- Email: [email protected]
[46] Labeling of the active site of l -aspartate β-decarboxylase with β-chloro-l -alanine
[45]
L-ASPARTATE ~-DECARBOXYLASE
427
with [14C]chloroketone enabled localization of the glutamine binding site on the heavy subuni~ of the enzyme. After inactivation with about 1 mole of [1~C] chloroketone, dissociation of the subunits was accomplished by boiling the protein in a solution containing 1% SDS and 1% 2-mercaptoethanol at pH 8.5 followed by gel electrophoresis; after disintegration of the gel in 30% H,_,02 substantial amounts of radioactivity were found associated only with the heavy subunit. ~1 A cysteine that reacted with chloroketone or iodoacetamide was found in the active site27 Comments Studies with glutamine analogs have greatly increased our understanding of the action of glutamine-utilizing enzymes. Considerable evidence indicates that separate sites exist for glutamine and ammonia and that a 7-glutamyl thioester is a central intermediate in the cleavage of glutamine. 1°,3',53 It has been suggested that amidotransferases may have developed by the association of a primordial glutaminase peptide, either in a separate subunit or covalently incorporated, with transferases specifically designed for different acceptors. 45,54 Comparison of peptides from glutamine binding sites of different enzymes is now possible and may lead to a better understanding of the evolution of this group of proteins. Inhibitors of glutamine utilization should also prove useful in studies of glutamine transport. 55H. Nagano, H. Zalkin, and E. J. Henderson, J. Biol. Chem. 245, 3810 (1970). 54H. C. Li and J. M. Buchanan, J. Biol. Chem. 246, 4713 (1971).
[46] L a b e l i n g o f t h e A c t i v e S i t e o f L - A s p a r t a t e ~ - D e c a r b o x y l a s e with fl-Chloro-L-alanine
By
NOEL M. RELYEA, SURESH S. TATE, and ALTON MEISTER
L-Aspartate-fl-decarboxylase, a pyridoxal 5'-phosphate enzyme, catalyzes the f~-decarboxyl.ation of L-aspartate to L-alanine [Eq. (1)] as L-Aspartate--~ Iralanine + CO2
(1)
well as a number of other reactions, 1 which include desulfination of Lcysteine sulfinate to L-alanine, 2 a-decarboxylation of aminomalonate to glycine, 3 decarboxylation of threo- and erythro-fl-hydroxyl-L-aspartate 1 S. S. Tate and A. Meister, Adv. Enzymol. 35, 503 (1971). 5 K. Soda, A. Novogrodsky, and A. Meister, Biochemistry 3, 1450 (1964). 5 A. G. Palekar, S. S. Tate, and A. Meister, Biochemistry 9, 2310 (1970).
428
ENZYMES, ANTIBODIES, AND OTHER PROTEINS
[46]
to L-serine, 4 various transamination reactions, 5-7 and q,fl-elimination reactions such as t h a t of fl-chloro-L-alanine s [Eq. (2) ]. CH2C]
CH3
I I COO-
CHNH~" + H20
~
I I COO-
C-'-O + NH,~ + H + + C I -
(2)
When the enzyme is incubated with fl-chloro-L-alanine, the rate of the catalytic reaction declines rapidly as the enzyme becomes irreversibly inhibited. Such inactivation is associated with the binding of close to 1 mole of the 3-carbon chain of the substrate analog per mole of active site. s The enzyme, after inactivation by treatment with fl-chloro-L[l*C]alanine, was treated with cyanogen bromide and a 14C-labeled peptide was then isolated. T r e a t m e n t of the labeled peptide with mild alkali leads to release of fl-hydroxy-[~4C]pyruvate, suggesting an ester linkage. Ammonolysis of the labeled peptide leads to release of the label, and amino acid analyses of enzymic hydrolyzates of the peptide before and after ammonolysis show formation of an equivalent amount of glutamine 9 [Eq. (3)]. The findings indicate that the labeled derivative - Glu
-Glu-
I I 0 I Ctt2 I
NH3
O~-C
C--O
I
COO-
-
I
O--C--NH 2
CH20H
I I COOC=O
(fl-hydroxypyruvate) is bound by an ester linkage to a glutamate residue at the active center of the enzyme. Preparation of/~-Chloro-L-[14C]alanine fl-Chloro-L-alanine labeled with 14C m a y be prepared from any of the various preparations of [14C]serine t h a t are commercially available E. Miles and A. Meister, Biochemistry 6, 1734 (1967). s S. S. Tate and A. Meister, Biochemistry 9, 2626 (1970). A. Novogrodsky, J. S. Nishimura, and A. Meister, J. Biol. Chem. 238, PC 1179 (1963). ' A. Novogrodsky and A. Meister, J. Biol. Chem. 239, 879 (1964). s S. S. Tate, N. Relyea, and A. Meister, Biochemistry 8, 5016 (1969). 9N. M. Relyea, S. S. Tate, and A. Meister, J. Biol. Chem. 249, 1519 (1974).
[46]
L-ASPARTATE ~-DECA.RBOXYLASE
429
according to the procedure of Fischer and Raske 1° as modified, 1~,12 which involves chlorination of serine methylester with phosphorus pentachloride. In this procedure it is of the utmost importance to maintain scrupulously dry conditions. L-[14C]Serine-HC1 (1.6 ~moles) and 75 mg (0.53 mmole) of unlabeled L-serine-HC1 are dissolved in 3 ml of dry methanol. Dry hydrogen chloride gas (dried by bubbling through concentrated H2SO4) is bubbled through the methanolic solution of serine at 0 ° for 30 min. The solution is allowed to warm to room temperature and then refluxed for 30 rain. After cooling, the solution is flash-evaporated to dryness and the residue is suspended in dry benzene; the benzene is removed by flashevaporation. The benzene step is repeated. The crystalline residue is dissolved in 1 ml of dry methanol, and 50 ml of dry diethyl ether are added slowly at 25 °. After chilling in ice for several hours, crystals of serine methylester hydrochloride are collected and washed with dry diethyl ether (m.p. 129°-131°). Chloroform is passed through an alumina column (Woelm) and stored over a Linde 4A molecular sieve for not more than 2 hr before use. Phosphorus pentachloride (135 mg) is added to 2.5 ml of dry chloroform and dissolved by stirring at 26 ° for 30 min. The solution is then chilled on ice and 105 mg of the serine methyl ester hydrochloride (prepared as described above) is added in small portions over a 30-min period with stirring. The solution is stirred at 26 ° for 2 hr until it clarifies; crystals then begin to form. The mixture is placed at 0 ° for 18 hr; dry petroleum ether (b.p. 700-90 o) is added and the crystals of fl-chloro-L-alanine methylester hydrochIoride are collected and washed with dry petroleum ether. The crystals (92 rag) are suspended in 3 ml of 6 M HC1 and refluxed for 1 hr. The solution is flash-evaporated, and the residue is dissolved in water and flash-evaporated again. This procedure is repeated once with water and then twice with benzene.
Labeling of the Enzyme The inactivation and labeling of L-aspartate-p-decarboxylase probably involves nueleophilie attack by a group on the enzyme on the fl-earbon atom of the ~-'aminoacrylate-Schiff base formed in the interaction of fl-chloro-L-alanine and enzyme-bound pyridoxal 5'-phosphate. s Labeling of the enzyme thus requires conditions under which the catalytic reaction can occur. An excess of fl-chloro-L-alanine is therefore required, since a substantial portion of the an.alog is converted to pyruvate before ~°E. Fischer and E. l~aske, Bet. Deut. Chem. Ges. 40, 3717 (1907). 11C. T. Walsh, A. Sehonbrunn, and R. H. Abeles, Y. Biol. Chem. 246, 6855 (1971). ~2j. p. Greenstein and M. Winitz, "Chemistry of the Amino Acids," p. 2677. Wiley, New York, 1961.
430
ENZYMES, ANTIBODIES, AND OTHER PROTEINS
[46]
the enzyme is completely inactivated. L-Aspartate-fl-decarboxylase (1 mg/ml) is incubated in 0.2 M sodium acetate buffer (pH 5.5) containing 10 mM fl-chloro-L-[14C]alanine for 20 min at 370.9 Under these conditions, the enzyme is completely inactivated. The labeled enzyme is precipitated at 5 ° by adding solid ammonium sulfate to 65% of ammonium sulfate saturation. The precipitated enzyme is recovered by centrifugation and dissolved in a small volume of 1 M sodium acetate at pH 6 .and then dialyzed exhaustively against water to remove salts; it is then lyophilized. Cleavage of the Labeled Enzyme to Yield a Labeled Peptide The labeled moiety introduced into the enzyme by treatment with fl-chloro-L- [14C] alanine is labile to alkali ; the following procedures are therefore carried out at acid values of pH. The labeled enzyme (5 mg/ml) is suspended in 70% formic acid and a 5-fold (w/w) excess of cyanogen bromide is added. The solution is stirred with a magnetic stirrer in a tightly stoppered flask for 24 hr at 25 °. The reaction is terminated by adding 10 volumes of cold water, and the solution is lyophilized. The labeled peptide is purified by cation-exchange chromatography13 as follows. 3_ water-jacketed column (45 X 0.9 cm) containing Technicon type P cation exchange resin is used at 50% The cyanogen bromide peptides are suspended in 2 ml of 0.2 M pyridine acetate at pH 3.1. After clarification by centrifugation, the solution is applied to the column and the peptides are eluted with a linear gradient established between 250 ml of 0.2 M pyridine acetate at pH 3.1, and 250 ml of 2 M pyridine acetate at pH 5.0, using a flow rate of 30 ml per hour; fractions of 2.5 ml are collected. Examination of the fractions for radioactivity and for amino acid content after hydrolysis (6 M HC1, 105 °, 18 hr) leads to identification of the first peak obtained from the column (fractions 4-6) as the major labeled cyanogen bromide peptide. When this peptide is subjected to acid hydrolysis followed by amino acid analysis, all the radioactivity elutes in an early ninhydrin-negative peak. Complete enzymic hydrolysis of the labeled peptide can also be achieved by cleavage with ~-chymotrypsin~4 followed by treatment with leucine aminopeptidase. 9 The amino acid composition found after complete enzymic hydrolysis is similar to but not the same as that found after acid hydrolysis. Thus, two aspartate residues are found after acid hydrolysis, and one aspartate and one asparagine residue are found after enzymic hydrolysis. On amino acid analysis after enzymic digestion, all the label appears in i~ R. T. Jones, "Methods of Biochemical Analysis" (D. Gliek, ed.), Vol. 18, pp. 205258. Wiley (Interscience), New York, 1970. ~4D. G. Smyth, this series, Vol. 11, p. 214 (1967).
[46]
L-ASPART&TE ~-DECARBOXVLASE
a ninhydrin-negative peak that elutes early. The nature of material may be established by paper chromatography using standardsg; thus, the labeled material moves with authentic pyruvate. It may also be identified as the corresponding phenylhydrazone. 15
431 the labeled appropriate fl-hydroxy2,4-dinitro-
Identification of the Protein Amino Acid Residue That Reacts with ~-Chloro-L-alanine
The alkali lability of the labeled enzyme derivative suggests that an ester of either glutamic acid or aspartie acid is formed by interaction of the enzyme with fl-chloro-L-alanine. Evidence derived from sequencing of the labeled peptide indicates that the label is not attached to the aspartate residue of the peptide, and thus suggests that the label is attached to a glutamate residue. Ammonolysis of esters to yield amides is a well known procedure, TM which may be usefully applied to the study of fl-chloro-L-alanine-labeled aspartate-B-decarboxylase. Thus, a sample of the labeled peptide (12 nmoles) is incubated in 0.5 ml of concentrated ammonium hydroxide (28% NH3) for 16 hr at 26 °. After flash-evaporation to dryness, the residue and a sample of the peptide not treated with ammonia are completely digested by successive treatment with chymotrypsin TM and leucine aminopeptidase. 9 In studies in which this procedure was followed, amino acid analysis showed that glutamine is formed and that glutamate disappears in the sample subjected to ammonolysis. The amount of glutamine formed is in close quantitative agreement with the molar amount of labeled residue bound to the peptide and released during ammonolysis. No glutamine is found after enzymic digestion of the sample of peptide which was not subjected to ammonolysis. As a further control, a sample of unlabeled peptide obtained by cyanogen bromide treatment of the unlabeled enzyme may be subjected to ammonolysis; no glutamine is found in the enzymic digest of this material2 Discussion
B-Chloro-L-alanine acts as a substrate and as an inhibitor of L-aspartate-fl-decarboxylase. The relatively rapid catalytic reaction is accompanied by a slower reaction in which the enzyme becomes inactivated. Several experimental approaches support the conclusion that fl-chloro-Lalanine is a true active site-directed inhibitor. (1) Only the L-isomer of fl-chloroalanine is a substrate and an inhibitor. (2) Treatment of the 15A. Meister and P. A. Abendsehein, Anal. Chem. 28, 171 (1956). 1~j. p. Greenstein and M. Winitz, "Chemistry of the Amino Acids," p. 1258. Wiley, New York, 1961.
432
ENZYMES, ANTIBODIES, AND OTHER PROTEINS
[47]
enzyme with fl-chloro-L-alanine leads to a spectral shift from a peak absorbance (native enzyme) at 355 nm to a peak at about 320 nm indicating that Schiff base formation is associated with inactivation. (3) When the apoenzyme or the 4'-deoxypyridbxine-5'-phosphate enzyme is treated with fl-chloro-L-alanine, neither inactivation nor binding of the substrate analog occurs. Cytoplasmic glutamate-aspartate transaminase also catalyzes a,flelimination of fl-chloro-L-alanine with concomitant inactivation of the enzyme. 1ms In this inactivation, there is evidence that the substrate analog binds to the c-amino group of the lysyl residue that is normally involved in binding pyridoxal 5'-phosphate. A number of reports have appeared on the esterification of specific protein carboxyl groups. ~9 Specificity may be due to enhanced reactivity of a particular enzyme carboxyl group or to the ability of the substrate analog to bind specifically, or to both. Several methods have been used to identify ester linkages. Takahashi et al. 2° found that inactivation of ribonuclease T by iodoacetate is accompanied by the incorporation of one carboxymethyl group per molecule of enzyme. An ester linkage was suggested by the finding that glycolic acid was released when the labeled enzyme was treated with hydroxylamine. The ester bond was stable during enzymic hydrolysis of the protein, thus facilitating isolation of the labeled residue, which was shown to be identical to authentic 7-carboxymethyl ester of glutamic acid. Studies on triosephosphate isomerase, in which 3-halogenoacetol phosphate 2~ and glycidol phosphate 2~,23 were used as active site-directed inhibitors, showed that a specific enzyme glutamic acid residue is esterified. l~y. Morino and M. Okamoto, Biochem. Biophys. Res. Commun. 47, 498 (1972). 18y. Morino and M. Okamoto, Biochem. Biophys. Res. Commun. 50, 1061 (1973). 19p. E. Wilcox, this series, Vol. 25, p. 596 (1972). 20K. Takahashi, W. H. Stein, and S. Moore, J. Biol. Chem. 242, 4682 (1967). 21F. C. Hartman, Biochemistry 10, 146 (1971). ~Ij. C. Miller and S. G. Waley, Biochem. J. 12'3, 163 (1971). 53S. G. Waley, J. C. Miller, I. A. Rose, and E. L. O'Connell, Nature (London) 227, 181 (1970).
[47] A c t i v e S i t e o f L - A s p a r a g i n a s e : Reaction with Diazo-4-oxonorvaline B y ROBERT E. HANDSCttUMACHER L-Asparaginase 1 from Escherichia coli catalyzes the conversion of the diazo ketone analog of L-asparagine, diazo-4-oxo-L-norvaline (DONV), ' J. C. Wriston, Jr. and T. O. YeIlin, Adv. Enzymol. 39, 185 (1973).
L-ASPARTATE ~-DECARBOXYLASE
427
with [14C]chloroketone enabled localization of the glutamine binding site on the heavy subuni~ of the enzyme. After inactivation with about 1 mole of [1~C] chloroketone, dissociation of the subunits was accomplished by boiling the protein in a solution containing 1% SDS and 1% 2-mercaptoethanol at pH 8.5 followed by gel electrophoresis; after disintegration of the gel in 30% H,_,02 substantial amounts of radioactivity were found associated only with the heavy subunit. ~1 A cysteine that reacted with chloroketone or iodoacetamide was found in the active site27 Comments Studies with glutamine analogs have greatly increased our understanding of the action of glutamine-utilizing enzymes. Considerable evidence indicates that separate sites exist for glutamine and ammonia and that a 7-glutamyl thioester is a central intermediate in the cleavage of glutamine. 1°,3',53 It has been suggested that amidotransferases may have developed by the association of a primordial glutaminase peptide, either in a separate subunit or covalently incorporated, with transferases specifically designed for different acceptors. 45,54 Comparison of peptides from glutamine binding sites of different enzymes is now possible and may lead to a better understanding of the evolution of this group of proteins. Inhibitors of glutamine utilization should also prove useful in studies of glutamine transport. 55H. Nagano, H. Zalkin, and E. J. Henderson, J. Biol. Chem. 245, 3810 (1970). 54H. C. Li and J. M. Buchanan, J. Biol. Chem. 246, 4713 (1971).
[46] L a b e l i n g o f t h e A c t i v e S i t e o f L - A s p a r t a t e ~ - D e c a r b o x y l a s e with fl-Chloro-L-alanine
By
NOEL M. RELYEA, SURESH S. TATE, and ALTON MEISTER
L-Aspartate-fl-decarboxylase, a pyridoxal 5'-phosphate enzyme, catalyzes the f~-decarboxyl.ation of L-aspartate to L-alanine [Eq. (1)] as L-Aspartate--~ Iralanine + CO2
(1)
well as a number of other reactions, 1 which include desulfination of Lcysteine sulfinate to L-alanine, 2 a-decarboxylation of aminomalonate to glycine, 3 decarboxylation of threo- and erythro-fl-hydroxyl-L-aspartate 1 S. S. Tate and A. Meister, Adv. Enzymol. 35, 503 (1971). 5 K. Soda, A. Novogrodsky, and A. Meister, Biochemistry 3, 1450 (1964). 5 A. G. Palekar, S. S. Tate, and A. Meister, Biochemistry 9, 2310 (1970).
428
ENZYMES, ANTIBODIES, AND OTHER PROTEINS
[46]
to L-serine, 4 various transamination reactions, 5-7 and q,fl-elimination reactions such as t h a t of fl-chloro-L-alanine s [Eq. (2) ]. CH2C]
CH3
I I COO-
CHNH~" + H20
~
I I COO-
C-'-O + NH,~ + H + + C I -
(2)
When the enzyme is incubated with fl-chloro-L-alanine, the rate of the catalytic reaction declines rapidly as the enzyme becomes irreversibly inhibited. Such inactivation is associated with the binding of close to 1 mole of the 3-carbon chain of the substrate analog per mole of active site. s The enzyme, after inactivation by treatment with fl-chloro-L[l*C]alanine, was treated with cyanogen bromide and a 14C-labeled peptide was then isolated. T r e a t m e n t of the labeled peptide with mild alkali leads to release of fl-hydroxy-[~4C]pyruvate, suggesting an ester linkage. Ammonolysis of the labeled peptide leads to release of the label, and amino acid analyses of enzymic hydrolyzates of the peptide before and after ammonolysis show formation of an equivalent amount of glutamine 9 [Eq. (3)]. The findings indicate that the labeled derivative - Glu
-Glu-
I I 0 I Ctt2 I
NH3
O~-C
C--O
I
COO-
-
I
O--C--NH 2
CH20H
I I COOC=O
(fl-hydroxypyruvate) is bound by an ester linkage to a glutamate residue at the active center of the enzyme. Preparation of/~-Chloro-L-[14C]alanine fl-Chloro-L-alanine labeled with 14C m a y be prepared from any of the various preparations of [14C]serine t h a t are commercially available E. Miles and A. Meister, Biochemistry 6, 1734 (1967). s S. S. Tate and A. Meister, Biochemistry 9, 2626 (1970). A. Novogrodsky, J. S. Nishimura, and A. Meister, J. Biol. Chem. 238, PC 1179 (1963). ' A. Novogrodsky and A. Meister, J. Biol. Chem. 239, 879 (1964). s S. S. Tate, N. Relyea, and A. Meister, Biochemistry 8, 5016 (1969). 9N. M. Relyea, S. S. Tate, and A. Meister, J. Biol. Chem. 249, 1519 (1974).
[46]
L-ASPARTATE ~-DECA.RBOXYLASE
429
according to the procedure of Fischer and Raske 1° as modified, 1~,12 which involves chlorination of serine methylester with phosphorus pentachloride. In this procedure it is of the utmost importance to maintain scrupulously dry conditions. L-[14C]Serine-HC1 (1.6 ~moles) and 75 mg (0.53 mmole) of unlabeled L-serine-HC1 are dissolved in 3 ml of dry methanol. Dry hydrogen chloride gas (dried by bubbling through concentrated H2SO4) is bubbled through the methanolic solution of serine at 0 ° for 30 min. The solution is allowed to warm to room temperature and then refluxed for 30 rain. After cooling, the solution is flash-evaporated to dryness and the residue is suspended in dry benzene; the benzene is removed by flashevaporation. The benzene step is repeated. The crystalline residue is dissolved in 1 ml of dry methanol, and 50 ml of dry diethyl ether are added slowly at 25 °. After chilling in ice for several hours, crystals of serine methylester hydrochloride are collected and washed with dry diethyl ether (m.p. 129°-131°). Chloroform is passed through an alumina column (Woelm) and stored over a Linde 4A molecular sieve for not more than 2 hr before use. Phosphorus pentachloride (135 mg) is added to 2.5 ml of dry chloroform and dissolved by stirring at 26 ° for 30 min. The solution is then chilled on ice and 105 mg of the serine methyl ester hydrochloride (prepared as described above) is added in small portions over a 30-min period with stirring. The solution is stirred at 26 ° for 2 hr until it clarifies; crystals then begin to form. The mixture is placed at 0 ° for 18 hr; dry petroleum ether (b.p. 700-90 o) is added and the crystals of fl-chloro-L-alanine methylester hydrochIoride are collected and washed with dry petroleum ether. The crystals (92 rag) are suspended in 3 ml of 6 M HC1 and refluxed for 1 hr. The solution is flash-evaporated, and the residue is dissolved in water and flash-evaporated again. This procedure is repeated once with water and then twice with benzene.
Labeling of the Enzyme The inactivation and labeling of L-aspartate-p-decarboxylase probably involves nueleophilie attack by a group on the enzyme on the fl-earbon atom of the ~-'aminoacrylate-Schiff base formed in the interaction of fl-chloro-L-alanine and enzyme-bound pyridoxal 5'-phosphate. s Labeling of the enzyme thus requires conditions under which the catalytic reaction can occur. An excess of fl-chloro-L-alanine is therefore required, since a substantial portion of the an.alog is converted to pyruvate before ~°E. Fischer and E. l~aske, Bet. Deut. Chem. Ges. 40, 3717 (1907). 11C. T. Walsh, A. Sehonbrunn, and R. H. Abeles, Y. Biol. Chem. 246, 6855 (1971). ~2j. p. Greenstein and M. Winitz, "Chemistry of the Amino Acids," p. 2677. Wiley, New York, 1961.
430
ENZYMES, ANTIBODIES, AND OTHER PROTEINS
[46]
the enzyme is completely inactivated. L-Aspartate-fl-decarboxylase (1 mg/ml) is incubated in 0.2 M sodium acetate buffer (pH 5.5) containing 10 mM fl-chloro-L-[14C]alanine for 20 min at 370.9 Under these conditions, the enzyme is completely inactivated. The labeled enzyme is precipitated at 5 ° by adding solid ammonium sulfate to 65% of ammonium sulfate saturation. The precipitated enzyme is recovered by centrifugation and dissolved in a small volume of 1 M sodium acetate at pH 6 .and then dialyzed exhaustively against water to remove salts; it is then lyophilized. Cleavage of the Labeled Enzyme to Yield a Labeled Peptide The labeled moiety introduced into the enzyme by treatment with fl-chloro-L- [14C] alanine is labile to alkali ; the following procedures are therefore carried out at acid values of pH. The labeled enzyme (5 mg/ml) is suspended in 70% formic acid and a 5-fold (w/w) excess of cyanogen bromide is added. The solution is stirred with a magnetic stirrer in a tightly stoppered flask for 24 hr at 25 °. The reaction is terminated by adding 10 volumes of cold water, and the solution is lyophilized. The labeled peptide is purified by cation-exchange chromatography13 as follows. 3_ water-jacketed column (45 X 0.9 cm) containing Technicon type P cation exchange resin is used at 50% The cyanogen bromide peptides are suspended in 2 ml of 0.2 M pyridine acetate at pH 3.1. After clarification by centrifugation, the solution is applied to the column and the peptides are eluted with a linear gradient established between 250 ml of 0.2 M pyridine acetate at pH 3.1, and 250 ml of 2 M pyridine acetate at pH 5.0, using a flow rate of 30 ml per hour; fractions of 2.5 ml are collected. Examination of the fractions for radioactivity and for amino acid content after hydrolysis (6 M HC1, 105 °, 18 hr) leads to identification of the first peak obtained from the column (fractions 4-6) as the major labeled cyanogen bromide peptide. When this peptide is subjected to acid hydrolysis followed by amino acid analysis, all the radioactivity elutes in an early ninhydrin-negative peak. Complete enzymic hydrolysis of the labeled peptide can also be achieved by cleavage with ~-chymotrypsin~4 followed by treatment with leucine aminopeptidase. 9 The amino acid composition found after complete enzymic hydrolysis is similar to but not the same as that found after acid hydrolysis. Thus, two aspartate residues are found after acid hydrolysis, and one aspartate and one asparagine residue are found after enzymic hydrolysis. On amino acid analysis after enzymic digestion, all the label appears in i~ R. T. Jones, "Methods of Biochemical Analysis" (D. Gliek, ed.), Vol. 18, pp. 205258. Wiley (Interscience), New York, 1970. ~4D. G. Smyth, this series, Vol. 11, p. 214 (1967).
[46]
L-ASPART&TE ~-DECARBOXVLASE
a ninhydrin-negative peak that elutes early. The nature of material may be established by paper chromatography using standardsg; thus, the labeled material moves with authentic pyruvate. It may also be identified as the corresponding phenylhydrazone. 15
431 the labeled appropriate fl-hydroxy2,4-dinitro-
Identification of the Protein Amino Acid Residue That Reacts with ~-Chloro-L-alanine
The alkali lability of the labeled enzyme derivative suggests that an ester of either glutamic acid or aspartie acid is formed by interaction of the enzyme with fl-chloro-L-alanine. Evidence derived from sequencing of the labeled peptide indicates that the label is not attached to the aspartate residue of the peptide, and thus suggests that the label is attached to a glutamate residue. Ammonolysis of esters to yield amides is a well known procedure, TM which may be usefully applied to the study of fl-chloro-L-alanine-labeled aspartate-B-decarboxylase. Thus, a sample of the labeled peptide (12 nmoles) is incubated in 0.5 ml of concentrated ammonium hydroxide (28% NH3) for 16 hr at 26 °. After flash-evaporation to dryness, the residue and a sample of the peptide not treated with ammonia are completely digested by successive treatment with chymotrypsin TM and leucine aminopeptidase. 9 In studies in which this procedure was followed, amino acid analysis showed that glutamine is formed and that glutamate disappears in the sample subjected to ammonolysis. The amount of glutamine formed is in close quantitative agreement with the molar amount of labeled residue bound to the peptide and released during ammonolysis. No glutamine is found after enzymic digestion of the sample of peptide which was not subjected to ammonolysis. As a further control, a sample of unlabeled peptide obtained by cyanogen bromide treatment of the unlabeled enzyme may be subjected to ammonolysis; no glutamine is found in the enzymic digest of this material2 Discussion
B-Chloro-L-alanine acts as a substrate and as an inhibitor of L-aspartate-fl-decarboxylase. The relatively rapid catalytic reaction is accompanied by a slower reaction in which the enzyme becomes inactivated. Several experimental approaches support the conclusion that fl-chloro-Lalanine is a true active site-directed inhibitor. (1) Only the L-isomer of fl-chloroalanine is a substrate and an inhibitor. (2) Treatment of the 15A. Meister and P. A. Abendsehein, Anal. Chem. 28, 171 (1956). 1~j. p. Greenstein and M. Winitz, "Chemistry of the Amino Acids," p. 1258. Wiley, New York, 1961.
432
ENZYMES, ANTIBODIES, AND OTHER PROTEINS
[47]
enzyme with fl-chloro-L-alanine leads to a spectral shift from a peak absorbance (native enzyme) at 355 nm to a peak at about 320 nm indicating that Schiff base formation is associated with inactivation. (3) When the apoenzyme or the 4'-deoxypyridbxine-5'-phosphate enzyme is treated with fl-chloro-L-alanine, neither inactivation nor binding of the substrate analog occurs. Cytoplasmic glutamate-aspartate transaminase also catalyzes a,flelimination of fl-chloro-L-alanine with concomitant inactivation of the enzyme. 1ms In this inactivation, there is evidence that the substrate analog binds to the c-amino group of the lysyl residue that is normally involved in binding pyridoxal 5'-phosphate. A number of reports have appeared on the esterification of specific protein carboxyl groups. ~9 Specificity may be due to enhanced reactivity of a particular enzyme carboxyl group or to the ability of the substrate analog to bind specifically, or to both. Several methods have been used to identify ester linkages. Takahashi et al. 2° found that inactivation of ribonuclease T by iodoacetate is accompanied by the incorporation of one carboxymethyl group per molecule of enzyme. An ester linkage was suggested by the finding that glycolic acid was released when the labeled enzyme was treated with hydroxylamine. The ester bond was stable during enzymic hydrolysis of the protein, thus facilitating isolation of the labeled residue, which was shown to be identical to authentic 7-carboxymethyl ester of glutamic acid. Studies on triosephosphate isomerase, in which 3-halogenoacetol phosphate 2~ and glycidol phosphate 2~,23 were used as active site-directed inhibitors, showed that a specific enzyme glutamic acid residue is esterified. l~y. Morino and M. Okamoto, Biochem. Biophys. Res. Commun. 47, 498 (1972). 18y. Morino and M. Okamoto, Biochem. Biophys. Res. Commun. 50, 1061 (1973). 19p. E. Wilcox, this series, Vol. 25, p. 596 (1972). 20K. Takahashi, W. H. Stein, and S. Moore, J. Biol. Chem. 242, 4682 (1967). 21F. C. Hartman, Biochemistry 10, 146 (1971). ~Ij. C. Miller and S. G. Waley, Biochem. J. 12'3, 163 (1971). 53S. G. Waley, J. C. Miller, I. A. Rose, and E. L. O'Connell, Nature (London) 227, 181 (1970).
[47] A c t i v e S i t e o f L - A s p a r a g i n a s e : Reaction with Diazo-4-oxonorvaline B y ROBERT E. HANDSCttUMACHER L-Asparaginase 1 from Escherichia coli catalyzes the conversion of the diazo ketone analog of L-asparagine, diazo-4-oxo-L-norvaline (DONV), ' J. C. Wriston, Jr. and T. O. YeIlin, Adv. Enzymol. 39, 185 (1973).