Some differences in turnover kinetics of penams and cephems catalyzed by classes A and C ?-lactamases

S. Adediran, Rex Praff
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Abstract

The differences in the kinetic mechanism of catalysis of class A and class C ?-lactamases are shown by the manner by which these enzymes catalyze the hydrolyses of the penicillins and the cephalosporins. The hydrolysis of cephalosporins that are good substrates of the class C P99 ?-lactamase, where deacylation of an acyl-enzyme intermediate is rate-determining, has been shown to involve a free enzyme isoform (Adediran et al, 2021). We describe here how reconversion of this isoform to the native enzyme is accelerated by bases (e.g. imidazole) and salts (e. g. sodium chloride). The hydrolysis of penicillins by the P99 enzyme, where deacylation is also rate-determining, is not affected by imidazole and sodium chloride, a result that suggests that an enzyme isoform does not accumulate as an intermediate in turnover of this class of substrate. In support of these conclusions, solvent deuterium kinetic isotope effects on kcat values were changed by the presence of imidazole for turnover of cephalothin by the P99 enzyme but unaffected for benzylpenicillin turnover. The hydrolyses of cephalosporins and penicillins by the class A TEM-2 ?-lactamase were not affected by imidazole and sodium chloride and thus also may not involve an accumulating free enzyme isoform. Solvent deuterium kinetic isotope effects and proton inventories on the class A PC1 and P99 ?-lactamase-catalyzed hydrolyses of benzylpenicillin at saturating concentrations showed the deacylation transition states of these two classes of enzymes to be different with respect to proton motion.
A类内酰胺酶和C ?-内酰胺酶催化槟兰和头孢的转化动力学差异
A类内酰胺酶和C类内酰胺酶催化青霉素类和头孢菌素类水解的方式表明了它们催化动力学机制的差异。头孢菌素是C类P99 -内酰胺酶的良好底物,其中酰基酶中间体的去酰化是决定速率的,已被证明涉及一种游离酶异构体(Adediran等,2021)。我们在这里描述了如何通过碱(如咪唑)和盐(如氯化钠)加速这种同工异构体向天然酶的再转化。P99酶对青霉素的水解,其中去酰化也是决定速率的,不受咪唑和氯化钠的影响,这一结果表明酶的异构体不会在这类底物的转化中作为中间体积累。为了支持这些结论,溶剂氘动力学同位素对kcat值的影响在P99酶转化头孢菌素时被咪唑的存在所改变,但对青霉素的转化没有影响。甲类TEM-2 -内酰胺酶对头孢菌素和青霉素的水解不受咪唑和氯化钠的影响,因此也可能不涉及积累的游离酶异构体。饱和浓度下A类PC1和P99 -内酰胺酶催化青霉素水解的溶剂氘动力学同位素效应和质子库存量表明,这两类酶的去酰化过渡态在质子运动方面是不同的。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
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