Specific Aims. For Pseudomonas HMG-CoA reductase: 1. To determine, by DNA sequencing and protein sequencing techniques, its complete primary structure. 2. To identify catalytic and substrate-binding residues present at the active site and to determine the primary structures of their associated peptides. 3. To prepare crystalline HMG-CoA reductase suitable for future X-ray diffraction analysis of its secondary-tertiary structure. This work will provide: 1. Detailed molecular information concerning the structure (both primary and at the active site) of a 4-electron oxidoreductase. 2. Knowledge of the structure and insight into the catalytic mechanism of Pseudomonas HMG-C0A reductase. This will be of importance in understanding both the Pseudomonas enzyme and HMG-CoA reductases from other species. 3. The primary sequence data and crystalline Pseudomonas HMG-CoA reductase will permit us to initiate determination of its complete 3-dimensional structure. Scientific Disciplines Involved: Biochemistry, structural chemistry of proteins, active site labelling with affinity reagents, DNA cloning and sequencing. Health Relatedness: Control of hypercholesterolemia (mammalian HMG-CoA reductase is a key regulatory point in cholesterogenesis). We anticipate that the insight gained concerning the mechanism of catalysis by Pseudomonas reductase will impact mammalian reductase (eg: in design of active-site directed inhibitors).
