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首页> 外文期刊>Biochemistry >INCREASE OF THE P-1 LYS/LEU SUBSTRATE PREFERENCE OF CARBOXYPEPTIDASE Y BY RATIONAL DESIGN BASED ON KNOWN PRIMARY AND TERTIARY STRUCTURES OF SERINE CARBOXYPEPTIDASES
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INCREASE OF THE P-1 LYS/LEU SUBSTRATE PREFERENCE OF CARBOXYPEPTIDASE Y BY RATIONAL DESIGN BASED ON KNOWN PRIMARY AND TERTIARY STRUCTURES OF SERINE CARBOXYPEPTIDASES

机译：基于已知的丝氨酸羧肽酶的初级和三级结构的理性设计提高羧肽酶Y的P-1 LYS / LEU碱基偏好

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摘要

The P-1 substrate preference of serine carboxypeptidases, as expressed by the Lys/Leu ratio, differs by up to 10(5)-fold, Predictions of the major determinants of this preference are made by correlating primary and tertiary structures to substrate preferences, In carboxypeptidase Y from yeast it is predicted that Trp312 constitutes such a determinant, reducing the P-1 Lys/Leu substrate preference of this enzyme. The predictions are tested by the construction and kinetic characterization of ten mutant enzymes of carboxypeptidase Y. All of these enzymes exhibit changes in their Pi substrate preference. Generally, small decreases in activity (k(cat)/K-m) are observed with substrates containing uncharged P-1 side chains. With substrates containing acidic P-1 side chains, i.e., FA-Glu-Ala-OH, the activity generally increases slightly, 7-fold in the case of W312K. The most dramatic effects of the Trp312 substitutions are observed with substrates containing basic P-1 side chains, i.e., k(cat)/K-m for the hydrolysis of FA-Lys-Ala-OH with W312E has increased 1150-fold, exclusively as a result of increased k(cat) values. Similar results have previously been obtained by mutational substitution at position 178 of carboxypeptidase Y. The construction and kinetic characterization of position 178+312 double mutants demonstrate that the kinetic effects of substitutions at these two positions are not additive. The P-1 Lys/Leu substrate preference of one double mutant, L178D+W312D, has changed 380000-fold as compared to the wild type enzyme, and the overall P-1 substrate preference of this enzyme closely resembles that of carboxypeptidase WII from wheat.
机译：丝氨酸羧肽酶的P-1底物偏好性（以Lys / Leu比表示）相差高达10（5）倍。通过将一级和三级结构与底物偏好性相关联，可以预测这种偏好性的主要决定因素，在来自酵母的羧肽酶Y中，预计Trp312构成了这种决定簇，从而降低了该酶的P-1 Lys / Leu底物偏好。通过十种羧肽酶Y突变酶的构建和动力学表征来检验这些预测。所有这些酶在Pi底物偏好方面均表现出变化。通常，在含有不带电的P-1侧链的底物上观察到活性的小幅下降（k（cat）/ K-m）。对于含有酸性P-1侧链（即FA-Glu-Ala-OH）的底物，活性通常略有增加，在W312K情况下为7倍。用含有碱性P-1侧链的底物，即用W312E水解FA-Lys-Ala-OH的k（cat）/ Km增加了1150倍，观察到了Trp312取代的最显着作用。 k（cat）值增加的结果。以前通过在羧肽酶Y的178位进行突变取代已经获得了相似的结果。178+ 312位双突变体的构建和动力学表征表明，在这两个位置上取代的动力学效应不是累加的。与野生型酶相比，一个双突变体L178D + W312D的P-1 Lys / Leu底物偏好已改变了380000倍，该酶的总体P-1底物偏好与小麦的羧肽酶WII相似。

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