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首页> 外文期刊>Biochemistry >ARAZOFORMYL DIPEPTIDE SUBSTRATES FOR THERMOLYSIN - CONFIRMATION OF A REVERSE PROTONATION CATALYTIC MECHANISM
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ARAZOFORMYL DIPEPTIDE SUBSTRATES FOR THERMOLYSIN - CONFIRMATION OF A REVERSE PROTONATION CATALYTIC MECHANISM

机译：芳甲酰二肽基质用于热溶菌素的确证-逆质子化催化机理。

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摘要

Cleavage by thermolysin of N-(4-methoxyphenylazoformyl)-L-leucyl-L-leucine plus some congeneric peptides provides a highly sensitive new kinetic assay for proteolytic activity. The pH dependence of Michaelis-Menten parameters k(cat) and K-m establishes kinetically a reverse protonation catalytic mechanism for this metalloprotease [Mock, W. L., & Aksamawati, M. (1994) Biochem. J. 302, 57-68]. An acidified water molecule (pK(a) of 5, seen in K-m) becomes displaced by substrate carboxamide from the hypercationic Zn2+ of the enzyme, yielding potent Lewis acid activation of the peptide linkage for subsequent hydrolysis. Conversion to product is induced by the side chain of enzymic residue His 231 (pK(a) of 8, seen in k(cat)), which provides general base catalysis for addition of H2O to the zinc-activated scissile carboxamide of the bound substrate. A previously described ''superactivation'' through chemical modification of the enzyme with acetylphenylalanyl-N-hydroxysuccinimide is nonexistent in the case of the new substrates, which indicates that their binding to thermolysin is largely productive, unlike normal peptides. Correct assignment of kinetically observed pK(a) values to active site residues, along with recognition of a predominantly nonproductive binding mode for ordinary substrates and thermolysin, forces reinterpretation of previous mechanistic formulations for the enzyme.
机译：N-（4-甲氧基苯基偶氮甲酰基）-L-亮氨酰-L-亮氨酸加上一些同类肽的热解酶裂解提供了高度敏感的蛋白水解活性新动力学测定。 Michaelis-Menten参数k（cat）和K-m的pH依赖性在动力学上建立了这种金属蛋白酶的反质子化催化机制[Mock，W. L.，＆Aksamawati，M.（1994）Biochem。 J. 302，57-68]。酸化的水分子（在K-m中见pK（a）为5，在K-m中可见）被底物羧酰胺从酶的高阳离子性Zn2 +置换而来，产生强力的路易斯酸活化肽键，以进行后续水解。酶残基His 231（pK（a）为8，在k（cat）中可见）的侧链可诱导产物转化，这为将H2O加至结合的底物的锌活化易裂羧酰胺上提供了一般的碱催化作用。 。在新底物的情况下，先前描述的通过用乙酰苯丙氨酰基-N-羟基琥珀酰亚胺化学修饰酶的“超活化”是不存在的，这表明它们与正常肽相比，与嗜热菌素的结合在很大程度上是有效的。动力学观察到的pK（a）值正确分配给活性位点残基，再加上对普通底物和嗜热菌蛋白酶的主要非生产性结合模式的认识，迫使人们重新解释了该酶以前的机械配方。

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