Yanchak M P, Taylor R A, Crowder M W
Department of Chemistry and Biochemistry, 112 Hughes Hall, Miami University, Oxford, Ohio 45056, USA.
Biochemistry. 2000 Sep 19;39(37):11330-9. doi: 10.1021/bi0010524.
In an effort to evaluate the roles of Lys184, Asn193, and Asp103 in the binding and catalysis of metallo-beta-lactamase CcrA from Bacteroides fragilis, site-directed mutants of CcrA were generated and characterized using metal analyses, CD spectroscopy, and kinetic studies. Three Lys184 mutants were generated where the lysine was replaced with alanine, leucine, and glutamate, and the analysis of these mutants indicates that Lys184 is not greatly involved in binding of cephalosporins to CcrA; however, this residue does have a significant role in binding of penicillin G. Three Asn193 mutants were generated where the asparagine was replaced with alanine, leucine, and aspartate, and these mutants exhibited <4-fold decrease in k(cat), suggesting that Asn193 does not play a large role in catalysis. However, stopped-flow visible kinetic studies showed that the Asn193 mutants exhibit a slower substrate decay rate and no change in the product formation rate as compared with wild-type CcrA. These results support the proposed role of Asn193 in interacting with and activating substrate during catalysis. Two Asp103 mutants were generated where the aspartate was replaced with serine and cysteine. The D103C and D103S mutants bind the same amount of Zn(II) as wild-type CcrA and exhibited a 10(2)-fold and 10(5)-fold decrease in activity, respectively. Results from solvent isotope, proton inventory, and rapid-scanning visible studies suggest that Asp103 plays a role in generating the enzyme intermediate but does not donate a proton to the enzyme intermediate during the rate-limiting step of the catalytic mechanism.
为了评估脆弱拟杆菌金属β-内酰胺酶CcrA中Lys184、Asn193和Asp103在结合和催化过程中的作用，构建了CcrA的定点突变体，并通过金属分析、圆二色光谱和动力学研究对其进行表征。构建了三个Lys184突变体，将赖氨酸分别替换为丙氨酸、亮氨酸和谷氨酸，对这些突变体的分析表明，Lys184在头孢菌素与CcrA的结合中作用不大；然而，该残基在青霉素G的结合中确实发挥着重要作用。构建了三个Asn193突变体，将天冬酰胺分别替换为丙氨酸、亮氨酸和天冬氨酸，这些突变体的催化常数（k(cat)）降低了不到4倍，表明Asn193在催化中作用不大。然而，停流可见动力学研究表明，与野生型CcrA相比，Asn193突变体的底物衰减速率较慢，产物形成速率没有变化。这些结果支持了Asn193在催化过程中与底物相互作用并激活底物的推测作用。构建了两个Asp103突变体，将天冬氨酸分别替换为丝氨酸和半胱氨酸。D103C和D103S突变体与野生型CcrA结合的锌离子量相同，活性分别降低了100倍和100000倍。溶剂同位素、质子总量和快速扫描可见研究的结果表明，Asp103在生成酶中间体中发挥作用，但在催化机制的限速步骤中不向酶中间体提供质子。
