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首页> 外文期刊>Biochemistry >Insight into the catlaysis iof hydrolysis of four newly synthesized substrates by papain:A proton inventory study
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Insight into the catlaysis iof hydrolysis of four newly synthesized substrates by papain:A proton inventory study

机译：木瓜蛋白酶水解四种新合成底物的催化作用的质子清单研究

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We synthesized the following four new peptide substrates, Suc—Phe—Leu—pNA, Suc—Phe— Leu—NMec, Suc—Phe—Leu—ONPh, and Pht—Phe-—Leu—pNA, and we applied the proton inventory method to their hydrolysis by papain. Useful relationships between the rate constants of the catalytic reaction have been established and contributed to the elucidation of the hydrolytic mechanism of papain. For all amide substrates, the parameter Ks and the rate constants k1, k—1, and k2 were estimated. Moreover. it was found that kcat/Km = k1 for all four substrates, while two exchangeable hydrogenic sites, one in the ground state and another in the transition state, generate an inverse isotope effect during the reaction governed by this parameter. The proton inventories of both k2 and k3 are essentially linear, whatever the acyl moiety and/or the leaving group of the substrate. The proton inventories of K5 are also essentiall~ linear for all amide substrates, while the observed large isotope effect of about 3 to 9 originates from a single hydrogenic site in the product state. This latter, in agreement to both the small transition state J fractionation factors found for kcat/Km (or k1) and the unit ground—state fractionation factors found forargues for the formation of a tetrahedral adduct during the reaction governed by the k1 parameter.1 Furthermore, papain acts as a one-proton catalyst during acylation or deacylation, both of which proce through similar concerted reaction pathways, where a nucleophilic attack is accompanied by the movemern of one proton.
机译：我们合成了以下四种新的肽底物：Suc-Phe-Leu-pNA，Suc-Phe-Leu-NMec，Suc-Phe-Leu-ONPh和Pht-Phe--Leu-pNA，并应用了质子清点方法被木瓜蛋白酶水解。已经建立了催化反应速率常数之间的有用关系，并有助于阐明木瓜蛋白酶的水解机理。对于所有酰胺底物，估计参数Ks以及速率常数k1，k-1和k2。此外。发现对于所有四种底物，kcat / Km = k1，而在该参数控制的反应过程中，两个可交换的氢位点（在基态下，另一个在过渡态下）产生反同位素效应。无论底物的酰基部分和/或离去基团如何，k2和k3的质子清单基本上都是线性的。对于所有酰胺底物，K5的质子清单也是基本线性的，而观察到的约3至9的大同位素效应则来自产物状态下的单个氢位。后者与针对kcat / Km（或k1）发现的较小过渡态J分馏因子和发现由k1参数控制的反应期间形成四面体加合物的单位基态分离因子一致。此外，木瓜蛋白酶在酰化或脱酰化过程中充当单质子催化剂，两者都通过相似的协同反应途径进行，亲核攻击伴随着一个质子的运动。

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