Kinetic and chemical mechanism of α-isopropylmalate synthase from Mycobacterium tuberculosis

Mycobacterium tuberculosis α-isopropylmalate synthase (MtIPMS) catalyzes the condensation of acetyl-coenzyme A (AcCoA) with α-ketoisovalerate (α-KIV) and the subsequent hydrolysis of α-isopropylmalyl-CoA to generate the products CoA and α- isopropylmalate (α-IPM). This is the first committed step in L-leucine biosynthesis. We have purified recombinant MtIPMS and characterized it using a combination of steady-state kinetics, isotope effects, isotopic labeling, and ¹H-NMR spectroscopy. The α-keto acid specificity of the enzyme is narrow, and the acyl-CoA specificity is absolute for AcCoA. In the absence of α-KIV, MtIPMS does not enolize the α protons of AcCoA but slowly hydrolyzes acyl-CoA analogues. Initial velocity studies, product inhibition, and dead-end inhibition studies indicate that MtIPMS follows a nonrapid equilibrium random bi-bi kinetic mechanism, with a preferred pathway to the ternary complex. MtIPMS requires two catalytic bases for maximal activity (both with pK_a values of ca. 6.7), and we suggest that one catalyzes deprotonation and enolization of AcCoA and the other activates the water molecule involved in the hydrolysis of α-isopropylmalyl-CoA. Primary deuterium and solvent kinetic isotope effects indicate that there is a step after chemistry that is rate-limiting, although, with poor substrates such as pyruvate, hydrolysis becomes partially rate-limiting. Our data is inconsistent with the suggestion that a metal-bound water is involved in hydrolysis. Finally, our results indicate that the hydrolysis of α-isopropylmalyl-CoA is direct, without the formation of a cyclic anhydride intermediate. On the basis of these results, a chemical mechanism for the MtIPMS-catalyzed reaction is proposed.