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.
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.
复制标题
一系列γ-谷氨酰苯胺底物类似物对大鼠肾γ-谷氨酰转肽酶的一般酸催化酰化中的非线性自由能关系。
作者：
A. Ménard;R. Castonguay;C. Lherbet;C. Rivard;Y. Roupioz;J. Keillor
从大鼠肾脏中纯化的γ -谷氨酰转肽酶（GGT）在Gly-Gly存在下与一系列8个准取代的l -谷氨酰基γ -苯胺发生反应，催化形成γ -glu -Gly-Gly （pH 8.0, 37℃）。通过不连续比色法测定释放的副取代苯胺的浓度来跟踪转肽化反应。进行稳态动力学研究以测量每个苯胺基物的k（cat）和k(M)值。每个苯胺基物的log（k(cat)）和sigma（-）参数的相关性所构建的Hammett图显示了统计上显著的向上曲率，这与一般酸催化的酰化机制一致，其中过渡态的几何形状随着对取代基的性质而变化。动力学同位素效应被测量，并与一个质子在限速过渡态飞行的反应相一致。pH值在7.0-9.5范围内呈钟形，其动力学pK(a)值可能归因于活性位点亲核试剂（或其通用碱催化伙伴）和活性位点通用酸。后者pK(a)值随温度的变化与铵离子作为一般酸的电离焓一致。对k（cat）作为温度函数的变化的检验给出了激活焓和熵的值，这些值类似于由类似的酰胺底物在凝乳胰蛋白酶酰化过程中形成的四面体中间体的一般酸催化分解所确定的值。
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.