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首页> 外文期刊>The FEBS journal >Characterization of recombinant prolidase from Lactococcus lactis - changes in substrate specificity by metal cations, and allosteric behavior of the peptidase
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Characterization of recombinant prolidase from Lactococcus lactis - changes in substrate specificity by metal cations, and allosteric behavior of the peptidase

机译：乳酸乳球菌重组脯氨酸酶的表征-金属阳离子对底物特异性的改变以及肽酶的变构行为

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摘要

The Lactococcus lactis NRRL B-1821 prolidase gene was cloned and overexpressed in Escherichia coli. Under suboptimum growth conditions, recombinant soluble and active prolidase was produced; in contrast, inclusion bodies were formed under conditions preferred for cell growth. Recombinant prolidase retained more than half its full activity between 30 and 60 degrees C, and was completely inactivated after 30 min at 70 degrees C. CD analysis confirmed that prolidase was inactivated at 67 degrees C. The enzyme was active under weak alkali to weak acidic conditions, and showed maximum activity at pH 7.0. Although these characteristics are similar to those for other reported prolidases, this prolidase was distinctive for two kinetic characteristics. Firstly, different substrate specificity was observed for its two preferred metal cations, zinc and manganese: Leu-Pro was preferred with zinc, whereas Arg-Pro was preferred with manganese. Secondly, the enzyme showed an allosteric response to changes in substrate concentrations, with Hill constants of 1.53 for Leu-Pro and 1.57 for Arg-Pro. Molecular modeling of this prolidase suggests that these unique characteristics may be attributed to a loop structure near the active site.
机译：乳酸乳球菌NRRL B-1821脯氨酸酶基因被克隆并在大肠杆菌中过表达。在最适生长条件下，产生了重组的可溶性和活性脯氨酸蛋白酶;相反，包涵体在细胞生长优选的条件下形成。重组蛋白水解酶在30至60摄氏度之间保留了其全部活性的一半以上，并且在70摄氏度30分钟后被完全灭活。CD分析证实，蛋白酶在67摄氏度下被灭活。该酶在弱碱至弱酸性下均具有活性在pH 7.0下显示最大活性。尽管这些特性与其他报道的蛋白酶有关，但该蛋白酶具有两个动力学特性。首先，对于其两种优选的金属阳离子，锌和锰，观察到不同的底物特异性：Leu-Pro对于锌是优选的，而Arg-Pro对于锰是优选的。其次，该酶表现出对底物浓度变化的变构反应，Leu-Pro的Hill常数为1.53，Arg-Pro的Hill常数为1.57。该蛋白质酶的分子模型表明，这些独特的特征可能归因于活性位点附近的环结构。

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