302
ENZYMES, ANTIBODIES, AND OTHER PROTEINS
[29]
Interaction of (I) with Other Mononucleotide-Utilizing Enzymes IMP dehydrogenase of mouse sarcoma 180 is inactivated by (I)7 in a manner similar in many respects to its inactivation of the same enzyme from A. aerogenes. Yeast adenylosuccinate AMP-lyase was not appreciably inactivated by 1 mM (I) under the usual assay conditions, nor was Escherichia coli IMP:L-aspartate ligase (GDP) appreciably inactivated by 200 ~M (I).S Affinity Labels Related to (I) 6-Thio-IMP and 6-thio-GMP under certain conditions inactivate IMP dehydrogenase4 and GMP reductase 2 of A. aerogenes, and evidence suggests that these effects could be associated with formation of disulfide bonds between the substrate analogs and a sulfhydryl group located near the substrate site of each enzyme.2,4,6 6-Thio-GMP and 6-thio-IMP do not appear to inactivate yeast adenylosuccinate AMP-lyase or E. coti IMP:L-aspartate ligase (GDP). 8 7j . H. Anderson and A. C. Sartorelli, Biochem. Pharmacol. 18, 2737 (1969). s A. Hampton, Fed. Proc., Fed. Am. Soc. Exp. Biol. 21, 370 (1962).
[29] C a r b o x y l i c - P h o s p h o r i c A n h y d r i d e s I s o s t e r i c w i t h Adenine Nueleotides By ALEXANDER HAMPTON, PETER d. HARPER, TAKUMA SASAKI, a n d PAUL HOWGATE
Adenine nucleotide analogs of this type are of interest as potential affinity labels because (1) they are structurally closely similar to the parent nucleotides, (2) after adsorption to the substrate site they can either acylate or phosphorylate amino acid residues, and (3) it is likely that binding of these analogs to the substrate site will frequently be accompanied by partial neutralization of negative charges on the phosphoryl groups that would increase the reactivity of the mixed anhydride function in the enzyme-bound analogs and thereby tend to increase the specificity of labeling of the substrate site. The present article describes the synthesis of carboxylic-phosphoric mixed anhydride analogs of AMP (I) ~,2 and ATP (II)2 and conditions under which they appear to react covalently at the adenine nucleotide sites of three enzymes. A. Hampton and P. J. Harper, Arch. Biochem. Biophys. 143, 340 (1971). A. Hampton, P. J. Harper, T. Sasaki, P. Howgate, and R. K. Preston, Biochem. Biophys. Res. Commun. 65, 945 (1975).
[29]
303
ISOSTERIC CARBOXYLIC-PItOSPHORIC ANHYDRIDES NH 2
NH2
II II
(HO)2 POC
N
(HO)2 POPOPOC HO OH
HO
OH
HO
(I)
OH
o[)
Synthesis of (I) A solution of sodium phosphate in aqueous 50% methanol is passed through a column containing a 15-fold excess of tri-n-butylammonium Dowex 50 ion-exchange resin. The solvent is removed under reduced pressure, and the residue is thrice coevaporated under reduced pressure with anhydrous pyridine; the product is dissolved in anhydrous pyridine to give a 0.5 M solution of tri-n-butylammonium phosphate. In order to obtain a homogeneous product it is necessary to carry out the following operations in a glove box in a dry nitrogen atmosphere with freshly prepared anhydrous reagents and solvents. To the sodium salt of 9-(fl-D-ribofuranosyluronic acid)adenine (0.5 mmole) 3 is added N,N-dimethylformamide (2 ml). Diphenyl phosphorochloridate (0.48 mmole) is added, and the suspension is stirred for 5 hr. The mixture is centrifuged to quantitatively remove unreacted sodium salt together with sodium chloride. The supernatant fluid is treated with diethyl ether (50 ml), and the precipitated diphenyl ester of (I) is collected by centrifugation and dissolved in dioxane (2 ml); the solution is clarified by centrifugation, and the sugernatant is treated with diethyl ether (50 ml). The precipitate is collected and freed of diethyl ether under reduced pressure. The purity and identity of this diphenyl ester can, if desired, be checked by chromatography after treating a portion with anhydrous ammonia-N,N-dimethylformamide.1 The diphenyl ester of (I) is not stable and is therefore immediately dissolved in 2 ml of the above 0.5 M tri-n-butylammonium phosphate solution. After 5 hr, the solution is clarified by centrifugati0n and treated with diethyl ether (50 ml). The precipitate is collected, dissolved in pyridine-N,N-dimethylformamide (4 ml of 1:1), and reprecipitated with ether (50 ml). That the product '~P. J. Harper and A. Hampton, J. Org. Chem. 35, 1688 (1970).
304
ENZYMES, ANTIBODIES, AND OTHER PROTEINS
[29]
is free of the diphenyl ester of (I) is shown by the absence of IR peaks at 690 and 782 cm -1 that would be due to monosubstituted phenyl groups; furthermore, the amination described below does not produce diphenyl phosphate as judged by chromatography in previously described systems. 1 The white precipitate (0.31 g), which contained a 90% yield (calculated from UV absorption) of the tri-n-butylammonium salt of (I) in admixture with about an equimolar amount of tri-n-butylammonium phosphate, is freed of ether under reduced pressure; IR 3250, 3150 sh, 1742, 1642, 1602 sh, 1200, and 1090 cm-1; UV spectra are identical with those of the starting acid2 The product is dissolved in the minimum of anhydrous N,N-dimethylformamide, and the solution [about 1% of (I)] is stored at --25 ° under nitrogen. The identity and homogeneity of the product may be further established by treatment of a portion of the solution under anhydrous conditions with dry ammonia, after which paper chromatography reveals a single ultraviolet-absorbing component of Rf 0.45 in ethanol-1 M ammonium acetate (7:3) and Rs 0.70 in nbutanol-acetic acid-water (5:2:3), corresponding to the authentic carl)oxamide l of the starting material. The acyl phosphate (I) hydrolyzes in Tris buffer at pH 7.7 at least 100-fold faster than does acetyl phosphate, as shown by the inability of (I) to inactivate adenylosuccinate-lyase after a 15-sec exposure to the buffer before addition of the enzyme. Compound (I) also lost its ability to inactivate AMP aminohydrolase after 15-sec hydrolysis at pH 6.5. 5 After the solution of (I) in N,N-dimethylformamide had been stored at --25 ° for 2 days, it showed UV spectral changes (upon dilution into aqueous solutions) indicative of N6-acylaminopurine nucleoside formation. In the case of AMP aminohydrolase, this change is associated with a marked reduction in the degree of enzyme inactivation, 2 and the use of freshly prepared solutions of (I) in studies of enzyme inactivation is thereforo indicated. Synthesis of (II) 2
A solution of (II) in N,N-dimethylformamide is prepared by the same procedure as described for (I) by substituting sodium tripolyphosphate for sodium phosphate. The identity and homogeneity of the product were established by treating a portion of the solution with dry ammonia gas when the only UV-absorbing paper chromatographic component obtained was the amide of the starting carboxylic acid. In addition, the reaction mixture was subjected to thin-layer chromatography on PEIcellulose using two developments with 4 M sodium formate of pH 3.4, and the plates were exposed to the vapor of concentrated HC1 for 3 min
[29]
ISOSTERIC CARBOXYLIC-PHOSPttORIC ANHYDRII)ES
305
and then sprayed with a molybdate-perchlorie acid solution. ~ Phosphatecontaining components were seen as white spots on a yellow background; this showed that the reaction mixture contained much tripolyphosphate (a streak at Rr 0.1-0.3) but no pyrophosphate (Rs 0.5) and a trace of phosphate (Rj 0.9). The latter was found to be formed in the same proportion by the action of ammonia on the stock solution of tri-n-butylammonium tripolyphosphate. Compound (II) is extensively hydrolyzed in 15 see at pH 7.6 and room temperature.'-' Reaction of (I) with Adenosine 5'-Phosphate (AMP) Aminohydrolase 2 In these experiments initial reaction rates were measured by the decrease in absorbance at 265 nm and calculated as nanomoles per minute from Ac -- 6600. The final volume of 0.90 ml contained 0.01 M potassium citrate (pH 6.5), 0.016 ~g of enzyme (Sigma Chemical Co., grade IV, from rabbit muscle), 0.005 M KC1, 60 t~M (I), and 50 t~M AMP. The order of addition was (a) buffer, (b) enzyme, (c) (I), followed 1 rain later by AMP. In control assays carried out in conjunction with the inactivation experiments, the order of addition was (a) buffer, (b) (I), followed 1 rain later by (c) the enzyme and (d) AMP. These control assays showed that hydrolyzed (I) was not inhibitory; the observed rates varied between 1.11 and 1.13 mnoles/min. A nominal initial level of 60 ~M of freshly prepared (I) caused 32% inactivation of the enzyme; when 5D uM AMP (12~. the Km of AMP) were present prior to the addition of (I) no inactivation occurred. The same amount of (I) was added to the enzyme in less buffer (about 0.1 mll to give a nominal initial level of 540 uM (I) and after 1 min the mixture was assayed by the standard procedure. Under these conditions, (I) caused 40% inactivation when added in one portion and 47% inactivation when added in 3 portions at 20-second intervals. The inactivation by (I) was abolished by as little as 15-second hydrolysis of (I) in the assay buffer prior to contact with the enzyme, thus indicating that inactivation is the result of acylation or phosphorylation of the enzyme by the mixed anhydride and that the reaction between (I) and the enzyme is extremely rapid. Reaction of (I) with Adenylosuccinate AMP-Lyase 1 The above solution of (I) was mixed at 22 ° with partially purified Escherichia coli adenylosueeinate AMP-lyase in 40 mM Tris chloride-10
4C. S. Hanes and F. A. Isherwood, Nature (Loudon) 164, 1107 (1949).
306
ENZYMES, ANTIBODIES, AND OTHER PROTEINS
[29]
mM sodium ethylenediamine tetraacetate (pH 7.7) to give 80 t ~ / ( I ) (and 80 gM tri-n-butylammonium phosphate); about 5 sec later ammonium adenylosuccinate (120 ~ final concentration) was added. UV absorbance measurements ~ showed 95-99% reduction in the rate (0.3 nmole/ min) of formation of AMP. This inhibition was abolished (a) by 30-sec hydrolysis of (I) in the buffer prior to successive addition of enzyme and substrate, (b) by prior addition to the enzyme of 120 ~ adenylosuccinate (Kin = 20 gM), and (c) by substituting 1 mM tri-n-butylammonium phosphate or 2 mM acetyl phosphate (20 rain interaction with enzyme) for (I). This and other findings indicated that (I) either acylates or phosphorylates the enzyme and that the reaction occurs at the nucleotide binding site. 1 Reaction of (II) with Pyruvate Kinase Initial reaction velocities were measured at 340 nm. For all experiments (except where noted) the final volume of 1.00 ml of 0.1 M Tris chloride (pH 7.6) contained 13 gg of rabbit muscle lactic dehydrogenase, 0.05 gg of rabbit muscle pyruvate kinase, 0.1 M KC1, 0.025 M MgSO4, 1.5 mM phosphoenolpyruvate (sodium salt), 0.25 mM ADP (sodium salt), and 0.25 mM NADH. The order of addition of the components was the same as described above for AMP aminohydrolase. When a nominal initial level of 100 tall// of (II) was added to the pyruvate kinase in three increments, about 50% inactivation occurred (see the table). The relationship between the degree of inactivation and the initial concentration of (II) was not examined further. Inactivation was prevented by 15-sec hydrolysis of (II) in the buffer prior to addition of enzyme. Pyruvate kinase inactivated by (II) did not regain activity when stored in the assay medium for 16 hr at 22% Protection of the enzyme from a 100 ~M nominal level of (II) was afforded by 100 ~M ATP, 2.5 mM ADP, or 1.5 mM phosphoenolpyruvate, the levels of the last two compounds being selected so as to be in excess of their enzymesubstrate dissociation constants (0.8 mM and 0.08 mM, respectively). Protection by these three substrates was concluded to imply that the action of (II) is probably ATP-site-directed. 2 Studies with Other Enzymes and Attempted Syntheses of Anhydride Analogs of Other Nucleotides AMP kinase of rabbit muscle was not inactivated by 1 mM nominal initial levels of (I) (a 2-day-old sample) or of (II) (freshly prepared). 5 C. E. Carter and L. It. Cohen, d. Biol. Chem. 222, 17 (1956).
[30]
307
THYMIDYLATE SYNTHETASE INACTIVATION OF RABBIT MUSCLE PYRUVATE KINASE BY (II)" R a t e × 10 ~ (hA340/min) Additions prior to (II) None None ~ None c 0.1 m M A T P 1.5 m M P E P 2.5 m M A D P d
E n z y m e plus hydrolyzed (II)
Enzyme plus (II)
Inactivation (%)
3.10 3.00 3.06 3.00 3.10 3.02
1.80 1.45 1.81 2.95 2.95 2.96
43 52 41 2 5 2
T h e nominal initial concentration of (II) was 100 tLM. b (II) added in 3 equal increments at 20-second intervals. ° After t r e a t m e n t with (II) the enzyme solution was stored at 22 ° for 16 hr before t h e velocity determination. T h e enzyme was exposed to 2.5 m M ADP, t h e n 0.1 m M (II), in 0.1 ml of buffer a n d assayed after dilution to t h e s t a n d a r d volume cf 1 ml.
The method used to convert adenosine-5'-carboxylic acid to (I) and (II) did not convert uridine- or thymidine-5'-carboxylic acids ~ to the analogous U M P or T M P carboxylic-phosphoric anhydrides. 6 G. P. Moss, C. B. Reese, K. Schofield, R, Shapiro, and A. R. Todd, 1. Chem. Soc. 1963, 1149 (1963).
[30] A c t i v e - S i t e L a b e l i n g o f T h y m i d y l a t e Synthetase with 5-Fluoro-2'-deoxyuridylate 1 By YUSUKE WATAYA a n d DANIEL V. SANTI
Thymidylate synthetase catalyzes the reductive methylation of 2'deoxyuridylate (dUMP) to thymidylate with concomitant conversion of 5,10-methylenetetrahydrofolate (CH~-H~folate) to 7,8-dihydrofolate. A number of studies of the thymidylate synthetase reaction have led to the proposal that a primary event in the catalytic sequence involves the addition of a nucleophilic group of the enzyme to the 6-position of the substrate dUMP, a required step for subsequent condensation of the 5-posi1 This work was supported by U.S. Public H e a l t h Service G r a n t CA-14394 from the National Cancer Institute. D. V. S. is a recipient of a National Institutes of Health Career D e v e l o p m e n t Award.
ENZYMES, ANTIBODIES, AND OTHER PROTEINS
[29]
Interaction of (I) with Other Mononucleotide-Utilizing Enzymes IMP dehydrogenase of mouse sarcoma 180 is inactivated by (I)7 in a manner similar in many respects to its inactivation of the same enzyme from A. aerogenes. Yeast adenylosuccinate AMP-lyase was not appreciably inactivated by 1 mM (I) under the usual assay conditions, nor was Escherichia coli IMP:L-aspartate ligase (GDP) appreciably inactivated by 200 ~M (I).S Affinity Labels Related to (I) 6-Thio-IMP and 6-thio-GMP under certain conditions inactivate IMP dehydrogenase4 and GMP reductase 2 of A. aerogenes, and evidence suggests that these effects could be associated with formation of disulfide bonds between the substrate analogs and a sulfhydryl group located near the substrate site of each enzyme.2,4,6 6-Thio-GMP and 6-thio-IMP do not appear to inactivate yeast adenylosuccinate AMP-lyase or E. coti IMP:L-aspartate ligase (GDP). 8 7j . H. Anderson and A. C. Sartorelli, Biochem. Pharmacol. 18, 2737 (1969). s A. Hampton, Fed. Proc., Fed. Am. Soc. Exp. Biol. 21, 370 (1962).
[29] C a r b o x y l i c - P h o s p h o r i c A n h y d r i d e s I s o s t e r i c w i t h Adenine Nueleotides By ALEXANDER HAMPTON, PETER d. HARPER, TAKUMA SASAKI, a n d PAUL HOWGATE
Adenine nucleotide analogs of this type are of interest as potential affinity labels because (1) they are structurally closely similar to the parent nucleotides, (2) after adsorption to the substrate site they can either acylate or phosphorylate amino acid residues, and (3) it is likely that binding of these analogs to the substrate site will frequently be accompanied by partial neutralization of negative charges on the phosphoryl groups that would increase the reactivity of the mixed anhydride function in the enzyme-bound analogs and thereby tend to increase the specificity of labeling of the substrate site. The present article describes the synthesis of carboxylic-phosphoric mixed anhydride analogs of AMP (I) ~,2 and ATP (II)2 and conditions under which they appear to react covalently at the adenine nucleotide sites of three enzymes. A. Hampton and P. J. Harper, Arch. Biochem. Biophys. 143, 340 (1971). A. Hampton, P. J. Harper, T. Sasaki, P. Howgate, and R. K. Preston, Biochem. Biophys. Res. Commun. 65, 945 (1975).
[29]
303
ISOSTERIC CARBOXYLIC-PItOSPHORIC ANHYDRIDES NH 2
NH2
II II
(HO)2 POC
N
(HO)2 POPOPOC HO OH
HO
OH
HO
(I)
OH
o[)
Synthesis of (I) A solution of sodium phosphate in aqueous 50% methanol is passed through a column containing a 15-fold excess of tri-n-butylammonium Dowex 50 ion-exchange resin. The solvent is removed under reduced pressure, and the residue is thrice coevaporated under reduced pressure with anhydrous pyridine; the product is dissolved in anhydrous pyridine to give a 0.5 M solution of tri-n-butylammonium phosphate. In order to obtain a homogeneous product it is necessary to carry out the following operations in a glove box in a dry nitrogen atmosphere with freshly prepared anhydrous reagents and solvents. To the sodium salt of 9-(fl-D-ribofuranosyluronic acid)adenine (0.5 mmole) 3 is added N,N-dimethylformamide (2 ml). Diphenyl phosphorochloridate (0.48 mmole) is added, and the suspension is stirred for 5 hr. The mixture is centrifuged to quantitatively remove unreacted sodium salt together with sodium chloride. The supernatant fluid is treated with diethyl ether (50 ml), and the precipitated diphenyl ester of (I) is collected by centrifugation and dissolved in dioxane (2 ml); the solution is clarified by centrifugation, and the sugernatant is treated with diethyl ether (50 ml). The precipitate is collected and freed of diethyl ether under reduced pressure. The purity and identity of this diphenyl ester can, if desired, be checked by chromatography after treating a portion with anhydrous ammonia-N,N-dimethylformamide.1 The diphenyl ester of (I) is not stable and is therefore immediately dissolved in 2 ml of the above 0.5 M tri-n-butylammonium phosphate solution. After 5 hr, the solution is clarified by centrifugati0n and treated with diethyl ether (50 ml). The precipitate is collected, dissolved in pyridine-N,N-dimethylformamide (4 ml of 1:1), and reprecipitated with ether (50 ml). That the product '~P. J. Harper and A. Hampton, J. Org. Chem. 35, 1688 (1970).
304
ENZYMES, ANTIBODIES, AND OTHER PROTEINS
[29]
is free of the diphenyl ester of (I) is shown by the absence of IR peaks at 690 and 782 cm -1 that would be due to monosubstituted phenyl groups; furthermore, the amination described below does not produce diphenyl phosphate as judged by chromatography in previously described systems. 1 The white precipitate (0.31 g), which contained a 90% yield (calculated from UV absorption) of the tri-n-butylammonium salt of (I) in admixture with about an equimolar amount of tri-n-butylammonium phosphate, is freed of ether under reduced pressure; IR 3250, 3150 sh, 1742, 1642, 1602 sh, 1200, and 1090 cm-1; UV spectra are identical with those of the starting acid2 The product is dissolved in the minimum of anhydrous N,N-dimethylformamide, and the solution [about 1% of (I)] is stored at --25 ° under nitrogen. The identity and homogeneity of the product may be further established by treatment of a portion of the solution under anhydrous conditions with dry ammonia, after which paper chromatography reveals a single ultraviolet-absorbing component of Rf 0.45 in ethanol-1 M ammonium acetate (7:3) and Rs 0.70 in nbutanol-acetic acid-water (5:2:3), corresponding to the authentic carl)oxamide l of the starting material. The acyl phosphate (I) hydrolyzes in Tris buffer at pH 7.7 at least 100-fold faster than does acetyl phosphate, as shown by the inability of (I) to inactivate adenylosuccinate-lyase after a 15-sec exposure to the buffer before addition of the enzyme. Compound (I) also lost its ability to inactivate AMP aminohydrolase after 15-sec hydrolysis at pH 6.5. 5 After the solution of (I) in N,N-dimethylformamide had been stored at --25 ° for 2 days, it showed UV spectral changes (upon dilution into aqueous solutions) indicative of N6-acylaminopurine nucleoside formation. In the case of AMP aminohydrolase, this change is associated with a marked reduction in the degree of enzyme inactivation, 2 and the use of freshly prepared solutions of (I) in studies of enzyme inactivation is thereforo indicated. Synthesis of (II) 2
A solution of (II) in N,N-dimethylformamide is prepared by the same procedure as described for (I) by substituting sodium tripolyphosphate for sodium phosphate. The identity and homogeneity of the product were established by treating a portion of the solution with dry ammonia gas when the only UV-absorbing paper chromatographic component obtained was the amide of the starting carboxylic acid. In addition, the reaction mixture was subjected to thin-layer chromatography on PEIcellulose using two developments with 4 M sodium formate of pH 3.4, and the plates were exposed to the vapor of concentrated HC1 for 3 min
[29]
ISOSTERIC CARBOXYLIC-PHOSPttORIC ANHYDRII)ES
305
and then sprayed with a molybdate-perchlorie acid solution. ~ Phosphatecontaining components were seen as white spots on a yellow background; this showed that the reaction mixture contained much tripolyphosphate (a streak at Rr 0.1-0.3) but no pyrophosphate (Rs 0.5) and a trace of phosphate (Rj 0.9). The latter was found to be formed in the same proportion by the action of ammonia on the stock solution of tri-n-butylammonium tripolyphosphate. Compound (II) is extensively hydrolyzed in 15 see at pH 7.6 and room temperature.'-' Reaction of (I) with Adenosine 5'-Phosphate (AMP) Aminohydrolase 2 In these experiments initial reaction rates were measured by the decrease in absorbance at 265 nm and calculated as nanomoles per minute from Ac -- 6600. The final volume of 0.90 ml contained 0.01 M potassium citrate (pH 6.5), 0.016 ~g of enzyme (Sigma Chemical Co., grade IV, from rabbit muscle), 0.005 M KC1, 60 t~M (I), and 50 t~M AMP. The order of addition was (a) buffer, (b) enzyme, (c) (I), followed 1 rain later by AMP. In control assays carried out in conjunction with the inactivation experiments, the order of addition was (a) buffer, (b) (I), followed 1 rain later by (c) the enzyme and (d) AMP. These control assays showed that hydrolyzed (I) was not inhibitory; the observed rates varied between 1.11 and 1.13 mnoles/min. A nominal initial level of 60 ~M of freshly prepared (I) caused 32% inactivation of the enzyme; when 5D uM AMP (12~. the Km of AMP) were present prior to the addition of (I) no inactivation occurred. The same amount of (I) was added to the enzyme in less buffer (about 0.1 mll to give a nominal initial level of 540 uM (I) and after 1 min the mixture was assayed by the standard procedure. Under these conditions, (I) caused 40% inactivation when added in one portion and 47% inactivation when added in 3 portions at 20-second intervals. The inactivation by (I) was abolished by as little as 15-second hydrolysis of (I) in the assay buffer prior to contact with the enzyme, thus indicating that inactivation is the result of acylation or phosphorylation of the enzyme by the mixed anhydride and that the reaction between (I) and the enzyme is extremely rapid. Reaction of (I) with Adenylosuccinate AMP-Lyase 1 The above solution of (I) was mixed at 22 ° with partially purified Escherichia coli adenylosueeinate AMP-lyase in 40 mM Tris chloride-10
4C. S. Hanes and F. A. Isherwood, Nature (Loudon) 164, 1107 (1949).
306
ENZYMES, ANTIBODIES, AND OTHER PROTEINS
[29]
mM sodium ethylenediamine tetraacetate (pH 7.7) to give 80 t ~ / ( I ) (and 80 gM tri-n-butylammonium phosphate); about 5 sec later ammonium adenylosuccinate (120 ~ final concentration) was added. UV absorbance measurements ~ showed 95-99% reduction in the rate (0.3 nmole/ min) of formation of AMP. This inhibition was abolished (a) by 30-sec hydrolysis of (I) in the buffer prior to successive addition of enzyme and substrate, (b) by prior addition to the enzyme of 120 ~ adenylosuccinate (Kin = 20 gM), and (c) by substituting 1 mM tri-n-butylammonium phosphate or 2 mM acetyl phosphate (20 rain interaction with enzyme) for (I). This and other findings indicated that (I) either acylates or phosphorylates the enzyme and that the reaction occurs at the nucleotide binding site. 1 Reaction of (II) with Pyruvate Kinase Initial reaction velocities were measured at 340 nm. For all experiments (except where noted) the final volume of 1.00 ml of 0.1 M Tris chloride (pH 7.6) contained 13 gg of rabbit muscle lactic dehydrogenase, 0.05 gg of rabbit muscle pyruvate kinase, 0.1 M KC1, 0.025 M MgSO4, 1.5 mM phosphoenolpyruvate (sodium salt), 0.25 mM ADP (sodium salt), and 0.25 mM NADH. The order of addition of the components was the same as described above for AMP aminohydrolase. When a nominal initial level of 100 tall// of (II) was added to the pyruvate kinase in three increments, about 50% inactivation occurred (see the table). The relationship between the degree of inactivation and the initial concentration of (II) was not examined further. Inactivation was prevented by 15-sec hydrolysis of (II) in the buffer prior to addition of enzyme. Pyruvate kinase inactivated by (II) did not regain activity when stored in the assay medium for 16 hr at 22% Protection of the enzyme from a 100 ~M nominal level of (II) was afforded by 100 ~M ATP, 2.5 mM ADP, or 1.5 mM phosphoenolpyruvate, the levels of the last two compounds being selected so as to be in excess of their enzymesubstrate dissociation constants (0.8 mM and 0.08 mM, respectively). Protection by these three substrates was concluded to imply that the action of (II) is probably ATP-site-directed. 2 Studies with Other Enzymes and Attempted Syntheses of Anhydride Analogs of Other Nucleotides AMP kinase of rabbit muscle was not inactivated by 1 mM nominal initial levels of (I) (a 2-day-old sample) or of (II) (freshly prepared). 5 C. E. Carter and L. It. Cohen, d. Biol. Chem. 222, 17 (1956).
[30]
307
THYMIDYLATE SYNTHETASE INACTIVATION OF RABBIT MUSCLE PYRUVATE KINASE BY (II)" R a t e × 10 ~ (hA340/min) Additions prior to (II) None None ~ None c 0.1 m M A T P 1.5 m M P E P 2.5 m M A D P d
E n z y m e plus hydrolyzed (II)
Enzyme plus (II)
Inactivation (%)
3.10 3.00 3.06 3.00 3.10 3.02
1.80 1.45 1.81 2.95 2.95 2.96
43 52 41 2 5 2
T h e nominal initial concentration of (II) was 100 tLM. b (II) added in 3 equal increments at 20-second intervals. ° After t r e a t m e n t with (II) the enzyme solution was stored at 22 ° for 16 hr before t h e velocity determination. T h e enzyme was exposed to 2.5 m M ADP, t h e n 0.1 m M (II), in 0.1 ml of buffer a n d assayed after dilution to t h e s t a n d a r d volume cf 1 ml.
The method used to convert adenosine-5'-carboxylic acid to (I) and (II) did not convert uridine- or thymidine-5'-carboxylic acids ~ to the analogous U M P or T M P carboxylic-phosphoric anhydrides. 6 G. P. Moss, C. B. Reese, K. Schofield, R, Shapiro, and A. R. Todd, 1. Chem. Soc. 1963, 1149 (1963).
[30] A c t i v e - S i t e L a b e l i n g o f T h y m i d y l a t e Synthetase with 5-Fluoro-2'-deoxyuridylate 1 By YUSUKE WATAYA a n d DANIEL V. SANTI
Thymidylate synthetase catalyzes the reductive methylation of 2'deoxyuridylate (dUMP) to thymidylate with concomitant conversion of 5,10-methylenetetrahydrofolate (CH~-H~folate) to 7,8-dihydrofolate. A number of studies of the thymidylate synthetase reaction have led to the proposal that a primary event in the catalytic sequence involves the addition of a nucleophilic group of the enzyme to the 6-position of the substrate dUMP, a required step for subsequent condensation of the 5-posi1 This work was supported by U.S. Public H e a l t h Service G r a n t CA-14394 from the National Cancer Institute. D. V. S. is a recipient of a National Institutes of Health Career D e v e l o p m e n t Award.
