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首页> 外文期刊>Antimicrobial agents and chemotherapy. >Understanding the molecular determinants of substrate and inhibitor specificities in the carbapenemase KPC-2: Exploring the roles of Arg220 and Glu276
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Understanding the molecular determinants of substrate and inhibitor specificities in the carbapenemase KPC-2: Exploring the roles of Arg220 and Glu276

机译：了解碳青霉烯酶KPC-2中底物和抑制剂特异性的分子决定因素：探索Arg220和Glu276的作用

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β-Lactamases are important antibiotic resistance determinants expressed by bacteria. By studying the mechanistic properties of β-lactamases, we can identify opportunities to circumvent resistance through the design of novel inhibitors. Comparative amino acid sequence analysis of class A β-lactamases reveals that many enzymes possess a localized positively charged residue (e.g., R220, R244, or R276) that is critical for interactions with β-lactams and β-lactamase inhibitors. To better understand the contribution of these residues to the catalytic process, we explored the roles of R220 and E276 in KPC-2, a class A β-lactamase that inactivates carbapenems and β-lactamase inhibitors. Our study reveals that substitutions at R220 of KPC-2 selectively impact catalytic activity toward substrates (50% or greater reduction in k cat/K m). In addition, we find that residue 220 is central to the mechanism of β-lactamase inhibition/inactivation. Among the variants tested at Ambler position 220, the R220K enzyme is relatively "inhibitor susceptible" (K i of 14 ± 1 μM for clavulanic acid versus K i of 25 ± 2 μM for KPC-2). Specifically, the R220K enzyme is impaired in its ability to hydrolyze clavulanic acid compared to KPC-2. In contrast, the R220M substitution enzyme demonstrates increased K m values for β-lactamase inhibitors (100 μM for clavulanic acid versus 25 ± 3 μM for the wild type [WT]), which results in inhibitor resistance. Unlike other class A β-lactamases (i.e., SHV-1 and TEM-1), the amino acid present at residue 276 plays a structural rather than kinetic role with substrates or inhibitors. To rationalize these findings, we constructed molecular models of clavulanic acid docked into the active sites of KPC-2 and the "relatively" clavulanic acid-susceptible R220K variant. These models suggest that a major 3.5-? shift occurs of residue E276 in the R220K variant toward the active S70 site. We anticipate that this shift alters the shape of the active site and the positions of two key water molecules. Modeling also suggests that residue 276 may assist with the positioning of the substrate and inhibitor in the active site. These biochemical and molecular modeling insights bring us one step closer to understanding important structure-activity relationships that define the catalytic and inhibitor-resistant profile of KPC-2 and can assist the design of novel compounds.
机译：β-内酰胺酶是细菌表达的重要抗生素抗性决定簇。通过研究β-内酰胺酶的机械特性，我们可以通过设计新型抑制剂来发现规避耐药性的机会。 A类β-内酰胺酶的比较氨基酸序列分析表明，许多酶具有局部带正电的残基（例如，R220，R244或R276），这对于与β-内酰胺和β-内酰胺酶抑制剂的相互作用至关重要。为了更好地理解这些残基对催化过程的贡献，我们探索了R220和E276在KPC-2（一种使碳青霉烯和β-内酰胺酶抑制剂失活的A类β-内酰胺酶）中的作用。我们的研究表明，KPC-2在R220处的取代选择性地影响了对底物的催化活性（k cat / K m降低50％或更多）。另外，我们发现残基220是β-内酰胺酶抑制/失活机制的中心。在Ambler位置220进行测试的变体中，R220K酶相对“易受抑制剂”影响（棒酸的K i为14±1μM，而KPC-2的K i为25±2μM）。具体而言，与KPC-2相比，R220K酶水解棒酸的能力受损。相比之下，R220M替代酶显示β-内酰胺酶抑制剂的K m值增加（棒酸的> 100μM，野生型[WT]的为25±3μM），这导致了抑制剂的耐药性。与其他A类β-内酰胺酶（SHV-1和TEM-1）不同，残基276上的氨基酸在底物或抑制剂中起着结构而非动力学的作用。为了合理化这些发现，我们构建了克拉维酸的分子模型，该分子模型停靠在KPC-2和“相对”克拉维酸敏感性R220K变体的活性位点中。这些模型表明，主要3.5-？ R220K变体中的残基E276向着活跃的S70位点转移。我们预计，这种转变会改变活性位点的形状和两个关键水分子的位置。模拟还表明，残基276可能有助于底物和抑制剂在活性位点中的定位。这些生化和分子建模的见解使我们更进一步地了解了重要的结构-活性关系，这些关系定义了KPC-2的催化和抗药性，并有助于新化合物的设计。

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