STRUCTURE AND FUNCTION OF ALLYLIC REARRANGEMENT ENZYMES

烯丙基重排酶的结构和功能

• 批准号: 3289953
• 负责人: John M. Schwab
• 金额: $ 18.89万
• 依托单位: PURDUE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1985
• 资助国家: 美国
• 起止时间: 1985-08-01 至 1994-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3289953
• 关键词: X ray crystallography; adduct; allyl compound; chemical hydration; drug design /synthesis /production; enzyme induction /repression; enzyme model; enzyme structure; hydrolase; molecular rearrangement; nuclear magnetic resonance spectroscopy; stereochemistry

Further studies on enzymes catalyzing allylic rearrangments are proposed. Beta-Hydroxydecanoyl thiol ester dehydrase (E. coli), which equilibrates thiol esters of (R)-3hydroxydecanoic acid, E-2-decenoic acid, and Z-3-decenoic acid, is the key enzyme in the biosynthesis of bacterial unsaturated fatty acids. Dehydrase is rapidly inactivated by the mechanism-based inactivator 3decynoyl-NAC (NAC =N-acetylcysteamine thiol ester), via dehydrase-catalyzed isomerization to 2,3decadienoyl-NAC. The amino acid sequence of dehydrase has recently been determined, and the residue that is modified by 3-decynoyl-NAC (and is therefore implicated as the active site base) was shown to be His-70. The long-term objective is to further knowledge of enzymw structure and function. Such knowledge is critical for the rational design of drup that act at the enzyme level. The specific aims of this proposal include (a) determination (by 15N NMR) of which of dehydrase's imidazole nitrogens is modified by 3-decynoyl-NAC; (b) synthesis and application of novel enzyme inactivators, with analysis of protein by peptide mapping and multinuclear NMR, in order to identify catalytically important residues other than His-70; (c) synthesis and evaluation of ketone analogs of dehydrase's thiol ester substrates as potential nonhydrolyzable substrates and inhibitors; (d) determinnation of the three-dimensional crystal structures of dehydrase that has been inactivated with novel 3-decynoic acid thiol esters, including the ACP thiol ester; (e) complete characterization of dehydrase by multinuclear NMR, allowing comparison of crystal- and solution-state samples and enabling the identification of dehydrase-ACP contact points; (f) generation of mutant dehydrases, with modified active site residues and modified acyl chain binding regions, to test the roles of amino acid residues that are suspected to be important in catalysis and to generate E. coli strains with altered unsaturated fatty acids; (g) reevaluation of the stereochemical course of the allylic rearrangement catalyzed by the Betahydroxydodecanoyl thiol ester dehydrase component of Brevibacterium ammoniagenes fatty acid synthetase (by 2H-decoupled 1H,13C chemical shift correlation spectroscopy), in order to test for mechanistic unifomity among enzymes catalyzing allylic rearrangements.

对催化烯丙基重排的酶进行了进一步的研究。