Yip Vivian L Y, Thompson John, Withers Stephen G
Department of Chemistry, University of British Columbia, 2036 Main Mall, Vancouver, BC, Canada V6T 1Z1.
Biochemistry. 2007 Aug 28;46(34):9840-52. doi: 10.1021/bi700536p. Epub 2007 Aug 4.
GlvA, a 6-phospho-alpha-glucosidase from Bacillus subtilis assigned to glycoside hydrolase family 4, catalyzes the hydrolysis of maltose 6'-phosphate via a redox-elimination-addition mechanism requiring NAD+ as cofactor. In contrast to previous reports and consistent with the proposed mechanism, GlvA is only activated in the presence of the nicotinamide cofactor in its oxidized, and not the reduced NADH, form. Significantly, GlvA catalyzes the hydrolysis of both 6-phospho-alpha- and 6-phospho-beta-glucosides containing activated leaving groups such as p-nitrophenol and does so with retention and inversion, respectively, of anomeric configuration. Mechanistic details of the individual bond cleaving and forming steps were probed using a series of 6-phospho-alpha- and 6-phospho-beta-glucosides. Primary deuterium kinetic isotope effects (KIEs) were measured for both classes of substrates in which either the C2 or the C3 protons have been substituted with a deuterium, consistent with C-H bond cleavage at each center being partially rate-limiting. Kinetic parameters were also determined for 1-[2H]-substituted substrates, and depending on the substrates and the reaction conditions, the measurements of kcat and kcat/KM produced either no KIEs or inverse KIEs. In conjunction with results of Brønsted analyses with both aryl 6-phospho-alpha- and beta-glucosides, the kinetic data suggest that GlvA utilizes an E1cb mechanism analogous to that proposed for the Thermotoga maritima BglT, a 6-phospho-beta-glucosidase in glycoside hydrolase family 4 (Yip, V.L.Y et al. (2006) Biochemistry 45, 571-580). The pattern of isotope effects measured and the observation of very similar kcat values for all substrates, including unactivated and natural substrates, indicate that the oxidation and deprotonation steps are rate-limiting steps in essentially all cases. This mechanism permits the cleavage of both alpha- and beta-glycosides within the same active site motif and, for activated substrates that do not require acid catalysis for cleavage, within the same active site, yielding the product sugar-6-phosphate in the same anomeric form in the two cases.
GlvA是一种来自枯草芽孢杆菌的6-磷酸-α-葡萄糖苷酶，属于糖苷水解酶家族4，它通过一种需要NAD⁺作为辅因子的氧化还原消除-加成机制催化6'-磷酸麦芽糖的水解。与之前的报道相反，并且与所提出的机制一致，GlvA仅在烟酰胺辅因子处于氧化形式（而非还原的NADH形式）时被激活。值得注意的是，GlvA催化含有活化离去基团（如对硝基苯酚）的6-磷酸-α-和6-磷酸-β-葡萄糖苷的水解，并且分别以异头构型的保留和翻转方式进行。使用一系列6-磷酸-α-和6-磷酸-β-葡萄糖苷探究了各个键断裂和形成步骤的机制细节。对两类底物测量了一级氘动力学同位素效应（KIEs），其中C2或C3质子已被氘取代，这与每个中心的C-H键断裂在一定程度上是限速步骤一致。还测定了1-[2H]-取代底物的动力学参数，并且根据底物和反应条件，kcat和kcat/KM的测量结果要么没有KIEs，要么产生反KIEs。结合对芳基6-磷酸-α-和β-葡萄糖苷的布伦斯特分析结果，动力学数据表明GlvA利用了一种类似于为嗜热栖热菌BglT（糖苷水解酶家族4中的一种6-磷酸-β-葡萄糖苷酶）所提出的E1cb机制（Yip, V.L.Y等人（2006年）《生物化学》45, 571 - 580）。所测量的同位素效应模式以及对所有底物（包括未活化和天然底物）非常相似的kcat值的观察表明，氧化和去质子化步骤在基本上所有情况下都是限速步骤。这种机制允许在相同的活性位点基序内裂解α-和β-糖苷，并且对于不需要酸催化裂解的活化底物，在相同的活性位点内进行，在两种情况下都以相同的异头形式产生产物糖-6-磷酸。
