Kogan R L, Fife T H
Biochemistry. 1985 May 21;24(11):2610-4. doi: 10.1021/bi00332a004.
The second-order rate constants k2/Km for acylation of alpha-chymotrypsin by a series of N-acylimidazole derivatives of aliphatic carboxylic acids have been determined at 30 degrees C by proflavin displacement from the active site. With cyclohexyl-substituted N-acylimidazoles, the rate constants increase with increasing chain length of the acyl group; i.e., k2/Km is in the order cyclohexylcarbonyl less than cyclohexylacetyl less than beta-cyclohexylpropionyl. The latter substrate has k2/Km = 1.2 X 10(6) M-1 s-1 at pH 8.0, which appears to be a maximum value for N-acylimidazole substrates. A further increase in the chain length of the acyl group with (gamma-cyclohexylbutyryl)imidazole results in a decrease in k2/Km. Hydrophobic effects of the hydrocarbon acyl groups are of predominant importance with regard to the relative values of k2/Km for aliphatic N-acylimidazole substrates. There is a linear correlation of the logarithms of the rate constants at pH 8.0 with the hydrophobic substituent constants, pi, having a slope of 1.71 (r = 0.90). On the other hand, there is little apparent correlation with the Taft steric effect constants, Es. A four-parameter equation including both pi and Es improved the correlation only slightly [log (k2/Km) = 1.88 pi + 1.01 Es + C]. In contrast, steric effects as reflected in the Es constants are the major influence in acylation of the enzyme by corresponding p-nitrophenyl esters. There are very likely significant differences in transition-state structure with the two types of substrates.
通过黄素从活性位点的取代作用，在30摄氏度下测定了一系列脂肪族羧酸的N - 酰基咪唑衍生物对α- 胰凝乳蛋白酶进行酰化作用的二级速率常数k2/Km。对于环己基取代的N - 酰基咪唑，速率常数随着酰基链长度的增加而增大；即，k2/Km的顺序为环己羰基＜环己乙酰基＜β-环己基丙酰基。后一种底物在pH 8.0时k2/Km = 1.2×10⁶ M⁻¹ s⁻¹，这似乎是N - 酰基咪唑底物的最大值。用（γ-环己基丁酰基）咪唑使酰基链长度进一步增加会导致k2/Km减小。对于脂肪族N - 酰基咪唑底物，烃酰基的疏水作用对k2/Km的相对值起主要作用。在pH 8.0时，速率常数的对数与疏水取代常数π呈线性相关，斜率为1.71（r = 0.90）。另一方面，与塔夫特空间效应常数Es几乎没有明显相关性。一个包含π和Es的四参数方程仅略微改善了相关性[log (k2/Km) = 1.88π + 1.01Es + C]。相比之下，Es常数所反映的空间效应是相应对硝基苯酯对该酶进行酰化作用的主要影响因素。这两种类型的底物在过渡态结构上很可能存在显著差异。
