beta-Secondary and solvent deuterium kinetic isotope effects on beta-lactamase catalysis
beta-Secondary and solvent deuterium kinetic isotope effects on beta-lactamase catalysis
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DOI：
10.1021/bi952107i
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发表时间：
1996-03-19
期刊：
影响因子：
2.9
通讯作者：
Pratt, RF
中科院分区：
文献类型：
--
作者：
Adediran, SA;Deraniyagala, SA;Pratt, RF
β-二级和溶剂氘动力学同位素效应已被确定为稳态动力学参数V/K和V的周转缩肽底物，间-[[（苯乙酰基）甘氨酰]氧基]苯甲酸，和β-内酰胺底物，青霉烷酸，由三个典型的A类β-内酰胺酶和C类β-内酰胺酶。这些底物的碱性水解的同位素效应已被用作参考框架。在确定β-二级同位素效应的基板的过渡态构象的效果已被明确考虑。逆β-二级同位素效应的V/K和V的A类酶与两种底物表明过渡状态的易裂键的羰基已成为四面体，因此反映了典型的酰基转移过渡状态。溶剂同位素效应表明，酶的脱酰作用（如金黄色葡萄球菌PC1 β-内酰胺酶的V所示）可能是一种经典的一般碱催化水解，但TEM β-内酰胺酶和蜡状芽孢杆菌β-内酰胺酶I的酶酰化过渡态（如V/K所示）中几乎没有质子运动。这些结果提供了动力学支持的推测，A类β-内酰胺酶采用不对称双位移机制的结构的理由，同位素效应的V/K的C类β-内酰胺酶的肠杆菌Clobacillus P99建议的酰基转移过渡态的青霉素，虽然，作为A类酶，没有显着的质子运动。另一方面，缩肽的V/K过渡态似乎不涉及共价化学。表明这一结论的是测得的β-次级同位素效应1.002 +/-0.012和逆溶剂同位素效应。这些结果提供了β-内酰胺酶对β-内酰胺和缩肽的周转动力学之间显著差异的实例。两种底物与P99 β-内酰胺酶的V过渡态可能涉及酰基转移（脱酰），其中酰基酶的构象受到严格限制。PC 1和P99 β-内酰胺酶的酰基酶的构象与一般碱催化剂在其活性位点的（不同）配置相关。
beta-Secondary and solvent deuterium kinetic isotope effects have been determined for the steady-state kinetic parameters V/K and V for turnover of a depsipeptide substrate, m-[[(phenylacetyl)glycyl]oxy]benzoic acid, and of a beta-lactam substrate, penicillanic acid, by three typical class A beta-lactamases and a class C beta-lactamase. The isotope effects on alkaline hydrolysis of these substrates have been used as a frame of reference. The effect of the transition state conformation of the substrates in determining the beta-secondary isotope effects has been explicitly considered. The inverse beta-secondary isotope effects on both V/K and V for the class A enzymes with both substrates indicate transition states where the carbonyl group of the scissile bond has become tetrahedral and therefore reflect typical acyl-transfer transition states. The solvent isotope effects indicate that enzyme deacylation (as reflected in V for the Staphylococcus aureus PC1 beta-lactamase) may be a classical general-base-catalyzed hydrolysis but that there is little proton motion in the enzyme acylation transition state (as revealed by V/K) for the TEM beta-lactamase and Bacillus cereus beta-lactamase I. These results provide kinetic support for the conjecture made on structural grounds that class A beta-lactamases employ an asymmetric double-displacement mechanism, The isotope effects on V/K for the class C beta-lactamase of Enterobacter cloacae P99 suggest an acyl-transfer transition state for the penicillin, although, as for the class A enzymes, without significant proton motion. On the other hand, the V/K transition state for depsipeptide does not seem to involve covalent chemistry. Suggestive of this conclusion are the measured beta-secondary isotope effect of 1.002 +/- 0.012 and the inverse solvent isotope effect. These results provide an example of a significant difference between the kinetics of turnover of a beta-lactam and a depsipeptide by a beta-lactamase. The V transition state for both substrates with the P99 beta-lactamase probably involves acyl-transfer (deacylation) where the conformation of the acyl-enzyme is closely restricted. The conformations of acyl-enzymes of the PC1 and P99 beta-lactamases correlate to the (different) dispositions of general base catalysts at their active sites.