| Inosine 5'-monophosphate dehydrogenase (IMPDH) is an enzyme critically positioned at the branchpoint of purine nucleotide biosynthesis. It catalyzes the NAD+-dependent oxidation of IMP to XMP, the penultimate step of guanine nucleotide biosynthesis. IMPDH makes use of a flexible loop containing a critical cysteine residue to carry out its catalytic function. The partitioning of the loop between productive and nonproductive conformations is believed to be an important feature of the kinetic mechanism and a determinant in the cation selectivity. In this thesis, the loop partitioning of several IMPDH isoforms is characterized. A clear difference in the loop partitioning among the isoforms is seen, and this difference correlates with changes in the rate of hydride transfer to NAD+. When K+, an essential cofactor, is omitted, the loop partitioning is affected. So orientation of the loop, aided by binding of the cofactor, appears to be an important step in the IMPDH mechanism.IMPDH is believed to be a moonlighting enzyme. The subdomain of IMPDH binds nucleic acid &sim30 A from the active site. Mutations in the subdomain cause the retinal disease autosomal dominant retinitis pigmentosa (adRP). These mutations disrupt nucleic acid binding, so the disease phenotype and nucleic acid binding are believed to be linked. IMPDH associates with polyribosomes, suggesting it may have a role in the regulation of translation. To further elucidate this potential moonlighting activity, the design and synthesis of a few biotinylated small molecule inhibitors of IMPDH to facilitate labeling of the enzyme in tissue extracts are described.Inhibition of IMPDH by the new compounds was characterized, and selective labeling of IMPDH in cell extracts by them was demonstrated. One of the inhibitors was used to isolate IMPDH-containing protein complexes from E. coli cell extracts, and the composition of the complexes was examined. A protocol for isolating crosslinked IMPDH, with the aid of one of the inhibitors, was also developed. |
