SITE DIRECTED MUTAGENESIS OF ASPARTATE AMINO TRANSFERASE

这项研究的目的和具体目的是 天冬氨酸氨基转移酶作用机制的研究 (AATase)主要通过定点突变-和通过 引入半胱氨酸残基的化学精制 通过相同的技术实现活动站点。这些突变的酶将是 以圆二向色性和差异性为物理特征 伯克利的扫描量热法；通过结晶学 协作和协作中的核磁共振动力学和机械论 将在伯克利继续进行快速和 传统的稳态动力学、荧光光谱和 动力学同位素效应，视每个突变体的情况而定。一名突变者 已经被设计成一种阳离子氨基酸- 特异性转氨酶将在这方面得到进一步的开发 方向由合理选择的第二位点突变增加 结合部位的负电荷密度，并通过选择 用该质粒转化的精氨酸营养缺陷菌 编码突变的AATase。有可能有选择性地 压力会产生意想不到的第二位点突变。这个 这项工作与健康有关的方面源于一般作用 磷酸吡哆醛依赖酶在氨基酸代谢中的作用 这些酶存在先天的新陈代谢错误，例如 同型半胱氨酸尿症、酪氨酸血症和缬氨酸血症。生理学 相邻酶在新陈代谢中的重要作用 路径将通过扰乱接触区域进行调查 化学修饰和定点突变。 对络合物稳定性的观察将由 差示扫描量热法；化学性质的研究 通过荧光、圆二色谱和 通过确定第一种酶的产物是否被引导 直接到达第二个的活动站点。更广泛的 这一研究方面的含义适用于一般 调节中间代谢的现象。

The objectives and specific aims of this research are to investigate the mechanism of action of aspartate aminotransferase (AATase) primarily through site-directed mutagenesis-and by chemical elaboration of cysteine residues introduced into the active site by the same technique. These mutant enzymes will be characterized physically by circular dichroism and differential scanning calorimetry in Berkeley; by crystallography in collaboration and by NMR in collaboration Kinetic and mechanistic characterization will be continued in Berkeley with rapid and conventional steady-state kinetics, fluorescence spectroscopy, and kinetic isotope effects as appropriate to each mutant. One mutant that has already been engineered to be a cationic amino acid- specific aminotransferase will be further developed in this direction by a rationally selected second-site mutation to increase the negative-charge density at the binding site, and by selecting arginine auxotrophs which have been transformed with the plasmid coding for the mutant AATase. It is possible that selective pressure will produce unanticipated second-site mutants. The health-related aspects of this work derive from the general role of pyridoxal phosphate-dependent enzymes in amino acid metabolism and of these enzymes in inborn errors of metabolism, such as homocystinuria, tyrosinemia, and valinemia. The physiological significant of the association of adjacent enzymes in metabolic pathways will be investigated by perturbing the contact areas by chemical modification and by site-directed mutagenesis. Observations on the stability of the complexes will be made by differential scanning calorimetry; and on the chemical properties of the associated AATase by fluorescence, circular dichroism, and by determining whether the product of the first enzyme is channeled directly to the active site of the second. The broader implications of the aspect of the research apply to the general phenomenon of the regulation of intermediary metabolism.