EFFECT OF REGULATORY LIGANDS AND THEIR BINDING SITES ON PEP CARBOXYLASES

这项研究将探索分子间的相互作用 磷酸烯醇式丙酮酸羧基酶及其调节配体。 实验的重点将集中在Crassula的酶上 阿根廷，涉及磷酸烯醇式丙酮酸的比较研究 由E.Coli和Z.Mays产生的羧基酶。新陈代谢抑制剂 苹果酸和活化剂葡萄糖6-磷酸是主要的 效应器有待研究。具体目标包括确定 哪些电离态对这些效应器来说是重要的，作用 金属离子在效应器结合中的作用，以及 效应器对于它们的调节活动是必不可少的。 苹果酸和葡萄糖6-磷酸之间的相互作用将是 已评估。相互排斥结合的动力学证据 将使用各种抑制剂来解决以下可能性 多个抑制物部位。 证据将从化学修饰研究中获得 参与效应器结合的氨基酸残基的类型，是否 效应器结合在真正的变构位置，以及有多少 存在不同类型的效应器部位。亲和力标签将 应该被证明是有用的探头 这种酶的调节位点，以及细菌和 哺乳动物酶与这两种酶中的任何一种具有结合位点 代谢中间体。

This research will explore the molecular interactions of phosphoenolpyruvate carboxylase with its regulatory ligands. Experimental emphasis will be focused on the enzyme from Crassula argentea, with comparative studies involving phosphoenolpyruvate carboxylase from E. coli and Z. mays. The metabolic inhibitor malate and the activator glucose 6-phosphate are the principal effectors to be studied. The specific aims include determining which ionization states are important for these effectors, the role of metal ions in effector binding, and what substructures of the effectors are essential for their regulatory activity. Interactions between malate and glucose 6-phosphate will be evaluated. Kinetic evidence of mutually exclusive binding of various inhibitors will be used to address the possibility of multiple inhibitor sites. Evidence will be obtained from chemical modification studies on the types of amino acid residues involved in effector binding, whether the effectors are binding at true allosteric sites, and how many distinct types of effector sites are present. Affinity labels will be developed which should prove to be useful probes of the regulatory sites of this enzymes, as well as bacterial and mammalian enzymes with binding sites for either of these two metabolic intermediates.