酶的天然结构变异作为研究机制的工具：以组织蛋白酶B和木瓜蛋白酶催化位点结构及特性的比较为例。牛脾脏和大鼠肝脏组织蛋白酶B与硫醇特异性双质子态探针（2,2'-二吡啶二硫化物）以及特异性合成底物（N-α-苄氧羰基-L-精氨酰-L-精氨酸2-萘酰胺）反应的pH依赖性动力学。
Natural structural variation in enzymes as a tool in the study of mechanism exemplified by a comparison of the catalytic-site structure and characteristics of cathepsin B and papain. pH-dependent kinetics of the reactions of cathepsin B from bovine spleen and from rat liver with a thiol-specific two-protonic-state probe (2,2'-dipyridyl disulphide) and with a specific synthetic substrate (N-alpha-benzyloxycarbonyl-L-arginyl-L-arginine 2-naphthylamide).
作者信息
Willenbrock F, Brocklehurst K
出版信息
Biochem J. 1984 Sep 15;222(3):805-14. doi: 10.1042/bj2220805.
Abstract
Cathepsin B (EC 3.4.22.1) from bovine spleen and the analogous enzyme from rat liver were investigated at 25 degrees C at I0.1 in acidic media by kinetic study of (a) the reactions of their catalytic-site thiol groups towards the two-protonic-state reactivity probe 2,2'-dipyridyl disulphide and (b) their catalysis of the hydrolysis of N-alpha-benzyloxycarbonyl-L-arginyl-L-arginine 2-naphthylamide. Reactivity-probe kinetics showed that nucleophilic character is generated in the sulphur atom of cathepsin B by protonic dissociation with pKa 3.4, presumably to form an S-/ImH+ ion-pair. Substrate-catalysis kinetics showed that ion-pair formation is not sufficient to generate catalytic competence in cathepsin B, because catalytic activity is not generated as the pH is raised across pKa 3.4 but rather as it is raised across pKa 5-6 (5.1 for kcat; 5.6 for kcat./Km for the bovine spleen enzyme and 5.8 for kcat./Km for the rat liver enzyme). The implications of these results and of known structural differences between the catalytic sites of the rat liver enzyme and papain (EC 3.4.22.2) for the mechanism of cysteine-proteinase-catalysed hydrolysis are discussed.
摘要
通过对（a）其催化位点硫醇基团与双质子态反应性探针2,2'-二吡啶二硫化物的反应以及（b）它们对N-α-苄氧羰基-L-精氨酰-L-精氨酸2-萘酰胺水解的催化作用进行动力学研究，在25℃、离子强度I0.1的酸性介质中对牛脾脏组织中的组织蛋白酶B（EC 3.4.22.1）和大鼠肝脏中的类似酶进行了研究。反应性探针动力学表明，组织蛋白酶B的硫原子通过pKa为3.4的质子解离产生亲核特性，推测形成S⁻/ImH⁺离子对。底物催化动力学表明，离子对的形成不足以使组织蛋白酶B产生催化活性，因为在pH值跨越pKa 3.4升高时不会产生催化活性，而是在pH值跨越pKa 5 - 6升高时产生催化活性（牛脾脏酶的kcat为5.1；kcat./Km为5.6；大鼠肝脏酶的kcat./Km为5.8）。讨论了这些结果以及大鼠肝脏酶和木瓜蛋白酶（EC 3.4.22.2）催化位点之间已知的结构差异对半胱氨酸蛋白酶催化水解机制的影响。
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本文引用的文献
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Computer programmes for processing enzyme kinetic data.用于处理酶动力学数据的计算机程序。
Nature. 1963 May 4;198:463-5. doi: 10.1038/198463a0.
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Human cathepsin B. Application of the substrate N-benzyloxycarbonyl-L-arginyl-L-arginine 2-naphthylamide to a study of the inhibition by leupeptin.人组织蛋白酶B。底物N-苄氧羰基-L-精氨酰-L-精氨酸2-萘酰胺在亮抑酶肽抑制作用研究中的应用。
Biochem J. 1980 Sep 1;189(3):447-53. doi: 10.1042/bj1890447.
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Half-time analysis of the integrated Michaelis equation. Simulation and use of the half-time plot and its direct linear variant in the analysis of some alpha-chymotrypsin, papain- and fumarase-catalysed reactions.米氏积分方程的半衰期分析。半衰期图及其直接线性变体在某些α-糜蛋白酶、木瓜蛋白酶和延胡索酸酶催化反应分析中的模拟与应用。
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Cathepsin B, Cathepsin H, and cathepsin L.组织蛋白酶B、组织蛋白酶H和组织蛋白酶L。
Methods Enzymol. 1981;80 Pt C:535-61. doi: 10.1016/s0076-6879(81)80043-2.
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Differences in the interaction of the catalytic groups of the active centres of actinidin and papain. Rapid purification of fully active actinidin by covalent chromatography and characterization of its active centre by use of two-protonic-state reactivity probes.猕猴桃蛋白酶和木瓜蛋白酶活性中心催化基团相互作用的差异。通过共价色谱法快速纯化完全活性的猕猴桃蛋白酶，并使用双质子态反应性探针表征其活性中心。
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Evidence for a two-state transition in papain that may have no close analogue in ficin. Differences in the disposition of cationic sites and hydrophobic binding areas in the active centres of papain and ficin.木瓜蛋白酶中可能在无花果蛋白酶中没有类似情况的双态转变的证据。木瓜蛋白酶和无花果蛋白酶活性中心中阳离子位点和疏水结合区域分布的差异。
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Current problems in mechanistic studies of serine and cysteine proteinases.丝氨酸蛋白酶和半胱氨酸蛋白酶机制研究中的当前问题。
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Biochem J. 1982 Jul 1;205(1):205-11. doi: 10.1042/bj2050205.
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Homology of amino acid sequences of rat liver cathepsins B and H with that of papain.大鼠肝脏组织蛋白酶B和H的氨基酸序列与木瓜蛋白酶氨基酸序列的同源性。
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