+* +Aldolase Dihydroxyace- Aldolasetonephosphate Aldolase

Removal of the COOH-terminal tyrosine with carboxypeptidaseA converts aldolase to a form, CP-aldolase,l which is character-ized by a decreased rate o f cleavage of fructose 1,6-diphosphate(FDP) and an unaltered rate of cleavage of fructose l-phosphate(FlP) (2). Several other chemical modifications of the enzymehave been found to produce a similar ef fec t on its act ivi ty. Par-tial proteolysis with chymotrypsin or with subtilisin A or B (3),iodination of tyrosines (4), hydroxylation of tyrosines withmushroom polyphenol oxidase (5), and photo-oxidation of histi-

* This work was supported by Grants GB7146 and GB27440from the National Science Foundation. This paper is dedicatedto Professor A. E. Braunstein, who has been a personal and scien-tific inspiration, on the occasion of his seventieth birthday.

1 The abbreviations used are: CP-aldolase, rabbit muscle aldol-ase modified by treatment with carboxypeptidase A to remove allCOOH-terminal tyrosines and variable am ounts of subsequentamino acids; FDP, fructose 1,6-diphosphate; FlP, fructosel-phosphate; HDP, a mixture of mannitol- and sorbitol 1,6-di-phosphates.

dines (6) are examples. Each type of modification slows therate of Step 2 above without affecting the rate of Step 1. Todate no chemical agent has been found to produce the reverseeffect . These results have led to the postulation that a basicresidue on the enzyme facilita tes Step 2 only and that participa-tion of this base in the catalytic mechanism is prevented by eachof the above modifications. Although positive identification ofactive site amino acid residues awaits the determination of theprimary (7) and secondary (8) structure of aldolase, characteriza-tions of the chemical structures of the enzymes modified in eachof the above ways would facilitate an understanding of theenzymatic mechanism.We report here the identification of the peptide bonds splitby alpha-chymotrypsin in producing a stable product with CP-aldolase-like properties.

MATERIALS AND METHODSRabbit muscle aldolase was prepared by the method of Taylor

et al. (9) from 6- to &pound rabbits, and each preparation wascrystallized a total of four times. The preparations used hadspecif ic activities ranging from 15 to 18 units per mg of protein.(A unit is defined as the amount of enzyme required to cleave 1pmole of FDP per min at pH 8 and 25.) Enzyme act ivi ty as-says were done by observing the absorbance change at 340 nm ina Cary 15 spectrophotometer. A standard assay contained10m2 M FlP or 10m3 M FDP, 0.05 M Tris, 0.1 mg of DPNH, 50pg of a-glycerophosphate dehydrogenase (Sigma), 20 pg oftriose phosphate isomerase (Sigma), and a limiting amount ofaldolase in a total volume of 1 ml. Protein concentrations weremeasured spectrophotometrically using an extinction coeff icientat 280 nm of 1.066 ml per cm per mg, determined by measure-ments in a Cary 15 spectrophotometer on aldolase samples withconcentrations calculated from total nitrogen determinationsusing the Kjeldahl method.Amino acid analyses were carried out by the method of Spack-man et csl. (10) using an automatic amino acid analyzer con-structed in our laboratory. The machine was built essentiallyas described by Spackman et ~1. except that Beckman AA-15and AA-27 resins and an Eagle Signal Co. (Davenport, Ia.)multigang 12 cam timer were used. The last was chosen becauseof its potential for programming an automatic sample-injectionassembly. NHz-terminal end groups were dansylated and iden-tified as described by Labouesse and Gros (11). Proteolytichydrolysis of peptide bonds was estimated in a Radiometer pHStat using a water-jacketed, closed vessel. It was assumed that

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Issue of June 10, 1972 C. F. Midelfort and A. H. Mehler 3619

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5 10 20 30 40 50TIME (MINUTES)

F IG . 1. Chymotryptic hydrolysis of muscle aldolase undervarious conditions. A solution of aldolase (10 mg per ml) in 0.02M KC1 was mixed with chymotrypsin at a final concentration of0.05 mg per ml. A, change in aldolase enzymatic act ivi ty withtime. Open symbols refer to assays using FlP as substrate; closedsymbols refer to assays using FDP as substrate. A and A, 25,pH 8.0, 10e3 M HDP; 0 and n , 25, pH 8.0, no HDP; 0 and l ,37,pH 8.0, 1O-3 M HDP;V andV, 25, pH 9.0, 10e3 M HDP. B, pHStat titration of HzOf ions liberated during peptide bond hy-drolysis . Conditions and symbols are the same as above.the new free amino groups had average pKs of 7.5. High vol-tage paper electrophoresis was done in a Gilson model D Electro-phorator. The ninhydrin color of column eluates was measuredafter alkaline hydrolysis by the method o f Him (12). The polyolanalogue of FDP, hexitol 1,6-diphosphate, (HDP) was preparedfrom FDP by sodium borohydride reduction (13). Carboxy-methylated aldolase was prepared by the method of Crestfieldet al. (14).

RESULTSReaction Conditions-Conditions were sought in which c~-

chymotrypsin produced an aldolase that still retained 100% ofits enzymatic act ivi ty toward FlP but had lost most of its activ-ity toward FDP. Fig. 1 shows that the act ivi ty toward FlPwas stable when a saturating concentration of FDP or HDP waspresent, when the pH was kept below 8.5, and when the tempera-ture was kept below 30. In the absence of FDP or HDP, theproteolysis appeared to be quite different in nature than in thepresence o f a diphosphate compound since the FlP and FDPcleavage activities fell at about the same rates. Measurementsof the HaO+ ions released at pH 8.0 in a Radiometer pH Statshowed that only a small number of peptide bonds were split inproducing the stable product with CP-aldolase-like properties(Fig. 1B).Stoichiometry of Limited Hydrolysis-Unambiguous correlationbetween the cleavage of a certain peptide bond and an eff ect on

T ABLE IAmino acid compositions of trichloroacetic acid supernatantstaken at various times after the addition of a-chymotrypsin toan aldolase solution containing lo+ M HDP and 0.05 M KC1 atpH 8 and 25.

T i me Initial enzymeactivity His /^n,+i Ser 1 Ala / Ile 1 b 1 ,, &! !,

min % moles amino acid/mo le aldolase0 100 0.0 IO.2 0.1 0.3 0.0 0.0


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3620 Modificat ion of Aldolase by Chymotrypsin Vol. 247, No. 11

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NATIVE ALDOLASEMARKER

20 40 60 80 100 120TUBE NUMBER (4ml FRACTIONS)

FIG. 2. Sephadex G-75 chromatography of a chymotrypsin-aldolase reaction mixture prepared at 25, pH 8.0, and 10eaM HDP.The column (2 X 100 cm) was equilibrated with 0.02 M KC1 at roomtemperature and run at a flow rate of 20 ml per hour. A lo-mlsample of 10 mg per ml of protein was applied to the column. Thecolumn was assayed for ninhydrin color by hydrolyzing O.l-mlsamples in 1 N NaOH for 20 min in an autoclave. The eluate wasalso monitored for total organic phosphate to determine theposition of the HDP. In a separate experiment, 1.5 ml of nativealdolase were applied to the same column to determine its elutionposition.TABLE II

Carboxypeptidase A digestion of electrophoretically separatedpeptides taken from second peak of Sephadex columna

Amin o acid Neutral peptide Basic peptide

Histidine. . . . . Trace 0.040 pmoleAspartic acid. Trace TraceSerine + asparagine. 0.007 pmole 0.019 pmoleAlanine.. . . . . 0.043 pmole 0.051 pmoleIsoleucine . None TraceTyrosine . 0.045 pmole 0.050 rmole

a Peptide (0.1 pmole) and carboxypeptidase A (0.25 mg) in 1ml; 26 hour digest.ninhydrinreactive spots, one more cationic than the other, werevisualized and eluted. Both yielded on ly dans-Ile after reactionwith dansyl chloride, and both contained equal molar amountsof the same six amino acids as the starting material. Theytherefore appear to represent corresponding portions of theCOOH-terminal regions of the OLand /3 subunits as described byLai et al. (15). To confirm this conclusion 0.1 pmole of eachpeptide was treated with 0.25 mg of carboxypeptidase A for 2hours, and the free amino acids released were determined. Thesedata (Table I I) show that the more neutral peptide containedthe carboxypeptidase A-resistant His-Asp peptide bond and thatthe more basic peptide contained the His-Asn bond. Theseresults show that the action o f cr-chymotrypsin in producing analdolase with cataly tic properties similar to CP-aldolase involvesrelease of the terminal hexapeptides from the following structures.

a-Chy-(Leu ,Phe) J, Ile-Ser-Asn-His-Ala-Tyr-COOH LY ubunit- (Leu ,Phe) 1 Ile-Ser-Asp-His-Ala-Tyr-COOH p subunit

DISCUSSIONThese results confirm those of Lai et al. (15) and indicate that

the COOH-terminal sequence as published by Winsted and Wold(16) is in error. As pointed out by Lai et al. the reasons for thisare 2-fold: (o) the mistaken identification of an asparagine as aserine during amino acid analysis and (b) the unusual resistanceof the Asp-His peptide bond o f the @ subunit to carboxypeptidaseA hydro lysis. This bond is probably stabilized by an interactionbetween the side chains o f the 2 amino acid residues by means of acovalent N-acyl imidazole bond or of a stable ion pair.

FDP or HDP binding to aldolase plays an important role inrestricting the number of peptide bonds susceptible to chymo-tryp tic attack. As illustrated in Fig. lA, under various condi-tions o f temperature and pH, the presence o f HDP causes therate o f decline o f FDP cleavage a ctivity to be much faster thanthat of FlP cleavage activity, whereas in the absence of HDPboth activit ies of aldolase fall at the same rate. Measurementsof peptide bonds hydrolyzed show that extens ive digestion occursin the absence of HDP. In addition, sugar diphosphate ana-logues (ribitol 1 ,5-diphosphate, HDP, and octitol 1 ,&diphos-phate) protect both native and carboxypeptidase A modifiedaldolase from trypsin-catalyzed inactivation at 37O.2 The samebinding constants for each analogue (2 to 3 X 10-G M, 2 to 3 x10m6 M, and 5 X low6 M, respect ively) were found for the protec-tion of both native and CP-aldolase. It may be concluded thatthe diphosphate substrates and analogues that are bound toaldolase maintain the enzyme in a conformation in which thebulk of the protein is not accessible to certain proteases.

The susceptibili ty of the COOH-terminal region and the resist-ance of the rest of the protein to proteolysis under the conditionsdescribed in the text are observed not only when chymotrypsinis the proteolytic enzyme but also when subtilisin Novo is used.Experiments to be the subject o f a future communication indicatethat COOH-terminal oligopeptides are quantitat ively cleavedby subtilisin before any other proteolysis occurs, and that oli-gopeptides derived from up to 14 residues f rom the COOHterminus are released. The absence of lysine and arginine resi-dues in this region perhaps explains why aldolase is completelyresistant to trypsin digestion under the above conditions.

The question of whether the (Y and /3 subunits of aldolase rep-resent polypeptides with different genetic determinants orwhether the ,8 subunit arises by in viva deamidation of the asubunit has been raised by Koida et al. (17). Results fromHoreckers laboratory (15) appear to indicate that the Asn-Aspreplacement at the fourth residue in from the COOH terminus isthe only difference between the two types of subunits. Sincethe genetic codons for Asn and Asp are, respect ively, AAPy andGAPy, the gene for one could have arisen from that for the otherby a purine-purine base replacement. Gene duplication wouldthen allow both sequences to be expressed in the same animal.We are currently investigating these two possibilities.

REFERENCES1. ROSE, I. A., OCONNELL, E., ANDMEHLER A.H.(1965)J. Biol.Chem. 240, 17582. DRECHSLER, E. R.,BoYER, P.D., ANDKOWALSKY, A.G.(1959)

J. Biol. Chem. 234, 26273. MEHLER, A. H., AND VISWANATHA, T. (1961) Fed. Proc. 20, 2324. WASSARMAN, P.M., AND KAPLAN,N. 0. (1968) J. Biol. Chem.243, 720

2 C. F. Midelfort and A. H. Mehler, unpublished experiments.

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Issue of June 10, 1972 C. F. Midelfort and A. H. Mehbr5. CORY, J. G., AND FRIEDEN, E. (1967) Biochemistry 6, 1216. HOFFEE, P., LAI, C. Y., PUGH, E. L., AND HORECI~ER, B. L.,

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11. GROS, C., AND LABOUESSE, B. (1969) Eur. J. Biochem. 7, 46312. HIRS. W. H. C. (19671 Methods Enzumol. 11. 32513. GINS&JRG, A., AI&I M&ILEB, A. H. (1966) Biochemistry 6, 262314. CRESTFIELD, A. M., STEIN, W. H., AND MOORE, S. (1963) J.

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Biochem. Biophys. 134, 623

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a chymotrypsin-catalyzed modification of

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