Graham M N, Mantle T J
Department of Biochemistry, Trinity College, Dublin 2, Ireland.
Biochem J. 1989 Jun 15;260(3):705-10. doi: 10.1042/bj2600705.
Enterobacter cloacae strain 5822 expresses low levels of a class C beta-lactamase which can be induced 100-fold by imipenem. Mutants that constitutively express high levels of beta-lactamase can be selected on aztreonam or cefotaxime. The beta-lactamase from one such mutant (5822M2) has been purified to homogeneity and compared on the basis of subunit Mr, pI, substrate specificity, inhibitor sensitivity and immunological cross-reactivity with the enzyme from strains P99, GN7471 and 208, which have been studied previously. The enzyme from strain 5822M2 is clearly related to these other forms and is of the A-type according to the criteria of Seeberg, Tolxdorff-Neutzling & Wiedemann [Antimicrob. Agents Chemother. (1983) 23, 918-925]. The enzyme from the wild-type strain (5822) is shown to be identical to that found in the depressed strain (5822M2), indicating that the mutation is in a regulatory gene. A detailed analysis of the kinetics of the enzyme from strain 5822M2 shows that all of the beta-lactams studied are substrates and that a mechanism involving the formation of an acyl-enzyme is probably applicable in every case. The substrates however can clearly be grouped into two classes, i.e. 'good' substrates with kcat. values of 80-1200 s-1 and 'poor' substrates/good inhibitors with kcat. values of 0.009-0.00007 s-1. The permeability barrier to aztreonam is 4-fold less in the derepressed strain when compared with the wild-type strain. This is associated with significant changes in the expression of outer membrane porins. The observed resistance in the derepressed mutant appears to be linked to the elevated levels of beta-lactamase (3000-fold) rather than to the modest changes in the permeability barrier.
阴沟肠杆菌5822菌株表达低水平的C类β-内酰胺酶，亚胺培南可使其诱导100倍。可在氨曲南或头孢噻肟上筛选组成型表达高水平β-内酰胺酶的突变体。来自一个这样的突变体（5822M2）的β-内酰胺酶已被纯化至同质，并根据亚基分子量、等电点、底物特异性、抑制剂敏感性以及与先前已研究的P99、GN7471和208菌株的酶的免疫交叉反应性进行了比较。根据Seeberg、Tolxdorff-Neutzling和Wiedemann [《抗菌药物与化疗》（1983年）23卷，918 - 925页]的标准，来自菌株5822M2的酶与这些其他形式明显相关，属于A型。野生型菌株（5822）的酶与在去阻遏菌株（5822M2）中发现的酶相同，表明该突变存在于一个调节基因中。对来自菌株5822M2的酶的动力学详细分析表明，所研究的所有β-内酰胺都是底物，并且涉及形成酰基酶的机制可能在每种情况下都适用。然而，底物显然可以分为两类，即催化常数（kcat）值为80 - 1200 s⁻¹的“良好”底物和催化常数（kcat）值为0.009 - 0.00007 s⁻¹的“不良”底物/良好抑制剂。与野生型菌株相比，去阻遏菌株对氨曲南的通透屏障低4倍。这与外膜孔蛋白表达的显著变化有关。在去阻遏突变体中观察到的耐药性似乎与β-内酰胺酶水平升高（3000倍）有关，而不是与通透屏障的适度变化有关。
