a-Isopropylmalate synthase catalyzes the first step in the biosynthesis of L-leucine. The enzyme catalyzes a Claisen condensation between acetyl coenzyme A and a-ketoisovalerate followed by hydrolysis of the isopropylmalyl-CoA intermediate. The enzyme from Mycobacterium tuberculosis (MtIPMS) is subject to V-type allosteric feedback inhibition by L-leucine. Data from pre-steady state kinetic experiments indicate a burst phase in the first turnover of uninhibited MtIPMS consistent with a rate-limiting step following catalysis. In the presence of saturating L-leucine concentrations, MtIPMS exhibits linear product formation, suggesting that a chemical step is now rate-limiting. Solvent kinetic isotope effects were measured to identify the chemical step. The pL-independent value of D2Okcat increases from ~1 to 1.6�0.2 in the presence of saturating concentrations of L-leucine. These results suggest V-type allosteric inhibition in MtIPMS results in part from a reduction in the intrinsic rate of the hydrolytic step upon L-leucine binding.

Mechanistic investigation of <i>V</i>-type allosteric regulation in α-isopropylmalate synthase from<i> Mycobacterium tuberculosis</i>

245th National Meeting (2013)

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Mechanistic investigation of V-type allosteric regulation in α-isopropylmalate synthase from Mycobacterium tuberculosis