白色链霉菌Gβ-内酰胺酶的活性位点丝氨酸突变体
Active-site serine mutants of the Streptomyces albus G beta-lactamase.
作者信息
Jacob F, Joris B, Frère J M
机构信息
Centre d'lngénierie des Protéines, Université de Liège, Belgium.
出版信息
Biochem J. 1991 Aug 1;277 ( Pt 3)(Pt 3):647-52. doi: 10.1042/bj2770647.
Abstract
By using site-directed mutagenesis, the active-site serine residue of the Streptomyces albus G beta-lactamase was substituted by alanine and cysteine. Both mutant enzymes were produced in Streptomyces lividans and purified to homogeneity. The cysteine beta-lactamase exhibited a substrate-specificity profile distinct from that of the wild-type enzyme, and its kcat./Km values at pH 7 were never higher than 0.1% of that of the serine enzyme. Unlike the wild-type enzyme, the activity of the mutant increased at acidic pH values. Surprisingly, the alanine mutant exhibited a weak but specific activity for benzylpenicillin and ampicillin. In addition, a very small production of wild-type enzyme, probably due to mistranslation, was detected, but that activity could be selectively eliminated. Both mutant enzymes were nearly as thermostable as the wild-type.
摘要
通过定点诱变，将白色链霉菌Gβ-内酰胺酶的活性位点丝氨酸残基用丙氨酸和半胱氨酸取代。两种突变酶均在变铅青链霉菌中产生并纯化至同质。半胱氨酸β-内酰胺酶表现出与野生型酶不同的底物特异性谱，其在pH 7时的kcat./Km值从未高于丝氨酸酶的0.1%。与野生型酶不同，突变体的活性在酸性pH值下增加。令人惊讶的是，丙氨酸突变体对苄青霉素和氨苄青霉素表现出微弱但特异的活性。此外，检测到极少量野生型酶的产生，可能是由于错译，但该活性可以被选择性消除。两种突变酶的热稳定性几乎与野生型相同。
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