Nonlinear free energy relationship in the general-acid-catalyzed acylationof rat kidney gamma-glutamyl transpeptidase by a series of gamma-glutamyl anilide substrate analogues

The gamma -glutamyl transpeptidase (GGT) purified from rat kidney reacts wi th a series of eight parasubstituted L-glutamyl gamma -anilides, in the pre sence of Gly-Gly, catalyzing the formation of gamma -Glu-Gly-Gly (pH 8.0, 3 7 degreesC). The transeptidation reaction was followed through the disconti nuous colorimetric determination of the concentration of released parasubst ituted aniline. Steady-state kinetic studies were performed to measure k(ca t) and K-m values for each anilide substrate. A Hammett plot constructed by the correlation of log(k(cat)) and the sigma (-) parameter for each anilid e 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. Kine tic isotope effects were measured and are consistent with a reaction involv ing a proton in flight at the rate-limiting transition state. The pH-rate p rofiles measured over pH 7.0-9.5 are bell-shaped with kinetic pK(a) 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 l atter pK(a) value as a function of temperature is consistent with an enthal py of ionization expected for an ammonium ion acting as a general acid. Exa mination of the variation of k(cat) as a function of temperature gave value s for the enthalpy and entropy of activation that are similar to those dete rmined for the general-acid-catalyzed breakdown of the tetrahedral intermed iate formed during acylation of chymotrypsin by similar amide substrates.