羧酸盐
三氟甲基
部分
立体化学
化学
活动站点
基质(水族馆)
非竞争性抑制
螯合作用
恶臭假单胞菌
晶体结构
衬底模拟
水解酶
催化作用
酶
结晶学
有机化学
地质学
海洋学
烷基
作者
Mitesh Nagar,A.D. Lietzan,Martin St. Maurice,Stephen L. Bearne
出处
期刊:Biochemistry
[American Chemical Society]
日期:2014-02-10
卷期号:53 (7): 1169-1178
被引量:22
摘要
Mandelate racemase (MR) from Pseudomonas putida catalyzes the Mg2+-dependent 1,1-proton transfer that interconverts the enantiomers of mandelate. Because trifluorolactate is also a substrate of MR, we anticipated that replacing the phenyl rings of the competitive, substrate-product analogue inhibitor benzilate (Ki = 0.7 mM) with trifluoromethyl groups might furnish an inhibitor. Surprisingly, the substrate-product analogue 3,3,3-trifluoro-2-hydroxy-2-(trifluoromethyl)propanoate (TFHTP) was a potent competitive inhibitor [Ki = 27 ± 4 μM; cf. Km = 1.2 mM for both (R)-mandelate and (R)-trifluorolactate]. To understand the origins of this high binding affinity, we determined the X-ray crystal structure of the MR–TFHTP complex to 1.68 Å resolution. Rather than chelating the active site Mg2+ with its glycolate moiety, like other ground state analogues, TFHTP exhibited a novel binding mode with the two trifluoromethyl groups closely packed against the 20s loop and the carboxylate bridging the two active site Brønsted acid–base catalysts Lys 166 and His 297. Recognizing that positioning a carboxylate between the Brønsted acid–base catalysts could yield an inhibitor, we showed that tartronate was a competitive inhibitor of MR (Ki = 1.8 ± 0.1 mM). The X-ray crystal structure of the MR–tartronate complex (1.80 Å resolution) revealed that the glycolate moiety of tartronate chelated the Mg2+ and that the carboxylate bridged Lys 166 and His 297. Models of tartronate in monomers A and B of the crystal structure mimicked the binding orientations of (S)-mandelate and that anticipated for (R)-mandelate, respectively. For the latter monomer, the 20s loop appeared to be disordered, as it also did in the X-ray structure of the MR triple mutant (C92S/C264S/K166C) complexed with benzilate, which was determined to 1.89 Å resolution. These observations indicate that the 20s loop likely undergoes a significant conformational change upon binding (R)-mandelate. In general, our observations suggest that inhibitors of other enolase superfamily enzymes may be designed to capitalize on the recognition of the active site Brønsted acid–base catalysts as binding determinants.
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