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STRUCTURE AND FUNCTION OF ALLYLIC REARRANGEMENT ENZYMES

STRUCTURE AND FUNCTION OF ALLYLIC REARRANGEMENT ENZYMES
烯丙基重排酶的结构和功能
批准号:
3289949
负责人:
John M. Schwab
金额:
$17.49万
依托单位:
依托单位国家:
美国
项目类别:
财政年份:
1985
资助国家:
美国
项目状态:
已结题
起止时间:
1985-08-01 至 1994-11-30

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中文摘要
翻译
提出了酶催化烯丙基重排反应的进一步研究方向。 β-羟基十二烷基硫醇酯脱水酶(E.Coli),平衡 (R)-3-羟基癸酸、E-2-癸烯酸的硫醇酯,以及 Z-3-癸烯酸是细菌生物合成的关键酶。 不饱和脂肪酸。脱水酶被快速失活 基于机理的失活剂3decynoyl-NAC(NAC=N-乙酰半胱胺硫醇 酯),通过脱水酶催化异构化生成2,3-十二烯基-乙酸乙酯。这个 最近测定了脱水酶的氨基酸序列,并对其进行了序列分析。 被3-十烷基-NAC修饰的残基(因此隐含为 活性位点碱基)显示为His-70。 长期目标是进一步了解酶的结构和 功能。这些知识对于DRUP的合理设计至关重要 在酶的水平上行动。这项建议的具体目标包括:(A) 用~(15)N核磁共振法测定脱水酶的咪唑类氮基 (B)新型酶的合成及应用 用肽谱和多核方法分析蛋白质的失活剂 核磁共振,以便识别除 His-70;(C)脱水酶硫醇类似物的合成与鉴定 作为潜在的非水解性底物和抑制剂的酯类底物; (D)脱水酶三维晶体结构的测定 它已经被新型的3-二十二酸硫醇酯灭活, 包括ACP硫醇酯;(E)脱水酶的完整特性 通过多核核磁共振,可以比较晶体和溶液状态 样品和能够识别脱水酶-ACP接触点; (F)产生具有修饰的活性中心残基的突变脱水酶和 修饰的酰链结合区,以测试氨基酸的作用 被怀疑在催化和产生 不饱和脂肪酸发生改变的大肠杆菌菌株;(G)重新评估 催化烯丙基重排的立体化学过程 短杆菌β-羟基十二烷基硫醇酯脱水酶组分 氨基脂肪酸合成酶(通过2H-去偶联的1H,13C化学位移 相关光谱学),以测试 催化烯丙基重排的酶。
英文摘要
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.
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SUICIDE SUBSTRATES IN SECONDARY METABOLISM
  • 批准号:
    3292824
  • 项目类别:
  • 资助金额:
    $8.2万
  • 财政年份:
    1988
  • 负责人:
    John M. Schwab
  • 依托单位:
SUICIDE SUBSTRATES IN SECONDARY METABOLISM
  • 批准号:
    3292825
  • 项目类别:
  • 资助金额:
    $7.58万
  • 财政年份:
    1988
  • 负责人:
    John M. Schwab
  • 依托单位:
SUICIDE SUBSTRATES IN SECONDARY METABOLISM
  • 批准号:
    3292826
  • 项目类别:
  • 资助金额:
    $7.83万
  • 财政年份:
    1988
  • 负责人:
    John M. Schwab
  • 依托单位:
STUDIES ON MECHANISTICALLY CRYPTIC ENZYMATIC REACTIONS
  • 批准号:
    3289946
  • 项目类别:
  • 资助金额:
    $5.05万
  • 财政年份:
    1985
  • 负责人:
    John M. Schwab
  • 依托单位:
海外基金