1. Isopropylmalate Synthase Amino acid analogs

2. Amino Acid Analogs Analogs – resemble naturally occurring amino acids Inhibit Wild Type – Why? What happens if incorporated into protein? Why are normal amino acids not synthesized? Natural Amino AcidAnalog LeucineFluoroleucine TryptophanMethyl-Tryptophan HistidineThiazole Alanine LysineAmino Ethyl Cysteine

3. Expose Wild Type Salmonella to Fluoroleucine Expected Minimal Media containing Fl-leu What is nature of variants that grow? Observed

4. Salmonella Strain Comparison Grown on regular Minimal Media Straing minutes Y glc μg/2 mg glc Secretes Leucine Relative IPMS Activity IPMS Inhibition by Leucine Wild type56900No1Yes Mutant 1960~900Trace10Yes Mutant 19160~900Trace1No CV24185500500 μg/ml10No Isopropyl Malate Synthase – First committed enzyme in the synthesis of leucine

5. Nature of Mutants MutantBehavior; Interpretation 19Excess amount enzyme, inhibited by leucine, but will produce excess leucine because of sheer amount of enzyme; Mutant in regulation of IPMS synthesis – non- allosteric 191Normal amount of enzyme, Not inhibited by Leucine, slightly overproduces leucine; Mutant in allosteric site – can’t shut enzyme off CV241Excess enzyme and no allosteric control, wastefully overproduces leucine Double mutant in allosteric site and expression – all messed up