REGULATION OF CARBOHYDRATE METABOLISM IN ORAL BACTERIA

口腔细菌中碳水化合物代谢的调节

• 批准号: 3963676
• 负责人: C L WITTENBERGER
• 金额: --
• 依托单位: NATIONAL INSTITUTE OF DENTAL & CRANIOFACIAL RESEARCH
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/3963676
• 关键词: Streptococcus salivarius; carbohydrate metabolism; dental caries; enzyme induction /repression; fatty acid biosynthesis; fructose biphosphatase; glutamate ammonia ligase; hydrogen peroxide; lactate dehydrogenases; microorganism immunology; molecular pathology; oral bacteria; oxidoreductase; proteolysis; pyruvate kinase; superoxides; tissue /cell culture; transferase

One aspect of this investigation involves attempts to delineate the biochemical reactions involved in the inactivation and proteolytic degradation of a cell-associated fructosyltransferase (FT) produced by Streptococcus salivarius. We are focusing on the FT inactivation step because this reaction appears to 'mark' the enzyme for subsequent proteolysis. In an in vitro system, it has been demonstrated that a partially purified NADH oxidase is responsible for catalyzing a copper-dependent oxidative inactivation of FT. The reaction products of the NADH oxidase are hydrogen peroxide and superoxide anion. A second NADH oxidase has also been isolated that produces only water and this enzyme is completely ineffective in the in vitro inactivation of FT. Both superoxide and hydrogen perioxide are required in addition to copper for FT inactivation. Our results suggest that FT inactivation by these two dioxygen reduction products may occur in a site-specific manner by a metal-catalyzed Haber-Weiss reaction. We suggest that FT first binds cupric ions at or near the catalytic site. The bound cupric ions could then be reduced by superoxide anions and subsequently reoxidized by hydrogen peroxide with the generation locally of hydroxyl radicals. Hydroxyl radicals are most likely involved in the inactivation, since neither hydrogen peroxide nor superoxide anions alone have any effect on FT activity. A second aspect of our current studies deals with the mechanism of activation of streptococcal lactate dehydrogenases (LDHs) by fructose 1,6-biophosphate (FBP). We showed previously that FBP mediates a conformational change in the enzyme which results in a marked increase in its affinity for substrate and coenzyme. We now find that FBP also affects the quaternary structure of these LDHs. A partially purified LDH from S. salivarius exists as a tetramer in the presence of FBP. When FBP was removed from the enzyme, it rapidly lost activity and was converted to a diamer. Addition of FBP back to the inactive diamer resulted in a restoration of activity that was accompanied by a return to the tetrameric form.

这项调查的一个方面涉及试图描绘 参与灭活和蛋白水解的生化反应 细胞相关果糖基转移酶（FT）的降解， 唾液链球菌。 我们专注于FT失活步骤 因为这种反应似乎为随后的酶“标记”， 蛋白水解 在体外系统中，已经证明， 部分纯化的NADH氧化酶负责催化 FT的铜依赖性氧化失活。 的反应产物 NADH氧化酶是过氧化氢和超氧阴离子。 第二个NADH 还分离出了仅产生水的氧化酶， 在FT的体外失活中完全无效。 都是超氧化物 和过氧化氢的需要，除了铜的FT 失活 我们的研究结果表明，FT失活这两个 分子氧还原产物可以以位点特异性的方式发生， 金属催化的Haber-Weiss反应 我们认为，FT首先结合 在催化位点处或附近的铜离子。 结合的铜离子可以 然后被超氧阴离子还原，随后被 过氧化氢，局部产生羟基自由基。 羟基自由基最有可能参与失活，因为 过氧化氢和超氧阴离子对FT均无影响 活动 我们目前研究的第二个方面涉及 果糖对链球菌乳酸脱氢酶的激活作用 1，6-生物磷酸盐（FBP）。 我们之前已经表明，FBP介导了一个 在酶的构象变化，导致显着增加， 其对底物和辅酶亲和力。 我们现在发现，FBP也影响 这些LDH的四级结构。 从S. 唾液肌在存在FBP时作为四聚体存在。 当FBP 从酶中除去后，它迅速失去活性，并转化为 钻石。 将FBP添加回非活性二聚体中， 恢复活动，伴随着返回到四聚体 form.