Tang Wei, Li Huiying, Doud Emma H, Chen Yunqiu, Choing Stephanie, Plaza Carla, Kelleher Neil L, Poulos Thomas L, Silverman Richard B
†Department of Chemistry, Department of Molecular Biosciences, Chemistry of Life Processes Institute, and Center for Molecular Innovation and Drug Discovery, Northwestern University, 2145 Sheridan Road, Evanston, Illinois 60208-3113, United States.
‡Departments of Molecular Biology and Biochemistry, Chemistry, and Pharmaceutical Sciences, University of California, Irvine, California 92697-3900, United States.
J Am Chem Soc. 2015 May 13;137(18):5980-9. doi: 10.1021/jacs.5b01202. Epub 2015 May 5.
Nitric oxide synthase (NOS) catalyzes the conversion of l-arginine to l-citrulline and the second messenger nitric oxide. Three mechanistic pathways are proposed for the inactivation of neuronal NOS (nNOS) by (S)-2-amino-5-(2-(methylthio)acetimidamido)pentanoic acid (1): sulfide oxidation, oxidative dethiolation, and oxidative demethylation. Four possible intermediates were synthesized. All compounds were assayed with nNOS, their IC50, KI, and kinact values were obtained, and their crystal structures were determined. The identification and characterization of the products formed during inactivation provide evidence for the details of the inactivation mechanism. On the basis of these studies, the most probable mechanism for the inactivation of nNOS involves oxidative demethylation with the resulting thiol coordinating to the cofactor heme iron. Although nNOS is a heme-containing enzyme, this is the first example of a NOS that catalyzes an S-demethylation reaction; the novel mechanism of inactivation described here could be applied to the design of inactivators of other heme-dependent enzymes.
一氧化氮合酶（NOS）催化L-精氨酸转化为L-瓜氨酸和第二信使一氧化氮。有人提出了三种机制途径来解释（S）-2-氨基-5-（2-（甲硫基）乙酰亚氨基）戊酸（1）使神经元型一氧化氮合酶（nNOS）失活的过程：硫化物氧化、氧化脱巯基和氧化脱甲基。合成了四种可能的中间体。所有化合物都用nNOS进行了测定，得到了它们的半数抑制浓度（IC50）、抑制常数（KI）和失活速率常数（kinact）值，并确定了它们的晶体结构。对失活过程中形成的产物的鉴定和表征为失活机制的细节提供了证据。基于这些研究，nNOS失活最可能的机制涉及氧化脱甲基，生成的硫醇与辅因子血红素铁配位。尽管nNOS是一种含血红素的酶，但这是一氧化氮合酶催化S-脱甲基反应的首个例子；这里描述的新型失活机制可应用于设计其他血红素依赖性酶的失活剂。
