DETERMINATION OF ENZYME MECHANISMS BY KINETIC STUDIES

我们的目标是开发用于研究酶机制的动力学工具，以及 将它们应用于具有代表性的酶。具体目标为：1) DPN烟酰胺环N-1位15N同位素效应的测定 及其对乙醇、甲酸盐和葡萄糖-6-P脱氢酶的类似物 为了确认这一建议，将环变形为船的形式 负责诱导氢化物转移到C-4。13C同位素效应 将在C-4进行测量，以完成初级和 这些体系中的次级同位素效应和过渡态的推导 结构作为核苷酸的氧化还原电势的函数。 2)测定羧基转移的机理 在小苏打、生物素和其他底物之间含有 生物素。我们将测量~(13)C、~(18)O和~(13)H的同位素效应 反应，并将尝试从二氧化碳和 磷酸盐。 3)次级18-O同位素对磷酰化转移影响的测量 推断这些机制是关联的还是非关联的。分子 在非桥位置标记的18-O包括葡萄糖-6-P(TO 与己糖激酶和磷酸葡萄糖变位酶一起使用)和ATP(与之一起使用 激酶和ATPase)。我们还将使用uPA测量这种同位素效应 和核糖核酸酶，以及β-环糊精-联咪唑，这是一种 核糖核酸酶模型。 4)类似物的动力学和化学性质的测定 含硫或氮的磷酸化代谢中间体 碳和磷之间的桥梁。即将发生的酶反应 将研究糖酵解和葡萄糖-6-P转化为 核酮糖-双-P，加上后者的羧基酶。这样做的目的是 研究是要确定硫与氧的异构化反应 或者氮，以及磷酸转移酶处理这种类似物的能力(哪种 与磷酰化转移机制有关)。 5)预苯酯的变构抑制动力学研究 酪氨酸。希望能发展出速率方程和理论来描述 这种相互作用既使用正常的底物、预苯醚，也使用 环上缺少一个双键的类似物(以及 侧链)，它是可逆氧化的，没有脱羧基。

Our purpose is to develop kinetic tools for studying enzyme mechanisms, and to apply them to representative enzymes. Specific aims will be: 1) Measurement of 15N isotope effects at N-1 of the nicotinamide ring of DPN and its analogs for alcohol, formate and glucose-6-P dehydrogenases in order to confirm the proposal that deformation of the ring to a boat form is responsible for inducing hydride transfer to C-4. 13C isotope effects will be measured at C-4 in order to complete the measurement of primary and secondary isotope effects in these systems and deduce transition state structures as a function of the redox potential of the nucleotides. 2) Determination of the mechanism by which carboxyl groups are transfered between bicarbonate, biotin and other substrates in enzymes containing biotin. We will measure 13C, 18-O and deuterium isotope effects on these reactions, and will attempt to synthesize carboxyphosphate from CO2 and phosphate. 3) Measurement of secondary 18-O isotope effects on phosphoryl transfer to deduce whether the mechanisms are associative or dissociative. Molecules to be labeled in the non-bridge positions with 18-O include glucose-6-P (to be used with hexokinase and phosphoglucomutase) and ATP (to be used with kinases and ATPases). We will also measure such isotope effects with UPA and ribonuclease, and with Beta-cyclodextrinyl-bisimidazole, which is a ribonuclease model. 4) Determination of the kinetic and chemical properties of analogs of phosphorylated metabolic intermediates containing sulfur or nitrogen in the bridge between carbon and phosphorus. The enzymatic reactions to be studied will be those of glycolysis and the conversion of glucose-6-P to ribulose-bis-P, plus the carboxylase for the latter. The purpose of this study is to determine how isosteric the replacement of oxygen with sulfur or nitrogen is, and how well phosphotransferases handle such analogs (which bears on the phosphoryl transfer mechanism). 5) Investigation of the kinetics of allosteric inhibition of prephenate by tyrosine. The hope is to develop rate equations and theory to describe this interaction using both the normal substrate, prephenate, and the analog lacking one double bond in the ring (as well as the keto group in the side chain), which is oxidized reversibly without decarboxylation.