TETHERED DOMAINS AS REGULATORY ELEMENTS

本建议旨在研究3- 磷酸甘油酸脱氢酶，一种酶， 从头丝氨酸生物合成。 丝氨酸是一种负变构抑制剂。 晶体结构表明调节位点位于一个结构域中 与其他两个结构域分开，核苷酸和底物结合 域. 假设是核苷酸的结构域运动和 必须存在底物结合域以进行催化。 丝氨酸在调节位点的结合阻止了这种结构域运动。 此外，丝氨酸在调节位点的结合增加了蛋白质的表达。 两个调控位点之间的相互作用。 初步实验 表明交联相邻的调节结构域导致 抑制，而定点诱变实验削弱了 亚基之间的相互作用防止丝氨酸的抑制。 这 建议使用各种荧光方法，并继续 晶体学的努力，以确定机制的变构 控制

This proposal is to study the regulatory mechanism of 3- phosphoglycerate dehydrogenase, an enzyme that is the committed step in de novo serine biosynthesis. Serine is a negative allosteric inhibitor. The crystal structure indicates that the regulatory site is in a domain separated from other two domains, the nucleotide and substrate binding domains. The hypothesis is that a domain motion of the nucleotide and substrate binding domains has to occur for catalysis to take place. Binding of serine at the regulatory site prevents this domain motion. Further, the binding of serine at the regulatory site increases the interaction between two regulatory sites. Preliminary experiments indicate that cross-linking the adjacent regulatory domains results in inhibition, whereas site directed mutagenesis experiments weakening the interaction between subunits prevents inhibition by serine. This proposal uses a variety of fluorescence methodologies and continued crystallographic efforts to determine the mechanism of allosteric control.