MICROENVIRONMENTAL GEOMETRY OF C-C LYASE ACTIVE SITES

C-C裂解酶活性位点的微环境几何结构

• 批准号: 3289727
• 负责人: HENRY P. MELOCHE
• 金额: $ 12.55万
• 依托单位: UNIVERSITY OF MIAMI SCHOOL OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 1984
• 资助国家: 美国
• 起止时间: 1984-12-01 至 1986-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3289727
• 关键词: aldehyde lyase; carbon carbon lyase; carboxylation; catalyst; chemical structure function; crosslink; deoxyglucose; drug design /synthesis /production; ketamine; lysine; pyruvates

Renewed support for the study of structure as related to catalysis using the enzyme 2-keto-3-deoxygluconate6P aldolase of Ps. putida is requested. Experiments will be carried out to determine whether Glu-56, labeled by bromopyruvate, reductively cross-links Lys-144, the Schiff's base-forming lysine. Also experiments will ask whether this reductive cross-linking is inter or intra subunit. Further experiments will be carried out in which the Gamma-carboxylate of Glu-56 is converted to the hydroxymate. This chemically mutated enzyme species will be tested as to whether it can form the ketimine with substrates while catalytic ketimine/eneamine turnover is disallowed. Such studies will implicate Glu-56 in acid/base catalysis. In addition, alkylation of Glu-56 will be attempted with an analog of GaP. This would confirm the single-base mechanism which implicates Glu-56 participation in proton activations necessary for pyruvate and/or KDPG turnover catalyzed by the enzyme. Other experiments will probe the enzyme's kinetic mechanism in the attempt to determine the rate limiting step(s) in catalysis. Coupled to these studies will be experiments designed to reveal the pKs pertinent to catalysis, viz. carbinolamine/ketimine (complex) formation and ketimine/eneamine (catalysis) interconversion. The studies will reveal the role of the bromopyruvate-sensitive Glu-56 in catalysis, establish acid/base participation in C-C synthesis, and contribute to concepts of rational drug design.

重新支持与催化使用有关的结构研究