The Mechanism of Action of Glycerol-3-Phosphate Dehydrogenase and Orotidine 5'-Monophosphate Decarboxylase: Insights from Studies of Enzyme Variants

• Main Author: Cristobal, Judith R
• Format: Dissertation
• Language: English
• Published: ProQuest Dissertations & Theses 01-01-2022
• Subjects: Biochemistry; Chemistry
• ISBN: 9798790657566

The role of a global, substrate-driven enzyme conformational change, in enabling the large rate acceleration for orotidine 5'-monophosphate decarboxylase (OMPDC)-catalyzed decarboxylation of orotidine 5'-monophosphate (OMP) and for glycerol-3-phosphate dehydrogenase (GPDH)-catalyzed reduction of dihydroxyacetone phosphate (DHAP), is examined in experiments that focus on the interactions between the proteins and the respective substrates. We are interested in obtaining high resolution X-ray crystal structures of wild-type and variant GPDH that would provide a more accurate description of the position of the amino acid side chains at the GPDH enzyme active site to support the analysis of our on-going mutagenesis studies. We have examined the protein–substrate interactions in GPDH by studying the effect of mutations on the critical amino acid residues that interact with the phosphodianion and carbonyl portion of the substrate, DHAP. We report here the kinetic parameters for the reduction of DHAP and small neutral molecules glycolaldehyde (GA) and acetaldehyde (AcA) catalyzed by either wild-type or variant GPDH enzymes. We employed protocols to rescue the activity of truncated mutant enzymes and of truncated alternative substrates by small molecule analogs of the deleted enzyme or substrate piece. We aim to provide insights into the mechanism for small molecule activation of enzyme activity that is analogous to allosteric activation, while enabling practical uses of chemical rescue in the activation of enzymes for catalysis. We also focused on the hypothesis that protein motions in OMPDC, which produce contacts between critical side chains and the OMP hydroxyl groups, activate OMPDC for catalysis of decarboxylation at the orotate ring.