ACTIVE
S I T E OF
YEAST TRIOSE PHOSPHATE ISOMERASE
Heck, H. d’A., and Truffa-Bachi, P. (1970), Biochemistry 9, 2776, Janin, J., and Cohen, G . N. (1969), Eur. J . Biochem. 11,520. Janin, J., and Iwatsubo, M. (1969), Eur. J . Biochem. 11, 530. Janin, J., van Rapenbusch, R., Truffa-Bachi, P., and Cohen, G. N. (1969), Eur. J . Biochem. 8,128. Mankovitz, R., and Segal, H. L. (1969), Biochemistry 8, 3775. National Research Council (1926), in International Critical Tables Vol. 5, p 17. Ogilvie, J. W., Sightler, J. H., and Clark, R. B. (1969), Biochemistry 8, 3557. Patte, J.-C., LeBras, G., Lovings, T., and Cohen, G. N. (1963), Biochim. Biophys. Acta 67,16. Patte, J.-C., Truffa-Bachi, P., and Cohen, G. N. (1966), Biochim. Biophys. Acta 128,426. Roark, D., and Yphantis, D. (1969), Ann. N . Y . Acad. Sci. 164,245. Starnes, W. L., Munk, P., Mau, S. B., Cunningham, G. N., COX,D. J., and Shive, W. (1972), Biochemistry 11,677.
Szentirmai, A., Szentirmai, M., and Umbarger, H. E. (1968), J . Bacteriol. 95, 1672. Truffa-Bachi, P., and Heck, H. d’A. (1971), Biochemistry I O , 2700. Truffa-Bachi, P., Le Bras, G., and Cohen, G. N. (1966a), Biochim. Biophys. Acta 128,440. Truffa-Bachi, P., Le Bras, G., and Cohen, G. N. (1966b), Biochim. Biophys. Acta 128,450. Truffa-Bachi, P., van Rapenbusch, R., Janin, J., Gros, C., and Cohen, G. N. (1968), Eur. J. Biochem. 5,73. Truffa-Bachi, P., van Rapenbusch, R., Janin, J., Gros, C., and Cohen, G. N. (1969), Eur. J . Biochem. 7,401. Tung, J.-S., and Knight, C. A. (1971), Biochem. Biophys. Res. Commun. 42,1117. Wampler, D. E. (1971), Anal. Biochem. 44,528. Wampler, D. E., Takahashi, M., and Westhead, E. W. (1970), Biochemistry 9,4210. Wampler, D . E., and Westhead, E. W. (1968), Biochemistry 7,1661. Weber, K., and Osborn, M. (1969), J. Biol. Chem. 244,4406.
Haloacetol Phosphates. A Comparative Study of the Active Sites of Yeast and Muscle Triose Phosphate Isomeraset 1. L. Norton and F. C. Hartman*
A procedure is described for purifying triose phosphate isomerase from Bakers’ yeast to homogeneity in 35-40 yields. This enzyme, like the corresponding enzyme from rabbit muscle, is inactivated by the substrate analog, 3-chloroacetol phosphate, cia selective esterification of a single glutamyl y-carboxylate. The amino acid sequence around ABSTRACT:
H
aloacetol phosphates are active-site-specific reagents for triose phosphate isomerase. In the case of the isomerase from both rabbit and chicken muscle, inactivation results from a highly selective esterification of a single glutamyl y-carboxylate (Hartman, 1970a; Coulson et a/., 1970). The unusual reactivity of this carboxylate and the observation that chloroacetol phosphate inactivates triose phosphate isomerase from diverse species led Hartman (1971) to postulate that the carboxylate is probably functional in catalysis. Comparative studies on enzymes with the same function but from different species can be pertinent to ascertaining whether a given amino acid residue is catalytically essential. The validity of this statement resides in numerous examples supporting the concept that active-site regions of enzymes have been conserved during evolution ; Le., residues involved
t From the Biology Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37830. Receiued July 3, 1972. This is paper V in a series; paper I V (Paterson et al., 1972) appeared in Biochemistry 11, 2070. A preliminary account of this work has appeared (Hartman and Norton, 1972). Research sponsored by the U . S. Atomic Energy Commission under contract with the Union Carbide Corp.
this reactive residue (Ala-Tyr-Glu-Pro-Val-Trp) is identical with that found in the rabbit muscle enzyme. Sequence homologies between the segments containing the active-site glutamyl residue in these two diverse species, rabbit and yeast, suggest that the carboxyl group is functional in catalysis.
in catalysis are species invariant, and the primary sequences adjacent to these residues are highly homologous (Smith, 1970). Therefore, as a further test of the functional significance of the active-site glutamyl residue, we have characterized the site of yeast triose phosphate isomerase that is modified by 3-chloroacetol phosphate. Experimental Section
Materials. NADH, DL-glyceraldehyde 3-phosphate (as the dimethyl ketal), and glycerophosphate dehydrogenase were obtained from Boehringer Mannheim Corp. Trypsin treated with ~-(tosylamido-2-phenyl)ethyl chloromethyl ketone, DFPtreated carboxypeptidase, and pepsin were products of Worthington Biochemical Corp. 32P0C13and NaB3H4were purchased from Ammersham-Searle Corp. Ultra-Pure biological grades of ammonium sulfate and guanidine hydrochloride were purchased from Schwarz-Mann. Phenyl isothiocyanate and trifluoroacetic acid were obtained from Aldrich Chemical Co. Chloroacetol phosphate was synthesized as described previously (Hartman, 1970b) ; the 32P-labeledreagent (initial specific radioactivity of 1.28 x 106 cpm/pmole) was prepared BIOCHEMISTRY,
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TABLE I:
Purification of Yeast Triose Phosphate Isomerase.
-
Fraction
Total Sp Act. Vol Protein Total Act. (Units/ Yield (ml) (mg) (Units) mg) (%I
2650 53,000" Extract 60430% ammon- 375 22,500' ium sulfate precipitate DEAE-cellulose 140 3,800' chromatography (PH 8.0) CM-cellulose 37.5 587b chromatography DEAE-cellulose 84 428 chromatography (PH 7.5)
11,900,000 10,600,000
230 100 410 89
7,600,000 2,000
63
5,500,000 9,300
46
4,500,000 10,500
38
Based on Lowry determinations. (see Experimental Section).
Based on A at 280 nm
by use of 32Poc13in the phosphorylation step, which was scaled down tenfold. Protein Determinations. The method of Lowry et ul. (1951), with human serum albumin as the standard, or spectrophotometric measurements at 280 nm were used to determine protein concentrations. The at 280 nm of purified yeast triose phosphate isomerase is 10.0 (see below). Extinction CoefJicient. Two samples of yeast triose phosphate isomerase at about 8 mg/ml were dialyzed exhaustively (three changes during 48 hr) against 0.01 M ammonium bicarbonate (pH 8.1). Aliquots of the dialyzed samples were diluted 20-fold into 0.1 M Tris-hydrochloride (pH 8.0 and 7.4), 0.02 M triethanolamine hydrochloride (pH 7.9), and 0.01 hi sodium phosphate (pH 8.0); the absorbancies at 280 nm of these solutions were then determined and found to be identical. Samples (2 ml) of the dialyzed, undiluted protein solutions were lyophilized to dryness in tared vials, dissolved in 3 ml of water, and again lyophilized. The vials were then heated overnight at 110" and cooled in a vacuum desiccator to constant weight. The two determinations yielded at 280 nm of 9.99 and 10.0. Enzyme Assaj~s.Triose phosphate isomerase activity was measured by the method of Beisenherz (1955) as modified by Norton er ul. (1970), in which the glycerophosphate dehydrogenase catalyzed reduction of dihydroxyacetone phosphate (formed from D-glyceraldehyde 3-phosphate in the isomerase reaction) by NADH is monitored at 340 nm. A Beckman Acta V recording spectrophotometer was used in these determinations. Electrophoresis. Analytical polyacrylamide gel electrophoresis was carried out with the instrument from Canal Industrial Corp. according to the instructions provided. The acrylamide concentration used was 7 %. After electrophoresis, protein was detected with Amido Schwarz, and excess stain was removed electrophoretically. Peplide Mapping and Autoradiography. Peptide maps on Whatman No. 3MM were prepared essentially by the method of Katz el al. (1959) except that electrophoresis preceded chromatography. The experimental conditions used were identical to those published previously (Hartman, 1971).
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Autoradiograms were prepared with Kodak No-Screen Medical X-Ray film; exposure times varied from 1 to 5 days depending upon the quantity of radioactivity present. Amino Acid Analyses. Samples were hydrolyzed with 6 N HC1 at 110" for 21 hr in sealed, evacuated (
