DETERMINATION OF ENZYME MECHANISMS BY KINETIC STUDIES

点击翻译按钮获取中文摘要

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.