MICROENVIRONMENTAL GEOMETRY OF C-C LYASE ACTIVE SITES

对与催化作用有关的结构研究的新支持 Ps的2-酮基-3-脱氧胆酸6P醛缩酶。putida要求。 将进行实验以确定是否存在Glu-56，其由 溴丙酮酸，还原性交联Lys-144，席夫碱形成 赖氨酸 此外，实验将询问这种还原性交联是否 亚基间或亚基内。 将进行进一步的实验， Glu-56的γ-羧酸盐转化为羟基酸盐。 这 化学突变的酶物种将被测试，以确定它是否可以形成 酮亚胺与底物，而催化酮亚胺/烯胺周转， 不允许。 这些研究将涉及酸/碱催化中的Glu-56。 在 此外，将尝试用GaP类似物烷基化Glu-56。 这将证实涉及Glu-56的单碱基机制 参与丙酮酸和/或KDPG所必需的质子活化 由酶催化的周转。 其他实验将探索 酶的动力学机制，试图确定速率限制 催化步骤。 与这些研究相结合的是 旨在揭示与催化有关的pKs，即 甲醇胺/酮亚胺（复合物）形成和酮亚胺/烯胺 （催化）相互转化。 这些研究将揭示 溴代戊酸敏感的Glu-56催化，建立酸/碱 参与C-C合成，并为合理药物的概念做出贡献 设计

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.