Fundamental questions addressed by this proposal relate to determining the basis for the presence of multiple genetic loci which encode acetohydroxy acid synthase (AHAS) activity in Escherichia coli. The AHAS reaction condenses two moles of pyruvate to one mole of form 2-acetolactate (AL), and one mole of pyruvate with one mole of 2-ketobutyrate to form one mole of 2- aceto-2-hydroxybutyrate (AHB). AL and AHB are biosynthetic precursors to L-valine and L-isoleucine. Isozymes of AHAS partition, or channel, pyruvate carbons to AL or AHB according to a specific partitioning bias, in response to 2-ketobutyrate levels. The Distinct partitioning bias, measured by a constant R, is a distinct property of an AHAS isozyme, and appears to be an important determinant of survival under different conditions of nutrient supply. AHAS II from two different alleles of ilvG, ilvG468 and ilvG474, differently partition pyruvate carbons such that the R-values are significantly different. We propose to determine the molecular basis for the difference in R-values, and to test hypotheses about the mechanism(s) of pyruvate partitioning. By a molecular genetic study of mutant alleles of ilvG, and steady state kinetic measurement, of pyruvate partitioning by resultant allozymes of AHAS II, we propose to identify structural domains which harbor amino acid residues that constitute the active site(s) of AHAS II. By use of a computer program (PARCOR) developed for this study, we have identified conserved structural domains common to AHAS isozymes I, II and III. Spontaneous mutations in ilvG468 will be selected to change the partitioning R-value to that of ilvG474, and alleles screened to identify mutations that alter R-values without perturbation of local secondary structure of resultant allozymes. Screens for structural changes will be accomplished by use of the PARCOR program. Saturation mutagenesis of coding regions for conserved structural domains will be done to seek all possible amino acid changes that alter partitioning with out perturbing local protein structure. Every ilvG allele will be tested for its ability singularly to support growth of E. coli on a rich and poor carbon source. By these approaches we expect to identify AHAS active site residues that are involved in differential partitioning of pyruvate carbons.
