Functional and regulatory mechanisms in alpha-isopropylmalate synthases

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The allosteric regulation of a protein is where the binding of a molecule at a distal site affects the physical and chemical properties at the binding site. A model system for studying allosteric mechanism is isopropylmalate synthase isolated from Mycobacterium tuberculosis (MtIPMS). MtIPMS catalyzes a Claisen-like condensation between acetyl-CoA and ketoisovalerate to form the products isopropylmalate and CoA, which is the first committed step in the biosynthesis of L-leucine. L-Leucine acts as a slow-onset feedback inhibitor binding 50 Å from the active site in the regulatory domain. Structural studies of MtIPMS indicate that a flexible loop becomes more ordered upon L-leucine binding. Alternate amino acid inhibitors and site-directed mutagenesis results indicate this flexible loop plays a role in the slow-onset mechanism of MtIPMS. Kinetically, L-leucine acts as a V-type inhibitor, lowering V_max for the reaction while K_m values remain relatively unchanged. A decrease in V_max could be caused by a decrease in the rate of a chemical step or product release. Results from rapid-reaction kinetics and kinetic isotope effects indicate that the rate-limiting step shifts from product release to hydrolysis upon the binding of L-leucine. Hydrogen/deuterium exchange experiments indicated that upon L-leucine binding a helix in the active site cavity undergoes a conformational change suggesting that it could be involved in the allosteric mechanism of MtIPMS. The results from site-directed mutagenesis studies indicate that this active site helix is not involved in the allosteric mechanism of MtIPMS. Isopropylmalate synthase isolated from Francisella novicida (FnIPMS) shares a sequences identity of 26% with MtIPMS over 526 residues. This is the first report of a monomeric IPMS to date. The kinetic parameters of FnIPMS are comparable to that of MtIPMS. However, the K_i value is approximately 150-fold higher than that of MtIPMS. Kinetic isotope effects also indicate that hydrolysis is the rate-limiting step in the presence of L-leucine.

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Chemistry, Biochemistry