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化酶及其调节配体。 实验的重点将集中在来自厚壳属的酶 Argentea，包括磷酸烯醇丙酮酸的比较研究 羧化酶; coli和Z.梅斯 述代谢抑制剂 苹果酸和激活剂葡萄糖6-磷酸是主要的 效应器进行研究。 具体目标包括确定 哪些电离状态对这些效应器很重要， 金属离子在效应器结合中的作用，以及 效应子对于它们的调节活性是必不可少的。 苹果酸和葡萄糖6-磷酸之间的相互作用将是 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 多个抑制剂位点。 证据将从化学改性研究中获得， 参与效应子结合的氨基酸残基类型， 效应物在真正的变构位点结合， 存在不同类型的效应位点。 亲和标签将 这将被证明是有用的探针的发展， 这种酶的调节位点，以及细菌和 哺乳动物的酶与这两种酶的结合位点 代谢中间产物

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.