MECHANISTIC STUDIES OF THERMOLYSIN

To probe structural features of the rate-limiting transition state and the mechanistic origins of substrate specificity, we determined temperature dependencies and solvent and 3-deuterium isotope effects for the thermolysin-catalyzed hydrolyses of FA-Gly-P', where FA is 3-(2-furyl)acrylate and P' is AlaNH2, AbuNH2, ValNH2, NvaNH2, PheNH2, LeuNH2, and Leu-Ala (cleavage occurs at Gly-P'). Important observations are (1) Eyring plots for kc/Km are nonlinear, with plateaus or decreases in activity at elevated temperatures. The following four reaction types are consistent with this result: (i) a simple reaction (i.e., a single rate-limiting transition state), with a negative heat capacity of activation; (ii) a complex reaction with partially rate-limiting, differentially temperature-sensitive sequential steps; (iii) a reaction involving a temperature-dependent equilibrium of two enzyme forms, where one form is inactive; and (iv) a reaction involving a temperature-dependent equilibrium of two enzyme forms, where both forms are active. Detailed analyses of the Eyring plots suggests that mechanism ii is most consistent with the data. (2) A plot of In [(kc/Km)T=5°C] vs In [(kc/Km)T=20°C] for the seven substrates of this study is linear and indicates that this series of reactions defines an isokinetic relationship. (3) kcKm correlates with the hydrophobicity of the P1' amino acid residue. (4) The β-deuterium isotope effect on kcKm is 0.97 ± 0.01 for the hydrolyses of both FA-Gly-(L, L)-LeuNH2 and FA-Gly-(L, L)-Leu-Ala (L = H, D) and suggests that the rate-limiting transition states for these reactions resemble the tetrahedral addition adduct of zinc-bound water and substrate. (5) For FA-Gly-LeuNH2, the β-deuterium isotope effect increases with increasing temperature. (6) Solvent deuterium isotope effects on kcKm for the hydrolyses of FA-Gly-P' are all near 0.74 and indicate an absence of general-acid/general-base catalysis in the rate-limiting step. Combined, the results of this study suggest a sequential mechanism for thermolysin in which the Michaelis complex changes conformation prior to the chemical steps of peptide bond hydrolysis. kcKm is rate-limited by both the conformational change and the hydrolysis step. The extent to which either of these steps limits kc/Km is dependent on temperature, with chemistry contributing more to rate limitation at lower temperature. The chemical step, which corresponds to formation and decomposition of a tetrahedral intermediate, is itself rate-limited by a reaction step that does not involve protolytic catalysis. Likely candidates for this step are addition of a zinc-bound hydroxide to the substrate carbonyl carbon and decomposition of a zwitterionic tetrahedral intermediate. © 1990, American Chemical Society. All rights reserved.