Carboxylation and Decarboxylation of Active Site Lys 84 Controls the Activity of OXA-24 尾-Lactamase of Acinetobacter baumannii: Raman Crystallographic and Solution Evidence

详细信息    查看全文

• 作者：Tao Che ; Robert A. Bonomo ; Sivaprakash Shanmugam ; Christopher R. Bethel ; Marianne Pusztai-Carey ; John D. Buynak ; Paul R. Carey
• 刊名：The Journal of the American Chemical Society
• 出版年：2012
• 出版时间：July 11, 2012
• 年：2012
• 卷：134
• 期：27
• 页码：11206-11215
• 全文大小：612K
• 年卷期：v.134,no.27(July 11, 2012)
• ISSN：1520-5126

文摘

The class D 尾-lactamases are characterized by the presence of a carboxylated lysine in the active site that participates in catalysis. Found in Acinetobacter baumannii, OXA-24 is a class D carbapenem hydrolyzing enzyme that exhibits resistance to most available 尾-lactamase inhibitors. In this study, the reaction between a 6-alkylidiene penam sulfone inhibitor, SA-1-204, in single crystals of OXA-24 is followed by Raman microscopy. Details of its reaction with SA-1-204 provide insight into the enzyme鈥檚 mode of action and help define the mechanism of inhibition. When the crystal is maintained in HEPES buffer, the reaction is fast, shorter than the time scale of the Raman experiment. However, when the crystal holding solution contains 28% PEG 2000, the reaction is slower and can be recorded by Raman microscopy in real time; the inhibitor鈥檚 Raman bands quickly disappear, transient features are seen due to an early intermediate, and, at approximately 2鈥?1 min, new bands appear that are assigned to the late intermediate species. At about 50 min, bands due to all intermediates are replaced by Raman signals of the unreacted inhibitor. The new population remains unchanged indicating (i) that the OXA-24 is no longer active and (ii) that the decarboxylation of Lys84 occurred during the first reaction cycle. Using absorbance spectroscopy, a one-cycle reaction could be carried out in aqueous solution producing inactive OXA-24 as assayed by the chromogenic substrate nitrocefin. However, activity could be restored by reacting aqueous OXA-24 with a large excess of NaHCO₃, which recarboxylates Lys84. In contrast, the addition of NaHCO₃ was not successful in reactivating OXA-24 in the crystalline state; this is ascribed to the inability to create a concentration of NaHCO₃ in large excess over the OXA-24 that is present in the crystal. The finding that inhibitor compounds can inactivate a class D enzyme by promoting decarboxylation of an active site lysine suggests a novel function that could be exploited in inhibitor design.