259 Table I: Kinetic Data o n the Cleavage of the Maleamic Acids, 25.0 "C, 1 M H,O in CH,CN 104k (s-') with HOAc-KOAc Buffera Comod
Mo."C
1O:l
1:lO
1:so
lb
151-152 118-120 129-136 123-125 107-110
75.3 45.5 41.6 28.8 20.1
-0.1 1.80 5.12 -0.07 0.866
0.0314 2.07d 4.55e 0.0238 0.959
26 3c 4b 5c
a Ratio 1O:l indicates the buffer ratio of HOAc/KOAc at a buffer concentration of 5 X M HOAc, 5 X M KOAc with 1 X 10- M substrate. 1:SO is 5 X M KOAc, with 1 X M substrate whose ammonium cation generates the HOAc. Rate constants are the average from at least three runs (except for the two approximate values for 1 and 4) and had standard deviations of 6-16%. bcharacterized by NMR spectra, and C, H, N analyses within 0.3% of theoretical. CCharacterized by NMR spectra, correct H, N (and Br) analyses, but low C analyses suggesting some hydration Or other impurity. dkH,O/kD,O is 1.47. ekH,O/kD,O is 2.27.
maleic anhydride, and again no catalytic effect was observed from the phenolic hydroxyls of compounds 2,3, or 5. However, the behavior of these compounds diverged in 1:lO HOAc/KOAc or 1:50 HOAc/KOAc. Compounds 1 and 4, without the phenolic hydroxyl, now underwent very slow hydrolysis, in parallel with the behavior of the free carboxylate ions in aqueous solution. In these (increasingly) more basic media, protonation of the leaving amino group is now a problem. By contrast, the phenolic compounds 2, 3, and 5 underwent a moderate decrease in rate in the 1:lO medium and no further decrease in the 1:50 medium. The phenolic hydroxyls can now supply the required acid proton, and acid from the medium is no longer required.8 As the data in Table I show, the catalytic effects of these hydroxyl groups are substantial. Comparing 2 with 1 in the neutral ( 1 5 0 ) solution, the acceleration is 66-fold. Thus, if carboxypeptidase actually cleaves esters and peptides by similar mechanisms, except that peptide hydrolysis is also assisted by a tyrosine hydroxyl, that assistance could bring the peptide rates up to those of ester^.^ An additional striking change on converting the medium from the 10: 1 acidic buffer to the neutral 1 : 10 or 1:SO buffer is that the substrate abandons the anhydride mechanism. Anhydride cannot be detected as a reaction intermediate with 2, 3, or 5 by spectroscopy or by trapping with added simple amines, although authentic dimethylmaleic anhydride can be detected in both these ways if it is added to the medium. We conclude that in this model system, the nucleophilic catalytic role played by the carboxylate ion at low pH is supplanted by another catalytic role, presumably general base delivery of water,* in the pH region corresponding to neutrality. Such a change in mechanism a t higher pH is known for other neighboring group catalysts,'0 and can be understood in terms of the energetics of the individual steps. In essence, a leaving group protonated by the weak phenolic hydroxyl cannot be ejected to form the high energy anhydride, only to form the more stable carboxylate ion. This implies that in the tetrahedral intermediate 6 a proton must next be removed from the hydroxyl, as well as added to the nitrogen. The phenolic group could assist in both of these processes. l '
k7$-
COu- H-0
I
6
Thus, these model systems utilize two of the three known functional groups of carboxypeptidase A to catalyze an amide hydrolysis. Furthermore, they use either of the two mechanisms generally considered for the enzyme, depending on the reaction conditions. This again calls attention to the necessity to resolve the mechanistic ambiguities with the enzyme itself. The accompanying communication'* indicates that the enzyme, at neutrality, apparently parallels our model system in utilizing nonnucleophilic catalysis by carboxylate. Acknowledgment. Financial assistance of this work by the National Institutes of Health is gratefully acknowledged. References and Notes (1) For reviews, see (a) E. T. Kaiser and E. L. Kaiser, Acc. Chem. Res., 5, 219 (1972):(b) W. N. Lipscomb, Tetrahedron, 30, 1725 (1974);F. A. Quiocho and W. N. Lipscomb, Adv. Protein Chem., 25, 1 (1971). (2)Reference l b and work cited therein. However, see also ref. 4a. (3) E. L. Vallee, J. F. Riordan, and J. E. Collman. Proc. Nat. Acad. Sci. U.S.A., 49, 109 (1963). (4)(a) J. T. Johnson and E. L. Vallee, Biochemistry, 14, 649 (1975);(b) E. 1.Kaiser, private communication. (c) Note that in our proposals in this and the accompanying communication the phenolic catalysis of proton transfer should be a subsequent fast step, not the ratedetermining step. Therefore conditions which simply slow that fast step, e.g., partial titration of the phenol, will not necessarily slow the overall rate. Of course blocking phenol catalysis entirely, by acetylation of the phenolic hydroxyl, could finally make the proton transfer step rate determining. (5)For the previous publication, see R. Breslow, D. E. McClure. R. S. Brown, and J. Eisenach, J. Am. Chem. SOC..97, 194 (1975). (6) A. J. Kirby and A. R. Fersht, Prog. Bioorg. Chem., 1, 28 (1971).Two recent studies of bifunctional catalysis of maleamic acid hydrolysis have been reported during the course of our work: (a) M. F. Aldersley, A. J. Kirby, P. W. Lancaster, R. S. McDonald, and C. R. Smith, J. Chem. SOC., Perkin Trans. 2, 1487 (1974);(b) A. J. Kirby, R. S. McDonald, and C. R. Smith, ibid., 1495 (1974).In this work the nucleophilic (anhydride)mechanism was maintained, at low pH, but proton transfers were catalyzed by an additional catalytic group. (7)Although "pH" can be read on a meter in such media, it seems best to consider the state of ionization of the catalytic groups. This should parallel the state of ionization of the buffer acid. (8) Two control reactions help establish this mechanism. KBenzoyl-o-hydroxybenzylamine is completely stable over many days in our medium: thus the carboxylate in 2 plays a role. Furthermore, reaction of dimethylmaleic anhydride with phenoxide ion produces the unstable phenyl monoester of dimethylmaleic acid: in the 1:50 HOAc/KOAc medium this rapidly re-forms the anhydride. Since hydrolysis of amide 2 does not proceed through the anhydride under these conditions, it must not be using the phenolic group as a nucleophile to form an intermediate phenyl ester. (9)Approximately lo3 would be needed if peptide substrates are to be brought to the reactivity of ester substrates. (10)E.g., A. R. Fersht and A. J. Kirby, J. Am. Chem. SOC.,90, 5826 (1968). describe the change from nucleophilic catalysis in aspirin acid to general base catalysis in its anion. (1 1) The somewhat greater reactivity of 3, the bromophenol, than of 2 indicates some net proton transfer from the phenolic group. Thus proton transfer to N is more advanced than proton recovery from the carbinol group in the tetrahedral intermediate. (12)R. Breslow and D. Wernick, J. Am. Chem. SOC..following paper in this issue.
Ronald Bredow,* David E. McClure Department of Chemistry, Columbia University New York, New York 10027 Received October 10. 1975
On the Mechanism of Catalysis by Carboxypeptidase A Sir:
Two general mechanisms have been proposed' for hydrolytic reactions catalyzed by bovine pancreatic carboxypeptidase A (CPA, E.C.3.4.12.2). In one the y-carboxylate of Glu-270 acts as a nucleophile at the scissile carbonyl, forming an anhydride intermediate;* in the other Glu-270 acts as a general base, delivering nucleophilic water instead. As the accompanying communication indicate^,^ we have model systems for both of these mechanisms. Work on the enzyme now allows us to choose between them. Communications to the Editor
260 Table I. Turnover Numbers for CPA Catalyzed I8O Exchanges N-Benzo ylplycine (niM)b 1.6 10 10 4.1 1.6 7.1 IO
8.3 7.6 1.6 7.6 7.6 8.4
Added component (mM)
Scheme I
VIE (min-')
Mo."C
1O:l
1:lO
1:so
lb
151-152 118-120 129-136 123-125 107-110
75.3 45.5 41.6 28.8 20.1
-0.1 1.80 5.12 -0.07 0.866
0.0314 2.07d 4.55e 0.0238 0.959
26 3c 4b 5c
a Ratio 1O:l indicates the buffer ratio of HOAc/KOAc at a buffer concentration of 5 X M HOAc, 5 X M KOAc with 1 X 10- M substrate. 1:SO is 5 X M KOAc, with 1 X M substrate whose ammonium cation generates the HOAc. Rate constants are the average from at least three runs (except for the two approximate values for 1 and 4) and had standard deviations of 6-16%. bcharacterized by NMR spectra, and C, H, N analyses within 0.3% of theoretical. CCharacterized by NMR spectra, correct H, N (and Br) analyses, but low C analyses suggesting some hydration Or other impurity. dkH,O/kD,O is 1.47. ekH,O/kD,O is 2.27.
maleic anhydride, and again no catalytic effect was observed from the phenolic hydroxyls of compounds 2,3, or 5. However, the behavior of these compounds diverged in 1:lO HOAc/KOAc or 1:50 HOAc/KOAc. Compounds 1 and 4, without the phenolic hydroxyl, now underwent very slow hydrolysis, in parallel with the behavior of the free carboxylate ions in aqueous solution. In these (increasingly) more basic media, protonation of the leaving amino group is now a problem. By contrast, the phenolic compounds 2, 3, and 5 underwent a moderate decrease in rate in the 1:lO medium and no further decrease in the 1:50 medium. The phenolic hydroxyls can now supply the required acid proton, and acid from the medium is no longer required.8 As the data in Table I show, the catalytic effects of these hydroxyl groups are substantial. Comparing 2 with 1 in the neutral ( 1 5 0 ) solution, the acceleration is 66-fold. Thus, if carboxypeptidase actually cleaves esters and peptides by similar mechanisms, except that peptide hydrolysis is also assisted by a tyrosine hydroxyl, that assistance could bring the peptide rates up to those of ester^.^ An additional striking change on converting the medium from the 10: 1 acidic buffer to the neutral 1 : 10 or 1:SO buffer is that the substrate abandons the anhydride mechanism. Anhydride cannot be detected as a reaction intermediate with 2, 3, or 5 by spectroscopy or by trapping with added simple amines, although authentic dimethylmaleic anhydride can be detected in both these ways if it is added to the medium. We conclude that in this model system, the nucleophilic catalytic role played by the carboxylate ion at low pH is supplanted by another catalytic role, presumably general base delivery of water,* in the pH region corresponding to neutrality. Such a change in mechanism a t higher pH is known for other neighboring group catalysts,'0 and can be understood in terms of the energetics of the individual steps. In essence, a leaving group protonated by the weak phenolic hydroxyl cannot be ejected to form the high energy anhydride, only to form the more stable carboxylate ion. This implies that in the tetrahedral intermediate 6 a proton must next be removed from the hydroxyl, as well as added to the nitrogen. The phenolic group could assist in both of these processes. l '
k7$-
COu- H-0
I
6
Thus, these model systems utilize two of the three known functional groups of carboxypeptidase A to catalyze an amide hydrolysis. Furthermore, they use either of the two mechanisms generally considered for the enzyme, depending on the reaction conditions. This again calls attention to the necessity to resolve the mechanistic ambiguities with the enzyme itself. The accompanying communication'* indicates that the enzyme, at neutrality, apparently parallels our model system in utilizing nonnucleophilic catalysis by carboxylate. Acknowledgment. Financial assistance of this work by the National Institutes of Health is gratefully acknowledged. References and Notes (1) For reviews, see (a) E. T. Kaiser and E. L. Kaiser, Acc. Chem. Res., 5, 219 (1972):(b) W. N. Lipscomb, Tetrahedron, 30, 1725 (1974);F. A. Quiocho and W. N. Lipscomb, Adv. Protein Chem., 25, 1 (1971). (2)Reference l b and work cited therein. However, see also ref. 4a. (3) E. L. Vallee, J. F. Riordan, and J. E. Collman. Proc. Nat. Acad. Sci. U.S.A., 49, 109 (1963). (4)(a) J. T. Johnson and E. L. Vallee, Biochemistry, 14, 649 (1975);(b) E. 1.Kaiser, private communication. (c) Note that in our proposals in this and the accompanying communication the phenolic catalysis of proton transfer should be a subsequent fast step, not the ratedetermining step. Therefore conditions which simply slow that fast step, e.g., partial titration of the phenol, will not necessarily slow the overall rate. Of course blocking phenol catalysis entirely, by acetylation of the phenolic hydroxyl, could finally make the proton transfer step rate determining. (5)For the previous publication, see R. Breslow, D. E. McClure. R. S. Brown, and J. Eisenach, J. Am. Chem. SOC..97, 194 (1975). (6) A. J. Kirby and A. R. Fersht, Prog. Bioorg. Chem., 1, 28 (1971).Two recent studies of bifunctional catalysis of maleamic acid hydrolysis have been reported during the course of our work: (a) M. F. Aldersley, A. J. Kirby, P. W. Lancaster, R. S. McDonald, and C. R. Smith, J. Chem. SOC., Perkin Trans. 2, 1487 (1974);(b) A. J. Kirby, R. S. McDonald, and C. R. Smith, ibid., 1495 (1974).In this work the nucleophilic (anhydride)mechanism was maintained, at low pH, but proton transfers were catalyzed by an additional catalytic group. (7)Although "pH" can be read on a meter in such media, it seems best to consider the state of ionization of the catalytic groups. This should parallel the state of ionization of the buffer acid. (8) Two control reactions help establish this mechanism. KBenzoyl-o-hydroxybenzylamine is completely stable over many days in our medium: thus the carboxylate in 2 plays a role. Furthermore, reaction of dimethylmaleic anhydride with phenoxide ion produces the unstable phenyl monoester of dimethylmaleic acid: in the 1:50 HOAc/KOAc medium this rapidly re-forms the anhydride. Since hydrolysis of amide 2 does not proceed through the anhydride under these conditions, it must not be using the phenolic group as a nucleophile to form an intermediate phenyl ester. (9)Approximately lo3 would be needed if peptide substrates are to be brought to the reactivity of ester substrates. (10)E.g., A. R. Fersht and A. J. Kirby, J. Am. Chem. SOC.,90, 5826 (1968). describe the change from nucleophilic catalysis in aspirin acid to general base catalysis in its anion. (1 1) The somewhat greater reactivity of 3, the bromophenol, than of 2 indicates some net proton transfer from the phenolic group. Thus proton transfer to N is more advanced than proton recovery from the carbinol group in the tetrahedral intermediate. (12)R. Breslow and D. Wernick, J. Am. Chem. SOC..following paper in this issue.
Ronald Bredow,* David E. McClure Department of Chemistry, Columbia University New York, New York 10027 Received October 10. 1975
On the Mechanism of Catalysis by Carboxypeptidase A Sir:
Two general mechanisms have been proposed' for hydrolytic reactions catalyzed by bovine pancreatic carboxypeptidase A (CPA, E.C.3.4.12.2). In one the y-carboxylate of Glu-270 acts as a nucleophile at the scissile carbonyl, forming an anhydride intermediate;* in the other Glu-270 acts as a general base, delivering nucleophilic water instead. As the accompanying communication indicate^,^ we have model systems for both of these mechanisms. Work on the enzyme now allows us to choose between them. Communications to the Editor
260 Table I. Turnover Numbers for CPA Catalyzed I8O Exchanges N-Benzo ylplycine (niM)b 1.6 10 10 4.1 1.6 7.1 IO
8.3 7.6 1.6 7.6 7.6 8.4
Added component (mM)
Scheme I
VIE (min-')
