Wong Brian J, Gerlt John A
Department of Biochemistry, University of Illinois, Urbana, Illinois 61801, USA.
J Am Chem Soc. 2003 Oct 8;125(40):12076-7. doi: 10.1021/ja037652i.
3-Hydroxyisobutyryl-CoA hydrolase (HICH), a member of the enoyl-CoA (crotonase) superfamily, catalyzes the hydrolysis of 3-hydroxyisobutyryl-CoA to 3-hydroxyisobutyrate. Like other members of the superfamily, the sequence of HICH contains conserved sequences for an oxyanion hole that stabilizes anionic intermediates. In contrast to most members of the superfamily, the reaction catalyzed by HICH does not proceed via formation of a thioester enolate anion; instead, evidence based on substrate deuterium isotope effects, the reactivity of substrate analogues that cannot form thioester enolate anions, single-turnover experiments in H218O, and the kinetic phenotypes of site-directed mutants provide evidence for a mechanism involving the formation of an anhydride intermediate involving Glu143 in the active site. In the reactions catalyzed by many members of the superfamily, homologues of Glu143 abstract the alpha proton of the thioester substrate to generate the thioester enolate anion intermediate. Presumably, one or more of the anionic tetrahedral intermediates on the HICH reaction coordinate are stabilized by the oxyanion hole. Thus, we conclude that the conserved oxyanion hole in this superfamily can be used to stabilize a variety of anionic intermediates.
3-羟基异丁酰辅酶A水解酶（HICH）是烯酰辅酶A（巴豆酸酶）超家族的成员之一，催化3-羟基异丁酰辅酶A水解为3-羟基异丁酸。与超家族的其他成员一样，HICH的序列包含用于稳定阴离子中间体的氧负离子洞的保守序列。与超家族的大多数成员不同，HICH催化的反应不是通过硫酯烯醇负离子的形成来进行的；相反，基于底物氘同位素效应、不能形成硫酯烯醇负离子的底物类似物的反应性、在H218O中的单周转实验以及定点突变体的动力学表型的证据，为一种涉及在活性位点形成涉及Glu143的酸酐中间体的机制提供了证据。在由超家族的许多成员催化的反应中，Glu143的同源物夺取硫酯底物的α质子以生成硫酯烯醇负离子中间体。据推测，HICH反应坐标上的一个或多个阴离子四面体中间体由氧负离子洞稳定。因此，我们得出结论，该超家族中保守的氧负离子洞可用于稳定多种阴离子中间体。
