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首页> 外文期刊>Biochemistry >Critical Role of Tryptophan 154 for the Activity and Stability of Class D β-Lactamases
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Critical Role of Tryptophan 154 for the Activity and Stability of Class D β-Lactamases

机译：色氨酸154对D类β-内酰胺酶活性和稳定性的关键作用

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The catalytic efficiency of the class D β-lactamase OXA-10 depends critically on an unusualncarboxylated lysine as the general base residue for both the enzyme acylation and deacylation steps ofncatalysis. Evidence is presented that the interaction between the indole group of Trp154 and the carboxylatednlysine is essential for the stability of the posttranslationally modified Lys70. Substitution of Trp154 by Gly,nAla, or Phe yielded noncarboxylated enzymes which displayed poor catalytic efficiencies and reduced stabilitynwhen compared to the wild-type OXA-10. The W154H mutant was partially carboxylated. In addition, thenmaximum values of kcat and kcat/KM were shifted toward pH 7, indicating that the carboxylation state ofnLys70 is dependent on the protonation level of the histidine.Acomparison of the three-dimensional structuresnof the different proteins also indicated that the Trp154 mutations did not modify the overall structures ofnOXA-10 but induced an increased flexibility of theΩ-loop in the active site. Finally, the deacylation-impairednW154A mutant was used to determine the structure of the acyl-enzyme complex with benzylpenicillin. Thesenresults indicate a role of the Lys70 carboxylation during the deacylation step and emphasize the importance ofnTrp154 for the ideal positioning of active site residues leading to an optimum activity
机译：D类β-内酰胺酶OXA-10的催化效率主要取决于不寻常的羧化赖氨酸作为催化酶的酰化和脱酰步骤的一般碱基残基。证据表明，Trp154的吲哚基团与羧化赖氨酸之间的相互作用对于翻译后修饰的Lys70的稳定性至关重要。与野生型OXA-10相比，Gly，nAla或Phe取代Trp154产生的非羧化酶显示出较差的催化效率并降低了稳定性。 W154H突变体被部分羧化。此外，然后kcat和kcat / KM的最大值移至pH 7，表明nLys70的羧化状态取决于组氨酸的质子化水平。对不同蛋白质的三维结构n的比较还表明Trp154突变确实不会改变nOXA-10的整体结构，但会引起活性位点Ω环的柔性增加。最后，使用脱酰-impairednW154A突变体来确定酰基青霉素与酰基酶复合物的结构。结果表明，Lys70羧化反应在脱酰步骤中发挥了作用，并强调了nTrp154对于活性位点残基的理想定位（导致最佳活性）的重要性。

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