Ornithine decarboxylase (ODC) activity is central to a major biochemical pathway, undergoes complex regulation, and is the target of drugs with potential therapeutic utility, but little is known of the enzyme's structure. We propose to use genetic and biochemical methods to enhance our understanding of the structure of ornithine decarboxylase and the relationship between that structure and its biological functions. Genetics 1) We will generate large numbers of random mutations throughout the structural gene and evaluate the effects of each of these on testable biochemical functions- 2) We will create specific structural mutations using site directed mutagenesis to test the function of particular amino acids or groups of amino acids, 3) Mouse, trypanosome and yeast ODC domain sequences and those from mutant genes generated as above will be exchanged to assemble novel ODC genes. 4) We will seek mutants with the following specific phenotypes: (a) resistance to a mechanism-dependent irreversible inhibitor (b) increased activity for decarboxylation of congeners of ornithine (c) dominant to wild type. We will also select and characterize revertants that restore enzymatic activity to mutants. Analysis of Mutants 1) ODC activity of lysates will be measured. Western blot analysis will be used under denaturing conditions to determine the level of expression of serologically detectable ODC. Western blot analyses using non-denaturing conditions will evaluate the efficiency of subunit assembly into dimers. 2) Mutant proteins will be purified and their enzymatic kinetic parameters determined, including specific activity and affinity for co-factor and substrate. 3) Pulse-chase experiments using metabolic labelling will be carried out to evaluate protein stability. 4) Mutant proteins will be subjected to solvent denaturation to measure the stability of their active conformation. Biochemical Determinants of ODC Function 1) The site or sites of modification by a suicide substrate inhibitor will be determined. 2) The site of co-factor binding will be determined. 3) The site or sites of phosphorylation will be determined.
