Elucidation of the chemical nature of the steady-state intermediates in the mechanism of carboxypeptidase A

Abstract

Cryospectrokinetic studies of zinc and cobalt carboxypeptidase A disclosed two intermediates in the hydrolysis of both peptides and depsipeptides and furnished all the rate and equilibrium constants for the reaction scheme E + S in equilibrium ES1 in equilibrium ES2---E + P [Auld, D. S., Galdes, A., Geoghegan, K. F., Holmquist, B., Martinelli, R. A., & Vallee, B. L. (1984) Proc. Natl. Acad. Sci. U.S.A. 81, 5041-5045]. Since the ES2 intermediate is the predominate enzyme species present at steady state, its chemical nature is deducible from subzero chemical quench studies done after steady state is established. Extrapolation of the product concentration to zero time, [P0], measures the concentration of the enzyme species in which bond cleavage has occurred. For peptides, the [P0]values are zero, indicating that no product is generated prior to turnover and therefore the ES2 intermediate involves a complex between enzyme and intact peptide substrate. For depsipeptides, [P0] values are 1 mol of produce per mole of enzyme over the entire temperature range -20 to -50 degrees C, indicating cleavage of the ester bond occurs prior to the rate-limiting step so that ES2 is more properly denoted by EP1P2, where P1 and P2 are the substrates for the reverse reaction. The rate-limiting step for depsipeptides thus involves release of the products which may occur directly or through a mandatory conformational change followed by rapid product release.

Conformational dynamics and solvent viscosity effects in carboxypeptidase‐A‐catalyzed benzoylglycylphenyllactate hydrolysis

“…However, the real intrinsic isotope effect is blurred by diffusion, reflected in the exponent 1 -6 z 0.4 in Eqn (5). The The combination of kinetic isotope and viscosity effects for the analogous Bz-Gly-Phe and Bz-Gly-0-Ph-Lac substrates offers interesting complementary clues to other observations on the two substrate classes based on structural [56,87,881 and cryokinetic investigations [59,89,901. Cryokinetic data for Dns-Ala-Ala-Phe (Dns, dansyl) and Dns-Ala-Ala-O-PhLac indicate that both reactions proceed through two inter-mediates where decay of the second is rate determining in either case.…”

“…Kinematic details are, however, different for peptide and ester hydrolysis [57,58]. In particular, cryokinetic investigations have revealed two intermediates for both peptide and ester [59], where second intermediate decay is rate determining for both. The decay involves chemical conversion for peptides, but product liberation for esters.…”

Diverse role of conformational dynamics in carboxypeptidase A‐driven peptide and ester hydrolyses: Disclosing the “Perfect Induced Fit” and “Protein Local Unfolding” pathways by altering protein stability

“…3,6,8,[23][24][25][26][27][28] This situation is rather distinct from some other classes of model enzymes such as, e.g., serine hydrolases for which the major mechanistic pattern has been firmly established. 29,30 Among other complexities, extremely diverse kinetic manifestations showing up through the pH profiles, [3][4][5][6]12 inhibition/activation patterns (due to substrates or other effectors), [3][4][5][6]12,13,22 cryospectroscopic (intermediate trapping) experiments, 6,14,16 and viscosity impact observations, [17][18][19] should be mentioned. Interestingly, rather diverse performance has been observed not only between two families of more natural yet synthetic (''probing'') peptide and the counterpart ester (depsi-peptide) substrates but also within the substrates of either of these types that may differ in the chain length and/or residue specificity.…”

“…A variety of catalytic mechanisms proposed for CPA so far can be grouped into two major patterns defined as the ''promoted water'' and ''anhydride intermediate'' pathways (vide infra). [4][5][6][12][13][14][15][16][20][21][22][23][24][25][26][27][28] Although, either of them can be attributed to both, the peptide and ester hydrolysis, because of the particular kinetic evidence accumulated so far, it is usually assumed that the first pattern is applicable for peptides, whilst another is effective for esters (see Results and Discussion). Indeed, the question regarding the diverse catalytic patterns of CPA for specific peptide and ester substrates appears to be the most conspicuous one.…”

“…Indeed, the question regarding the diverse catalytic patterns of CPA for specific peptide and ester substrates appears to be the most conspicuous one. [3][4][5][6][12][13][14][15][16][20][21][22][23][24][25][26][27] The ''promoted water'' pathway entails direct participation of Zn 21 -activated water in a rate-determining step as a nucleophile with the carboxylate group of Glu270 acting as a general base (Scheme 1), whereas the ''anhydride intermediate'' pathway implies direct nucleophilic attack by Glu270 (Scheme 2), with water to be involved in the following stage (for the detailed discussion, see Results and Discussion). Notwithstanding that the mechanistic difference for peptide and ester substrates has been discussed numerous times, [3][4][5][6]12,13,18,19,26,27 definitely this problem requires further clarification, implying both the adequate classification and theory-based description, on the grounds of matching current concepts (vide infra).…”