ATP-DEPENDENT PROTEIN KINASES IN STREPTOCOCCI

在革兰氏阳性菌如化脓性链球菌中，S。lactis和S. 磷酸烯醇丙酮酸：糖磷酸转移酶系统（PTS） 运输和磷酸化多种糖包括葡萄糖， 果糖、甘露醇、乳糖和蔗糖。 大量证据表明， 糖的摄取和排出速率由ATP依赖的 蛋白质磷酸化 一种磷酸化丝氨酰的HPr激酶 HPr中的一个残基和HPr（ser）-P磷酸酶已在体外鉴定 并与监管有关。 为了表征这种新的细菌调节机制， 建议采用直接的生物化学方法。 另外还 计划： 1.分离毫克量的P-（ser）HPr和游离HPr， 比较它们与PTS的其它蛋白质的相互作用（酶I， 酶III（glc）、酶III（lac）和膜相关酶II 复合物）。 2.纯化HPr激酶和HPr（ser）P磷酸酶。 3.探索与生理相关的糖-P磷酸酶 其由P（ser）HPr或由蛋白激酶催化的 磷酸化事件。 4.确定PTS的酶II催化的机制 游离糖的流出。 5.检查其他细菌的蛋白激酶， 碳水化合物的运输和代谢。 这些研究应该导致一个具体的生物化学概念， 蛋白激酶介导的细菌转运调节， 将遗传技术应用于该问题。

In Gram-positive bacteria such as Streptococcus pyogenes, S. lactis and S. mutans, the phosphoenolpyruvate:sugar phosphotransferase system (PTS) transports and phosphorylates a variety of sugars including glucose, fructose, mannitol, lactose and sucrose. Extensive evidence suggests that the rates of sugar uptake and efflux are controlled by ATP-dependent protein phosphorylation. An HPr-kinase which phosphorylates a seryl residue in HPr, and an HPr(ser)-P phosphatase have been identified in vitro and implicated in regulation. In order to characterize this novel bacterial regulatory mechanism we propose to take a straightforward biochemical approach. Specifically we plan to: 1. Isolate milligram quantities of P-(ser)HPr and free HPr so that we can compare their interactions with the other proteins of the PTS (Enzyme I, Enzyme III(glc), Enzyme III(lac), and the membrane-associated Enzyme II complexes). 2. Purify both the HPr kinase and the HPr(ser)P phosphatase. 3. Attempt to discover the physiologically relevant sugar-P phosphatase which is activated either by P(ser)HPr or by a protein kinase-catalyzed phosphorylation event. 4. Determine the mechanism by which the Enzymes II of the PTS catalyze efflux of free sugar. 5. Examine other bacteria for protein kinases which may regulate carbohydrate transport and metabolism. These studies should lead to a concrete biochemical concept of protein-kinase mediated transport regulation in bacteria and allow application of genetic techniques to the problem.