Regulation Of Sugar Transport And Metabolism In Oral Bacteria

GlvA是一种来自枯草芽孢杆菌的6-磷酸-α-葡糖苷酶，属于糖苷水解酶家族4。我们先前假设这种不寻常的酶通过需要NAD+作为辅因子的氧化还原-消除-加成机制催化麦芽糖6-磷酸的水解。在过去的一年里，我们的研究提供了物理化学和动力学证据来支持假设的假设。与以前的报道相反，并且与提出的机制一致，GlvA仅在烟酰胺辅因子以其氧化形式存在时被激活，而不是还原的NADH形式。值得注意的是，GlvA催化含有活化的离去基团如对硝基苯酚的6-磷酸-α-和6-磷酸-β-葡糖苷的水解，并且分别以端基异构体构型的保留和转化来进行。使用一系列6-磷酸-α-和6-磷酸-β-葡糖苷探测各个键断裂和形成步骤的机制细节。对于其中C2或C3质子已被氘原子取代的两类底物测量初级氘动力学同位素效应（KIE），这与每个中心处的C-H键裂解部分限速一致。动力学参数也被确定为1- 2 H-取代的基板，并根据基板和反应条件，kcat和kcat/KM的测量产生没有KIE或逆KIE。结合使用芳基6-磷酸-α-和β-葡糖苷的Bronsted分析的结果，动力学数据表明GlvA利用类似于针对海栖热袍菌BglT提出的E1 cb机制，海栖热袍菌BglT是一种6-磷酸-β-葡糖苷酶，其也包括在糖基水解酶超家族的家族4中（Yip，V.L.Y等人（2006）Biochemistry 45，571-580）。测量的同位素效应的模式，并观察到非常相似的kcat值的所有基板，包括未活化和天然基板，表明氧化和去质子化步骤是限速步骤，在基本上所有的情况下。这种催化机制允许α-和β-糖苷在相同的活性位点基序内裂解，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裂解。值得注意的是，糖-6-磷酸产物（葡萄糖-6P）在两种情况下具有相同的异头（α）形式。我们的研究结果的摘要最近发表在同行评审的期刊《生物化学》上。

GlvA, a 6-phospho-alpha-glucosidase from Bacillus subtilis, is assigned to glycoside hydrolase family 4. We have previously hypothesized that this unusual enzyme catalyzes the hydrolysis of maltose 6-phosphate via a redox-elimination-addition mechanism requiring NAD+ as cofactor. In the past year our studies have provided physico-chemical, and kinetic evidence to support the postulated hypothesis. 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 atom, 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 Bronsted 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 that is also included in family 4 of the glycosyl hydrolase superfamily (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 catalytic 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. Remarkably, the sugar-6-phosphate product (glucose-6P), has the same anomeric (alpha) form in the two cases. A summary of our findings has recently appeared in the peer-reviewed journal, Biochemistry.