Rate Enhancement in beta-Hydroxyacid Oxidative Decarboxylases

University Of Oklahoma Norman Campus, Norman OK

Investigators

Abstract

0091207 Cook Enzymes are powerful catalysts that increase the rate of a biochemical reaction up to 1017-times compared to the reaction in the absence of enzyme. To modulate an enzyme's activity or to design a new catalyst, it is of import to understand how enzymes catalyze their reaction. A number of enzymes catalyze their reaction via several steps. Thus, overlaying the catalytic role of the enzyme in each of the individual chemical steps is the ability of the enzyme to orchestrate the overall reaction as it proceeds from one step to the other. Research will be conducted to determine information on the advantages derived from acid and base catalysis, Lewis acid catalysis, and substrate binding energy for the metal ion dependent malic enzyme (ME) and the metal ion independent 6-phosphogluconate dehydrogenase (6PGDH). The overall goal will be achieved via the following specific aims. 1. The rates of individual steps along the reaction pathway will be estimated for both enzymes. 2. The analog 2-deoxy-6PG rapidly undergoes the first (but not the second) step of the 6PGDH reaction. It will be used to separate the 6PGDH reaction into individual steps. In addition, mutant 6PGDHs and MEs with impaired catalytic groups will be used to produce defects along the catalytic cycles of the respective enzymes, allowing isolation of each of the chemical steps. 3. Site-directed mutagenesis will be used to identify enzyme residues involved in catalysis and binding in the ME and 6PGDH reactions. For both enzymes, all mutants will be characterized with respect to their structural integrity, binding of reactants, and kinetic and chemical mechanism.