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).
[47]
L-&SPA RAGINASE
433
to 5-hydroxy-4-oxo-L-norvaline and is also inactivated by covalent attachment of the analog to a region in the active site. ~ In aqueous buffers the decomposition of DONV is so rapid (6 ~moles/min per milligram of enzyme) in comparison to the inactivation reaction (3 nmoles of enzyme inactivated per minute) that labeling of the active site is not technically reasonable. If, however, L-asparaginase is permitted to react with DONV in the presence of 50% dimethyl sulfoxide (DMSO), there is virtually no catalytic decomposition of DONV, but a 400-fold increased rate of inactivation by DONV. The enzyme itself is stable in 50% DMSO for several hours at 25 ° in the absence of DONV, and at least 85% activity can be regained within a minute by dilution with aqueous buffers. The K,n for the inactivation reaction is similar in aqueous (70 t~M) and DMS0 buffers (90 ~M). Under these inactivation conditions 4 moles of L-DONV bind to each tetrameric form of the enzyme. The specificity of the inactivation process can be demonstrated by the competitive inhibition caused by the natural substrate, a-asparagine, since in 50% DMSO the rate of hydrolysis of asparagine is less than 2% of that in aqueous buffers.
Synthesis of 5-[14C]DONV [l~C]Diazomethane is generated from N-methyl-[14C]N-nitroso-ptoluenesulfonamide (diazald) according to an adaptation of the procedure reported in A. I. Vogel. a [~4C]Diazald (214 rag, 1 mCi/Inmole, New England Nuclear) is placed in the distillation flask of a semimicro distillation apparatus and treated with an ice-cold solution of KOH (40 mg) in 95% ethanol (1 ml). After 5 rain the ethereal diazomethane is distilled under nitrogen. An ethereal solution of freshly prepared L-fl-methoxycarbonyl-fltrifluoroacetamidopropionyl chloride4 (110 mg in 5 ml of ether) is added dropwise at 0 ° to the ethereal solution of [~4C]diazomethane until only a pale yellow color remains with the reaction mixture, indicating a slight excess of diazomethane. After 20 min, the solvent and reagent are removed under reduced pressure, and the residual ester, a white solid, is dissolved in methanol (4 ml) and hydrolyzed with 0.2 N NaOH (8 ml) at --16 ° for 24 hr. The orange solution is adjusted to pH 6.2 with 0.1 N HC1 at 4 ° and lyophilized. The dark red residue is dissolved in H,.,O (1 ml), and 2R. C. Jackson and R. E. Handschumacher, Biochemistry 9, 3585 (1970). '~A. I. Vogel, "Practical Chemistry," 3rd ed., p. 971. Longmans, Green, New York, 1957. 4y. Liwsehitz,R. D. Irsay, and A. I. Vincze, J. Chem. Soc. (London) 1959, 1308.
434
ENZYMES, ANTIBODIES, AND OTHER PROTEINS
[47J
the solution and washings (5 X 1 ml) are applied to a column (1 X 20 cm) of charcoal (Darco G-60, Atlas Chemical Industries) and Celite 535 (1:1 by weight). The column is eluted with 1% aqueous acetone at 4 °. The fractions containing DONV are detected by the A274nm ~c274, m 1~cm __-11,000) and lyophilized to remove acetone. The yield is consistently 3550 mg of 5-[14C]DONV (1 mCi/mmole)2 The [~4C]DONV is diluted to give a specific activity of 0.34 mCi/mmole. Immediately before use, a 0.6 mM solution in sodium phosphate (0.05 M, pH 7.0) is filtered through an Amicon P-10 membrane to remove any polymeric material. Reaction Conditions
L-Asparaginase (100 rag) dissolved in 45 ml of sodium phosphate (0.05 M, pH 7.0) was mixed with an equal volume of DMSO, and the temperature was maintained at 25 ° by cooling in ice. Fifty milliliters of the freshly prepared solution of [14C]DONV (0.6 raM) were also diluted with an equal volume of DMSO. The [14C]DONV and enzyme solutions were mixed rapidly at 25 °, and appropriate samples were assayed for residual activity in the coupled enzyme method. If it is essential to minimize nonspecific labeling, a 10-fold excess of nonradioactive DONV (300 ~moles) in 20 ml of 50% DMSO:phosphate buffer can be added after 3 rain when 30-50% loss of activity will have occurred. To assure maximal inactivation (90-95%) the reaction was allowed to proceed for 30 min at 25 °. The reaction mixture was then dialyzed at 4 ° for 24 hr against the phosphate buffer (4 raM, pH 7.0) and lyophilized. Under these conditions, the amount of [14C]DONV bound to the enzyme is proportional to the degree of inactivation, and during the rapid inactivation phase 4 moles of DONV residue have been bound per mole of enzyme (135,000 daltons), a result consistent with the tetrameric structrue of identical subunits. Continued exposure to DONV will result in more nonspecific labeling at a much slower rate. The DONV residue is stable to trichloroacetic acid precipitation but is released by 1 N HC1 in 30 rain at 110 °. Using chymotryptic digestion, a decapeptide (Val-GlyAla-Met-Arg-Pro-Ser-Thr-Ser-Met) corresponding to residues 111 to 120 in the primary structure 6 has been isolated as the primary radioactive peptide. 7 The DONV residue is attached to either serine or threonine, presumably by a ke~o ether linkage. R. E. Handschumacher, C. J. Bates, P. K. Chang, A. T. Andrews, and G. A. Fischer, Science 161, 62 (1968). e T. Maita, K. Morokuma, and G. Matusuda, J. Biochem. 76, 1351 (1974). 7R. G. Peterson, F. F. Richards, and R. E. Handschumacher, J. Biol. Chem., in press (1977).
[48]
SE~Ur~ PREALBUMIN
435
Comments
The buffer used in the 50% DMSO solution can change the balance between catalytic decomposition of DONV and enzyme inactivation. If instead of sodium phosphate, Tris or ammonium phosphate is used as buffer, sufficient catalytic activity is retained in 50% DMSO to render active site labeling difficult because of the rapid decomposition of the [:~C]DONV. The extreme of this effect may be seen with hydroxylamine (0.1 M), which, in aqueous buffer, accelerates the catalytic decomposition of DONV 3-fold and eliminates covalent inactivation in the DMSO buffer system. Inactivation by the n-isomer of DONV can be accomplished at 27% of the rate with L-DONV. Although the enzyme exhibits considerable stereospecificity, •-asparagine provides a useful means of occupying the active site since it has a Km close to that of the L-isomer but is a poor substrate (Vm~ = 2% Vm~ for L-asparagine). 1 Attempts to label the active site with 5-chloro or 5-bromo-4-oxo-anorvaline have not been successful; these haloketones are also not substrafes for the enzyme to a discernible degree. Although aspartic acid semialdehyde is a potent inhibitor of the enzyme, s attempts to bind it to asparaginase covalently by NaBH4 reduction have not been successful. The specificity of the affinity label by DONV is seen in the lack of activity of the glutamine analog 6-diazo-5-oxo-L-norleueine (DON) as a covalent inaetivator or substrate for E. coli L-asparaginase. This is of some interest, since glutamine is hydrolyzed at about 3% of the rate with asparagine and the glutaminase activity is lost coincident with asparaginase activity as inactivation proceeds. By contrast, the glutaminase of E. coli is covalently inactivated by DON. This analog also can serve as a very poor substrate for the enzyme but appears to yield gluramie acid and formaldehyde (see this volume [45]). Several enzymes that readily accept both asparaginase and glutamine as substrates have also been studied, and comments on affinity labeling of their active sites may also be found in this volume [45]. J. 0. Westerik and R. Wolfenden, J. Biol. Chem. 249, 6351 (1974).
[48]
Labeling of Serum Prealbumin with N-Bromoacetyl-L- thyroxine B y SHEUE-'~ANN CItENG
Human prealbumin is involved in the transport of thyroid hormones in plasma. N-Bromoacetyl-L-thyroxine [BrAcTs; (II) in Scheme 2], an analog of L-thyroxine [T~; (I) in Scheme 2], differs from T4 only in the
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).
[47]
L-&SPA RAGINASE
433
to 5-hydroxy-4-oxo-L-norvaline and is also inactivated by covalent attachment of the analog to a region in the active site. ~ In aqueous buffers the decomposition of DONV is so rapid (6 ~moles/min per milligram of enzyme) in comparison to the inactivation reaction (3 nmoles of enzyme inactivated per minute) that labeling of the active site is not technically reasonable. If, however, L-asparaginase is permitted to react with DONV in the presence of 50% dimethyl sulfoxide (DMSO), there is virtually no catalytic decomposition of DONV, but a 400-fold increased rate of inactivation by DONV. The enzyme itself is stable in 50% DMSO for several hours at 25 ° in the absence of DONV, and at least 85% activity can be regained within a minute by dilution with aqueous buffers. The K,n for the inactivation reaction is similar in aqueous (70 t~M) and DMS0 buffers (90 ~M). Under these inactivation conditions 4 moles of L-DONV bind to each tetrameric form of the enzyme. The specificity of the inactivation process can be demonstrated by the competitive inhibition caused by the natural substrate, a-asparagine, since in 50% DMSO the rate of hydrolysis of asparagine is less than 2% of that in aqueous buffers.
Synthesis of 5-[14C]DONV [l~C]Diazomethane is generated from N-methyl-[14C]N-nitroso-ptoluenesulfonamide (diazald) according to an adaptation of the procedure reported in A. I. Vogel. a [~4C]Diazald (214 rag, 1 mCi/Inmole, New England Nuclear) is placed in the distillation flask of a semimicro distillation apparatus and treated with an ice-cold solution of KOH (40 mg) in 95% ethanol (1 ml). After 5 rain the ethereal diazomethane is distilled under nitrogen. An ethereal solution of freshly prepared L-fl-methoxycarbonyl-fltrifluoroacetamidopropionyl chloride4 (110 mg in 5 ml of ether) is added dropwise at 0 ° to the ethereal solution of [~4C]diazomethane until only a pale yellow color remains with the reaction mixture, indicating a slight excess of diazomethane. After 20 min, the solvent and reagent are removed under reduced pressure, and the residual ester, a white solid, is dissolved in methanol (4 ml) and hydrolyzed with 0.2 N NaOH (8 ml) at --16 ° for 24 hr. The orange solution is adjusted to pH 6.2 with 0.1 N HC1 at 4 ° and lyophilized. The dark red residue is dissolved in H,.,O (1 ml), and 2R. C. Jackson and R. E. Handschumacher, Biochemistry 9, 3585 (1970). '~A. I. Vogel, "Practical Chemistry," 3rd ed., p. 971. Longmans, Green, New York, 1957. 4y. Liwsehitz,R. D. Irsay, and A. I. Vincze, J. Chem. Soc. (London) 1959, 1308.
434
ENZYMES, ANTIBODIES, AND OTHER PROTEINS
[47J
the solution and washings (5 X 1 ml) are applied to a column (1 X 20 cm) of charcoal (Darco G-60, Atlas Chemical Industries) and Celite 535 (1:1 by weight). The column is eluted with 1% aqueous acetone at 4 °. The fractions containing DONV are detected by the A274nm ~c274, m 1~cm __-11,000) and lyophilized to remove acetone. The yield is consistently 3550 mg of 5-[14C]DONV (1 mCi/mmole)2 The [~4C]DONV is diluted to give a specific activity of 0.34 mCi/mmole. Immediately before use, a 0.6 mM solution in sodium phosphate (0.05 M, pH 7.0) is filtered through an Amicon P-10 membrane to remove any polymeric material. Reaction Conditions
L-Asparaginase (100 rag) dissolved in 45 ml of sodium phosphate (0.05 M, pH 7.0) was mixed with an equal volume of DMSO, and the temperature was maintained at 25 ° by cooling in ice. Fifty milliliters of the freshly prepared solution of [14C]DONV (0.6 raM) were also diluted with an equal volume of DMSO. The [14C]DONV and enzyme solutions were mixed rapidly at 25 °, and appropriate samples were assayed for residual activity in the coupled enzyme method. If it is essential to minimize nonspecific labeling, a 10-fold excess of nonradioactive DONV (300 ~moles) in 20 ml of 50% DMSO:phosphate buffer can be added after 3 rain when 30-50% loss of activity will have occurred. To assure maximal inactivation (90-95%) the reaction was allowed to proceed for 30 min at 25 °. The reaction mixture was then dialyzed at 4 ° for 24 hr against the phosphate buffer (4 raM, pH 7.0) and lyophilized. Under these conditions, the amount of [14C]DONV bound to the enzyme is proportional to the degree of inactivation, and during the rapid inactivation phase 4 moles of DONV residue have been bound per mole of enzyme (135,000 daltons), a result consistent with the tetrameric structrue of identical subunits. Continued exposure to DONV will result in more nonspecific labeling at a much slower rate. The DONV residue is stable to trichloroacetic acid precipitation but is released by 1 N HC1 in 30 rain at 110 °. Using chymotryptic digestion, a decapeptide (Val-GlyAla-Met-Arg-Pro-Ser-Thr-Ser-Met) corresponding to residues 111 to 120 in the primary structure 6 has been isolated as the primary radioactive peptide. 7 The DONV residue is attached to either serine or threonine, presumably by a ke~o ether linkage. R. E. Handschumacher, C. J. Bates, P. K. Chang, A. T. Andrews, and G. A. Fischer, Science 161, 62 (1968). e T. Maita, K. Morokuma, and G. Matusuda, J. Biochem. 76, 1351 (1974). 7R. G. Peterson, F. F. Richards, and R. E. Handschumacher, J. Biol. Chem., in press (1977).
[48]
SE~Ur~ PREALBUMIN
435
Comments
The buffer used in the 50% DMSO solution can change the balance between catalytic decomposition of DONV and enzyme inactivation. If instead of sodium phosphate, Tris or ammonium phosphate is used as buffer, sufficient catalytic activity is retained in 50% DMSO to render active site labeling difficult because of the rapid decomposition of the [:~C]DONV. The extreme of this effect may be seen with hydroxylamine (0.1 M), which, in aqueous buffer, accelerates the catalytic decomposition of DONV 3-fold and eliminates covalent inactivation in the DMSO buffer system. Inactivation by the n-isomer of DONV can be accomplished at 27% of the rate with L-DONV. Although the enzyme exhibits considerable stereospecificity, •-asparagine provides a useful means of occupying the active site since it has a Km close to that of the L-isomer but is a poor substrate (Vm~ = 2% Vm~ for L-asparagine). 1 Attempts to label the active site with 5-chloro or 5-bromo-4-oxo-anorvaline have not been successful; these haloketones are also not substrafes for the enzyme to a discernible degree. Although aspartic acid semialdehyde is a potent inhibitor of the enzyme, s attempts to bind it to asparaginase covalently by NaBH4 reduction have not been successful. The specificity of the affinity label by DONV is seen in the lack of activity of the glutamine analog 6-diazo-5-oxo-L-norleueine (DON) as a covalent inaetivator or substrate for E. coli L-asparaginase. This is of some interest, since glutamine is hydrolyzed at about 3% of the rate with asparagine and the glutaminase activity is lost coincident with asparaginase activity as inactivation proceeds. By contrast, the glutaminase of E. coli is covalently inactivated by DON. This analog also can serve as a very poor substrate for the enzyme but appears to yield gluramie acid and formaldehyde (see this volume [45]). Several enzymes that readily accept both asparaginase and glutamine as substrates have also been studied, and comments on affinity labeling of their active sites may also be found in this volume [45]. J. 0. Westerik and R. Wolfenden, J. Biol. Chem. 249, 6351 (1974).
[48]
Labeling of Serum Prealbumin with N-Bromoacetyl-L- thyroxine B y SHEUE-'~ANN CItENG
Human prealbumin is involved in the transport of thyroid hormones in plasma. N-Bromoacetyl-L-thyroxine [BrAcTs; (II) in Scheme 2], an analog of L-thyroxine [T~; (I) in Scheme 2], differs from T4 only in the
