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首页> 外文期刊>Biochemistry >Modeling Domino Effects in Enzymes: Molecular Basis of the Substrate Specificity of the Bacterial Metallo-beta-lactamases IMP-1 and IMP-6.
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Modeling Domino Effects in Enzymes: Molecular Basis of the Substrate Specificity of the Bacterial Metallo-beta-lactamases IMP-1 and IMP-6.

机译：酶中的Domino效应模拟：细菌金属β-内酰胺酶Imp-1和Imm-6的底物特异性的分子基。

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摘要

Metallo-beta-lactamases can hydrolyze a broad spectrum of beta-lactam antibiotics and thus confer resistance to bacteria. For the Pseudomonas aeruginosa enzyme IMP-1, several variants have been reported. IMP-6 and IMP-1 differ by a single residue (glycine and serine at position 196, respectively), but have significantly different substrate spectra; while the catalytic efficiency toward the two cephalosporins cephalothin and cefotaxime is similar for both variants, IMP-1 is up to 10-fold more efficient than IMP-6 toward cephaloridine and ceftazidime. Interestingly, this biochemical effect is caused by a residue remote from the active site. The substrate-specific impact of residue 196 was studied by molecular dynamics simulations using a cationic dummy atom approach for the zinc ions. Substrates were docked in an intermediate structure near the transition state to the binding site of IMP-1 and IMP-6. At a simulation temperature of 100 K, most complexes were stable during 1 ns of simulation time. However,at higher temperatures, some complexes became unstable and the substrate changed to a nonactive conformation. To model stability, six molecular dynamics simulations at 100 K were carried out for all enzyme-substrate complexes. Stable structures were further heated to 200 and 300 K. By counting stable structures, we derived a stability ranking score which correlated with experimentally determined catalytic efficiency. The use of a stability score as an indicator of catalytic efficiency of metalloenzymes is novel, and the study of substrates in a near-transition state intermediate structure is superior to the modeling of Michaelis complexes. The remote effect of residue 196 can be described by a domino effect: upon replacement of serine with glycine, a hole is created and a stabilizing interaction between Ser196 and Lys33 disappears, rendering the neighboring residues more flexible; this increased flexibility is then transferred to the active site.
机译：金属β-内酰胺酶可以水解广谱的β-内酰胺抗生素，从而赋予细菌的抗性。对于假单胞菌铜绿假单胞菌酶IMP-1，已经报道了几种变体。 IMP-6和IMP-1分别不同于单个残基（甘氨酸和196位的丝氨酸），但具有显着不同的基板光谱;虽然对两个变体的两个头孢菌素头孢菌素和头孢噻肟的催化效率相似，但是Imp-1比朝向Cephaloridine和头孢他啶的Imp-6更高的效率高达10倍。有趣的是，这种生化效果是由远离活跃部位的残留物引起的。通过使用锌离子的阳离子伪原子方法来研究残留物196的基质特异性影响。将底物靠近过渡状态的中间结构对接到IMP-1和IMP-6的结合位点。在100K的模拟温度下，在1NS的模拟时间内大多数复合物稳定。然而，在较高的温度下，一些配合物变得不稳定并且基板改变为非保持构象。为了模型稳定性，对所有酶底物复合物进行六种分子动力学模拟。通过计数稳定的结构，进一步加热至200和300k的稳定结构，我们衍生出稳定性排名分数，其与实验确定的催化效率相关。使用稳定性得分作为金属酶催化效率的指标是新颖的，并且在近过渡状态中间结构中的基材的研究优于迈克莱斯配合物的建模。残留物196的远程效果可以通过多米诺效应描述：用甘氨酸置换丝氨酸时，产生孔，Ser196和Lys33之间的稳定相互作用消失，使相邻的残留物更加柔韧;然后将这种增加的灵活性转移到活性位点。

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