Dorovska-Taran V, Momtcheva R, Gulubova N, Martinek K
Biochim Biophys Acta. 1982 Mar 18;702(1):37-53. doi: 10.1016/0167-4838(82)90025-5.
The method of 'added nucleophile' (methanol) was employed to study the steady-state kinetics of the alpha-chymotryptic hydrolysis of methyl esters of N-acetyl-L-amino acids of the RCH(NHCOCH3)-C(O)OCH3 type, which are derivatives of alpha-aminobutyric acid, norvaline, norleucine, phenylalanine, alpha-aminoheptanoic acid and alpha-aminooctanoic acid. In the 5 to 30 degrees C temperature range at pH 8.0 the elementary rate and binding constants for both hydrolysis (Ks, k2, k3) and methanolysis (k-2) of the intermediate acylenzyme have been determined. The following results are discussed: 1. The enthalpy-entropy compensation phenomenon, which has previously been established to take place in alpha-chymotryptic catalysis, is characterized by two values of isokinetic temperatures (Tc), i.e., about 200 K at the step of binding the substrate and 430--460 K at the chemical steps of the enzymatic reaction. 2. It is demonstrated how the free energy and enthalpy, as well as entropy, change along all the reaction coordinate. To explain the results obtained, a mechanism for enzyme-substrate interaction is suggested according to which the sorption of substrate group R on the enzyme is different in the metastable intermediates and in the transition states of the reaction: (i) in the enzyme-substrate complex and in the acylenzyme, substrate group R merely emerges from water to be buried in the protein. Two facts point to the hydrophobic nature of the E-R interaction. First, the change in the free energy is exactly delta GRextr, that is the free energy of the transfer (extraction) of substrate group R from water to the organic solvent. Second, the motive force of the E-R interaction is the change in the entropy only. (ii) In the transition state the situation is different, i.e., the E-R interaction becomes thermodynamically still more favourable (the free energy change is 2 delta GRtrans) and involves thermodynamically favourable changes not only in the entropy (as is the case in the intermediates), but also in the enthalpy. Hence the E-R interaction in the transition states is accompanied by conformational and solvational changes of the enzyme-substrate system and, first and foremost, of the entire protein.
采用“添加亲核试剂”（甲醇）的方法，研究了RCH(NHCOCH3)-C(O)OCH3型N-乙酰-L-氨基酸甲酯（α-氨基丁酸、正缬氨酸、正亮氨酸、苯丙氨酸、α-氨基庚酸和α-氨基辛酸的衍生物）的α-胰凝乳蛋白酶水解的稳态动力学。在pH 8.0、5至30℃的温度范围内，测定了中间酰基酶水解（Ks、k2、k3）和甲醇解（k-2）的基元速率和结合常数。讨论了以下结果：1. 先前已确定在α-胰凝乳蛋白酶催化中发生的焓-熵补偿现象，其特征在于两个等动力学温度（Tc）值，即在底物结合步骤约为200 K，在酶促反应的化学步骤为430 - 460 K。2. 证明了自由能、焓以及熵如何沿整个反应坐标变化。为了解释所得结果，提出了一种酶-底物相互作用机制，根据该机制，底物基团R在酶上的吸附在亚稳中间体和反应的过渡态中是不同的：（i）在酶-底物复合物和酰基酶中，底物基团R仅仅从水中出来并被埋入蛋白质中。两个事实表明E-R相互作用的疏水性质。第一，自由能变化恰好是ΔGRextr，即底物基团R从水转移（提取）到有机溶剂中的自由能。第二，E-R相互作用的驱动力仅是熵的变化。（ii）在过渡态情况不同，即E-R相互作用在热力学上变得更有利（自由能变化为2ΔGRtrans），并且不仅涉及热力学上有利的熵变化（如在中间体中那样），还涉及焓变化。因此，过渡态中的E-R相互作用伴随着酶-底物系统，首先是整个蛋白质的构象和溶剂化变化。
