Investigations of Mycobacterium tuberculosis alpha-isopropylmalate synthase

Abstract

Mycobacterium tuberculosis alpha-isopropylmalate synthase (MtIPMS) is a member of the Claisen-condensing enzymes which catalyze the AcCoA-dependent carboxymethylation of an alpha-keto acid. MtIPMS catalyzes the first committed step in the L-leucine biosynthetic pathway in mycobacteria, which is essential for intracellular survival and therefore a target for antitubercular drug design.;During the characterization of the feedback inhibition of Mt IPMS by L-leucine we observed that L-leucine inhibition is time dependent, characteristic of slow-onset inhibition. Analysis of the inhibition indicated that L-leucine is a noncompetitive inhibitor versus both substrates, alpha-ketoisovalerate and AcCoA, and the slow-onset could be explained by a rapid binding step followed by a slow isomerization.;MtIPMS is activated by monovalent cations (K +>Rb+>NH4+>Na +>Li+) and by divalent metals (Mg2+>Mn 2+>Co2+>Ni2+>Ca2+). Of note, Zn2+ proposed to be the catalytic metal, is a "submicromolar" inhibitor of MtIPMS. Part of the monovalent activation seems to be due to "tuning" of the divalent metal. Small, but significant, changes in the kinetic parameters for the substrates are seen when the identity of the divalent metal is varied, whereas no such changes are observed when the identity of the monovalent cation is varied.;The steady-state kinetic characterization of MtIPMS indicates that the enzyme follows a nonrapid equilibrium random kinetic mechanism, with a preferred pathway to the ternary complex. There is narrow substrate specificity for alpha-keto acid substrates and an almost absolute specificity for AcCoA. pH studies indicate that two enzymic bases are required for the catalyzed reaction and several other ionizable groups are required for substrate binding. Primary deuterium isotope effects measured with [2H 3-methyl]AcCoA were unity under a variety of conditions, clearly indicating that enolization of AcCoA is not the rate-limiting step in the Mt IPMS-catalyzed reactions. Solvent isotope effects were also unity with the physiological substrates. However, in the presence of pyruvate, significant solvent isotope effects (∼ 2.0) were measured, indicating that hydrolysis begins to become rate-limiting. Our 18O-labeling experiment revealed that no anhydride is released into solution, indicating that the hydrolysis of the intermediate occurs on the enzyme's surface and is regioselective, with or without formation of this anhydride.