Current problems in mechanistic studies of serine and cysteine proteinases.
Current problems in mechanistic studies of serine and cysteine proteinases.
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DOI：
10.1042/bj2070001
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发表时间：
1982-10
期刊：
影响因子：
--
通讯作者：
L. Polgár;P. Halász
中科院分区：
文献类型：
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作者：
L. Polgár;P. Halász
丝氨酸蛋白酶是研究最广泛的酶之一（最近的综述参见Blow，1976; Kraut，1977; Huber & Bode，1978）。它们最著名的代表，如胰蛋白酶和胰凝乳蛋白酶，是通过进化而相关的胰酶;丝氨酸蛋白酶还包括通过不同途径进化的细菌枯草杆菌蛋白酶，和参与血液凝固和补体激活级联反应的酶，以及在噬菌体成熟，受精，以及其他一些生物现象领域。大部分的机制研究已经进行了糜蛋白酶。这些研究确定了1960年代催化作用的基本特征（参见第二章）。Bender & Kezdy，1965）。图1总结了主要结果。可以看出，由“反应性”丝氨酸残基的羟基对底物的羰基碳原子的亲核攻击由作为通用碱的组氨酸残基催化。这导致四面体中间体和咪唑鎓离子的形成。中间体通过一般的酸催化分解成酰基酶、咪唑碱和醇或胺。酰基酶通过逆反应途径水解。当然，在水解中，水分子的羟基是亲核试剂，而不是丝氨酸残基的羟基。这种机制意味着四面体中间体和咪唑鎓离子之间的紧密接触，这抑制了质子在一般酸催化之前释放到溶剂中（Polgar，1971），并导致一次遭遇型反应（Polgar，1972）。对胰凝乳蛋白酶的X射线衍射研究开启了机理研究的新时代（马修斯等人，1967; Birktoft & Blow，1972）和其他丝氨酸蛋白酶（参见，Kraut，1977），这使得有可能用结构特征描述基元反应步骤。在这方面可能会提出几个有趣的问题。首先，由于酶特异性底物加合物不适用于当前的X射线衍射测量，什么是来自酶抑制剂或酶底物类似物复合物的立体化学机制的现实？丝氨酸-组氨酸-天冬氨酸催化三联体的机制作用是什么，这在过去十年中一直是一个争论的主题？四面体中间体的负氧原子周围的精致环境（氧阴离子空穴）对催化的贡献是什么？如何建立化学小分子，如立体电子理论和同位素效应，应用于酶催化？大多数与丝氨酸酶有关的上述问题也出现在半胱氨酸蛋白酶的情况下。这些广泛分布的酶的主角是木瓜蛋白酶，一种植物蛋白酶（Glazer & Smith，1971），直到最近其空间结构可用的唯一半胱氨酸蛋白酶（Drenth等人，1971 a，B）。其他相关巯基酶
Serine proteinases are among the most extensively studied enzymes (for recent reviews see Blow, 1976; Kraut, 1977; Huber & Bode, 1978). Their best known representatives, like trypsin and chymotrypsin, are pancreatic enzymes that are related through evolution; serine proteinases also include the bacterial subtilisins, which evolved through a different route, and enzymes that participate in the cascade reactions of blood clotting and complement activation, as well as enzymes that play an important role in phage maturation, fertilization, and in a number of other fields of biological phenomena. Most of the mechanistic studies have been performed with chymotrypsin. Those studies established the basic features of the catalytic action by the 1960's (cf. Bender & Kezdy, 1965). The principal results are summarized in Fig. 1. It is seen that the nucleophilic attack by the hydroxyl group of the 'reactive' serine residue on the carbonyl carbon atom of the substrate is catalysed by a histidine residue as a general base. This leads to the formation of the tetrahedral intermediate and an imidazolium ion. The intermediate breaks down by general acid catalysis to an acyl-enzyme, an imidazole base, and alcohol or amine. The acylenzyme is hydrolysed through the reverse reaction pathway. Of course, in hydrolysis the hydroxyl group of a water molecule is the nucleophile instead of the hydroxyl group of the serine residue. This mechanism implies a close contact between the tetrahedral intermediate and the imidazolium ion, which inhibits the release of proton into the solvent before general acid catalysis (Polgar, 1971), and leads to a one-encounter type reaction (Polgar, 1972). A new era of mechanistic investigations started with X-ray diffraction studies on chymotrypsin (Matthews et al., 1967; Birktoft & Blow, 1972) and other serine proteinases (cf. Kraut, 1977), which rendered it possible to clothe elementary reaction steps with structural features. Several intriguing questions may be raised in this respect. First of all, as enzyme-specific substrate adducts are not amenable to current X-ray diffraction measurements, what is the reality of the stereochemical mechanisms derived from enzyme-inhibitor or enzyme-substrate analogue complexes? What is the mechanistic role of the serine-histidine-aspartate catalytic triad, which has been a subject of debate over the past decade? What is the contribution to catalysis of the exquisite environment (oxyanion hole) around the negative oxygen atom of the tetrahedral intermediate? How can the established chemistry obtained on small molecules, e.g. stereoelectronic theory and isotope effects, be applied to enzyme catalysis? Most of the above questions related to the serine enzymes also emerge in the case of cysteine proteinases. The protagonist of these wide-spread enzymes is papain, a plant proteinase (Glazer & Smith, 1971), the only cysteine proteinase until recently whose steric structure was available (Drenth et al., 1971 a,b). Other related thiol enzymes