Nonlinear free energy relationship in the general-acid-catalyzed acylation of rat kidney gamma-glutamyl transpeptidase by a series of gamma-glutamyl anilide substrate analogues.
The gamma-glutamyl transpeptidase (GGT) purified from rat kidney reacts with a series of eight parasubstituted L-glutamyl gamma-anilides, in the presence of Gly-Gly, catalyzing the formation of gamma-Glu-Gly-Gly (pH 8.0, 37 degrees 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 sigma(-) 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.
Ménard
A
A
Département de chimie, Université de Montréal, C.P. 6128, Succursale centre-ville, Montréal, Québec, Canada H3C 3J7.
Castonguay
R
R
Lherbet
C
C
Rivard
C
C
Roupioz
Y
Y
Keillor
J W
JW
D000215:Acylation; D000818:Animals; D001665:Binding Sites; D002384:Catalysis; D003903:Deuterium / Q000737:chemistry / Q000378:metabolism; D017666:Deuterium Oxide / Q000737:chemistry / Q000378:metabolism; D004734:Energy Metabolism; D005973:Glutamine / Q000031:analogs & derivatives* / Q000138:chemical synthesis / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007668:Kidney / Q000201:enzymology*; D007700:Kinetics; D008297:Male; D011522:Protons; D051381:Rats; D017207:Rats, Sprague-Dawley; D013379:Substrate Specificity; D013696:Temperature; D005723:gamma-Glutamyltransferase / Q000737:chemistry / Q000378:metabolism*
D011522:Protons; D005973:Glutamine; C001300:gamma-glutamine-4-nitroanilide; D003903:Deuterium; D005723:gamma-Glutamyltransferase; D017666:Deuterium Oxide