The Different Inhibition Mechanisms of OXA-1 and OXA-24 β-Lactamases Are Determined by the Stability of Active Site Carboxylated Lysine
The Different Inhibition Mechanisms of OXA-1 and OXA-24 β-Lactamases Are Determined by the Stability of Active Site Carboxylated Lysine
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DOI：
10.1074/jbc.m113.533562
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发表时间：
2014-02-28
影响因子：
4.8
通讯作者：
Carey, Paul R.
中科院分区：
文献类型：
--
作者：
Che, Tao;Bethel, Christopher R.;Carey, Paul R.
背景：OXA-1和OXA-24是对临床常用抑制剂有抵抗力的D -内酰胺酶.结果如下：光谱方法和动力学测量表明，候选青霉烯类药物是OXA-1的良好抑制剂，但被OXA-24迅速水解。结论：OXA-24中的活性位点水有助于Lys-84的可逆羧化，使许多反应循环成为可能。重要性：了解D -内酰胺酶的作用机制对药物开发至关重要，D -内酰胺酶的催化效率关键取决于一种不常见的羧化赖氨酸作为酶的酰化和脱酰化步骤的一般碱基残基。对D -内酰胺酶OXA-1和OXA-24的微生物学和生物化学研究表明，当与两种6-亚甲基青霉烯类（青霉烯1和青霉烯3）反应时，这两种酶的行为不同：青霉烯类是OXA-1的良好抑制剂，但更像OXA-24的底物。紫外差光谱和拉曼光谱分析表明，它们的反应机理不同。青霉烯类在OXA-1中形成一种不寻常的中间体，即1，4-硫氮杂卓衍生物，并进行脱酰反应，然后使Lys-70脱羧，使OXA-1失去活性。这种失活不能通过加入100 mM NaHCO 3来逆转。在OXA-24中，在温和条件下（酶：抑制剂= 1：4），仅检测到水解产物，酶保持活性。然而，在苛刻条件下（酶：抑制剂= 1：2000），OXA-24通过Lys-84的脱羧作用被抑制;然而，该酶可以通过加入100 mM NaHCO 3重新活化。我们的结论是，OXA-24不仅脱羧困难，但也容易再羧;相比之下，OXA-1脱羧容易，但再羧困难。活性位点的结构分析表明，晶体水分子可能在OXA-24的羧化中起重要作用（在OXA-1中未发现类似的水分子），支持OXA-24活性位点中的水分子可以显著降低羧化的能垒的建议。
Background: OXA-1 and OXA-24 are class D -lactamases that resist clinically used inhibitors. Results: Spectroscopic methods and kinetic measurements show that penem drug candidates are good inhibitors of OXA-1 but are rapidly hydrolyzed by OXA-24. Conclusion: An active site water in OXA-24 aids the reversible carboxylation of Lys-84, enabling many reaction cycles. Significance: Understanding the mechanism of class D -lactamases is vital for drug development.The catalytic efficiency of class D -lactamases depends critically on an unusual carboxylated lysine as the general base residue for both the acylation and deacylation steps of the enzyme. Microbiological and biochemical studies on the class D -lactamases OXA-1 and OXA-24 showed that the two enzymes behave differently when reacting with two 6-methylidene penems (penem 1 and penem 3): the penems are good inhibitors of OXA-1 but act more like substrates for OXA-24. UV difference and Raman spectroscopy revealed that the respective reaction mechanisms are different. The penems form an unusual intermediate, a 1,4-thiazepine derivative in OXA-1, and undergo deacylation followed by the decarboxylation of Lys-70, rendering OXA-1 inactive. This inactivation could not be reversed by the addition of 100 mm NaHCO3. In OXA-24, under mild conditions (enzyme:inhibitor = 1:4), only hydrolyzed products were detected, and the enzyme remained active. However, under harsh conditions (enzyme:inhibitor = 1:2000), OXA-24 was inhibited via decarboxylation of Lys-84; however, the enzyme could be reactivated by the addition of 100 mm NaHCO3. We conclude that OXA-24 not only decarboxylates with difficulty but also recarboxylates with ease; in contrast, OXA-1 decarboxylates easily but recarboxylates with difficulty. Structural analysis of the active site indicates that a crystallographic water molecule may play an important role in carboxylation in OXA-24 (an analogous water molecule is not found in OXA-1), supporting the suggestion that a water molecule in the active site of OXA-24 can lower the energy barrier for carboxylation significantly.