PKA OF CATALYTIC BASE IN ENOLASE

烯醇酶催化基的 PKA

• 批准号: 7420560
• 负责人: GEORGE H REED
• 金额: $ 0.01万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-04-20 至 2007-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7420560
• 关键词: carboxylate; enzymes; hydrogen ions; mutant; yeasts

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. High resolution structural data (Larsen et al., 1996) and functional assays of site-directed mutant forms of yeast enolase (Poyner et al., 1996) indicate that Lys 345 serves as the catalytic base in the first step of the catalytic cycle of the enzyme. Lys 345 is positioned near the C-2 proton of the substrate, 2-phosphoglycerate (2-PGA), and mutant forms of enolase in which Lys 345 is changed to another residue have catalytic activities reduced by between 4 and 5 orders of magnitude relative to the wild type enzyme. Moreover, these mutant enzymes fail to catalyze exchange of the C-2 proton in D2O (Poyner et al., 1996). The crystallographically determined structure (Larsen et al., 1996) indicates that acidification of this carbon acid is accomplished by coordination of the carboxylate group to both required magnesium ions and a H-bonding interaction of one carboxylate oxygen to the ammonium group of Lys 396. It is well established that the pKa values of functional groups of amino acid side chains can be perturbed significantly in enzymic complexes (Highbarger et. al., 1996; Mcintosh et al., 1996), and interpretations of pH rate profiles are frequently problematic (Kyte, 1995). Nevertheless, understanding the energetics of the ionization at C-2 of 2-PGA in catalysis by enolase requires knowledge of the pKa of the side chain of Lys 345. The aim of this project is to determine the pKa of a gamma-thialysine form of Lys 345 in yeast enolase and its complexes with substrates by direct titration using 15N or 13C NMR with isotopically labeled forms of thialysine.

该子项目是利用NIH/NCRR资助的中心赠款提供的资源的许多研究子项目之一。子弹和调查员（PI）可能已经从其他NIH来源获得了主要资金，因此可以在其他清晰的条目中代表。列出的机构适用于该中心，这不一定是调查员的机构。高分辨率的结构数据（Larsen等，1996）和对酵母烯醇酶的位置突变形式的功能测定（Poyner等，1996）表明，LYS 345在酶的催化循环的第一步中是催化基础。 LYS 345位于底物的C-2质子附近，2-磷酸甘油酸（2-PGA）和烯醇酶的突变形式，其中将LYS 345更改为另一个残基的烯醇酶，相对于野生型酶，将催化活性降低了4至5幅度。此外，这些突变酶无法催化D2O中C-2质子的交换（Poyner等，1996）。晶体学确定的结构（Larsen等，1996）表明，该碳酸的酸化是通过将羧酸盐组协调到所需的镁离子和一个羧酸氧气的H键相互作用来实现的（Highbarger等，1996; McIntosh等，1996），pH率曲线的解释经常有问题（Kyte，1995）。然而，了解烯醇酶在催化催化中C-2的电离在2-PGA的电离的能量学需要了解Lys 345侧链的PKA。该项目的目的是确定lys 345的PKA lys-thia-thia ly- ly-在酵母中的伊斯特氏酶345的lys 345及其与酵母烯醇酶的形式及其建筑物的形式，并使用15N或13N或13N，或者使用15n或13NC，硫透析。