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

• Published in: Biochemistry (Easton) Vol. 40; no. 42; pp. 12678 - 12685
• Main Authors: Ménard, Annie, Castonguay, Roselyne, Lherbet, Christian, Rivard, Caroline, Roupioz, Yoann, Keillor, Jeffrey W
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 23-10-2001
• Subjects: Acylation; Animals; Binding Sites; Catalysis; Deuterium - chemistry; Deuterium - metabolism; Deuterium Oxide - chemistry; Deuterium Oxide - metabolism; Energy Metabolism; gamma-Glutamyltransferase - chemistry; gamma-Glutamyltransferase - metabolism; Glutamine - analogs & derivatives; Glutamine - chemical synthesis; Glutamine - metabolism; Hydrogen-Ion Concentration; Kidney - enzymology; Kinetics; Male; Protons; Rats; Rats, Sprague-Dawley; Substrate Specificity; Temperature
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi011234d

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 k cat and K M values for each anilide substrate. A Hammett plot constructed by the correlation of log(k cat) 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 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 latter pK a 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 k cat 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.