Substitution of Arg-244 by Cys or Ser in SHV-1 and SHV-5 β-lactamases confers resistance to mechanism-based inhibitors and reduces catalytic efficiency of the enzymes
Substitution of Arg-244 by Cys or Ser in SHV-1 and SHV-5 β-lactamases confers resistance to mechanism-based inhibitors and reduces catalytic efficiency of the enzymes
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DOI：
10.1111/j.1574-6968.1998.tb12889.x
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发表时间：
1998-03-01
影响因子：
2.1
通讯作者：
Tzouvelekis, LS
中科院分区：
文献类型：
--
作者：
Giakkoupi, P;Tzelepi, E;Tzouvelekis, LS
通过基于PCR的位点特异性突变程序，将SHV-1和SHV-5 β-内酰胺酶中保守残基Arg-244替换为较小的不带电荷的氨基酸Cys和Ser。突变型β-内酰胺酶显示对克拉维他的敏感性降低，对他唑巴坦和舒巴坦的敏感性降低程度较小。如在比较MIC测定中所示，R244 C和R244 S酶保留残余青霉素酶活性，而它们对头孢菌素的活性急剧降低。除氨曲南外，相应的SHV-5突变体不能水解羟亚氨基-β-内酰胺。突变体β-内酰胺酶的催化活性受损主要是由于对β-内酰胺底物的亲和力降低。以上变异为更多的突变体。这些结果提供了关于Arg-244参与（a）β-内酰胺酶抑制剂灭活和（B）β-内酰胺在SHV酶活性位点的正确定位的模式的信息。(C)1998年，欧洲微生物学会联合会。出版社：Elsevier Science B. V.
The conserved residue Arg-244 was substituted by the smaller uncharged amino acids Cys and Ser in SHV-1 and SHV-5 beta-lactamases by a PCR-based site-specific mutagenesis procedure. The mutant beta-lactamases displayed decreased susceptibility to clavulanate and, to a lesser extent, to tazobactam and sulbactam. As shown in comparative MIC determinations, R244C and R244S enzymes retained a residual penicillinase activity while their activity towards cephalosporins was drastically diminished. The respective SHV-5 mutants were unable to hydrolyze oxyimino-beta-lactams except aztreonam. The impaired catalytic activity of the mutant beta-lactamases was mainly due to the lowering of affinity for beta-lactam substrates. The above alterations were more mutants. These results provide information on the mode of involvement of Arg-244 in (a) inactivation by beta-lactamase inhibitors and (b) the proper positioning of beta-lactams in the active site of SHV enzymes. (C) 1998 Federation of European Microbiological Societies. Published by Elsevier Science B.V.