Nonlinear Free Energy Relationship in the General-Acid-Catalyzed Acylation of Rat Kidney γ-Glutamyl Transpeptidase by a Series of γ-Glutamyl Anilide Substrate Analogues†
journal contributionposted on 27.09.2001, 00:00 by Annie Ménard, Roselyne Castonguay, Christian Lherbet, Caroline Rivard, Yoann Roupioz, Jeffrey W. Keillor
The γ-glutamyl transpeptidase (GGT) purified from rat kidney reacts with a series of eight parasubstituted L-glutamyl γ-anilides, in the presence of Gly−Gly, catalyzing the formation of γ-Glu−Gly−Gly (pH 8.0, 37 °C). The transpeptidation reaction was followed through the discontinuous colorimetric determination of the concentration of released parasubstituted aniline. Steady-state kinetic studies were performed to measure kcat and KM values for each anilide substrate. A Hammett plot constructed by the correlation of log(kcat) and the σ- parameter for each anilide substrate displays statistically significant upward curvature, consistent with a general-acid-catalyzed acylation mechanism in which the geometry of the transition state changes with the nature of the para substituent. Kinetic isotope effects were measured and are consistent with a reaction involving a proton in flight at the rate-limiting transition state. The pH-rate profiles measured over pH 7.0−9.5 are bell-shaped with kinetic pKa values that may be attributed to the active site nucleophile (or its general-base catalytic partner) and the active-site general acid. The variation of the latter pKa value as a function of temperature is consistent with an enthalpy of ionization expected for an ammonium ion acting as a general acid. Examination of the variation of kcat as a function of temperature gave values for the enthalpy and entropy of activation that are similar to those determined for the general-acid-catalyzed breakdown of the tetrahedral intermediate formed during acylation of chymotrypsin by similar amide substrates.