Enzyme Structure-Function Relationships

提出了两种方法来研究柠檬酸合成酶(CS)和丝氨酸羧肽酶(CPD)这两种不同酶的底物专一性和催化机理。第一个将是对酶的结构进行X射线晶体分析，并比较相关或突变形式的酶的配体亲和力和催化活性。第二部分是通过理论计算研究静电场在这些机制中的作用。柠檬酸合成酶是少数几种能够直接形成碳键的酶之一。提出了部分反应的详细机理。构建了具有不同活性的CS定点突变体。它们的结构将通过结晶学技术、酶动力学和静电场计算进行分析，以研究选定的侧链的作用。丝氨酸羧肽酶是一种高度特异的蛋白水解酶，对多肽激素的成熟是必不可少的。它们与其他丝氨酸蛋白酶无关，但具有相同的活性部位群。对抑制剂结合的分析将确定底物专一性的基础。将对酵母CPD的选定突变体进行研究，以确定该反应不寻常的pH依赖性的基础，并测试其酶机制是否与其他丝氨酸蛋白酶的所建议的类似。

Two approaches are proposed to investigate substrate specificity and catalytic mechanisms of two different enzymes, citrate synthase (CS) and serine carboxypeptidase (CPD). The first will be an X-ray crystallographic analysis of the structures of the enzymes and to compare ligand affinities and catalytic activity of related or mutant forms of the enzymes. The second will be to investigate the role of electrostatic fields in the mechanisms by use of theoretical calculations. Citrate synthase is one of few enzymes capable of directly forming a carbon bond. A detailed mechanism for part of the reaction has been proposed. Site-directed mutants of CS with differing activity have been constructed. Their structures will be analyzed by crystallographic techniques, enzyme kinetics and electrostatic field calculations to study the roles of selected side chains. Serine carboxypeptidases are highly specific proteinases which are essential for the maturation of peptide hormones. They are unrelated to other serine proteinases, but have the same active site groups. Analysis of inhibitor binding will determine the basis for substrate specificity. Selected mutants of yeast CPD will be studied to determine the basis for the unusual pH dependence of the reaction and to test whether the enzymatic mechanism is similar to that proposed for other serine proteinases.