Effects of oligomeric state on catalysis by UDP‐glucose dehydrogenase

Abstract

Human UDP‐glucose dehydrogenase (UGDH) is an enzyme that catalyzes the conversion of UDP‐glucose to UDP‐glucuronate by two successive oxidation reactions. Previous studies have highlighted the importance of several conserved active site residues and many of the mechanistic details have been examined. Recent studies indicate that wild‐type UGDH exists as a hexamer, composed of a trimer of catalytically active dimers. Examination of the available UGDH crystal structure suggests that disruption of its oligomeric state may impact its enzymatic activity. To test this hypothesis, we have designed, expressed, and purified two UGDH mutants, E110A and T325A. Both mutants were found primarily in the dimeric state by gel filtration and sedimentation velocity experiments. Importantly, these engineered dimer mutants have kinetic constants comparable to wild‐type enzyme. The E110A and T325A mutants were subsequently used to examine the importance of the hexameric structure using cell culture models. Results of these studies suggest that the intact hexamer is required for optimal UGDH activity.