Kinetic and Chemical Mechanism of α–Isopropylmalate Synthase from Mycobacterium tuberculosis

Mycobacterium tuberculosis, the causative agent of Tuberculosis, is intrinsically resistant to many first line antibiotics used to treat other common bacterial infections and resistance to effective drugs is increasing. The characterization of new targets and the development of new drugs are urgently needed. Mycobacteria, like many other bacteria, synthesize the branched-chain amino acids L-valine and L –leucine, and pantothenic acid, from α-ketoisovalerate1 (α–KIV). Studies using transposon mutagenesis in mycobacteria have demonstrated that the L-leucine biosynthetic pathway is essential for Mycobacterium bovis both in vitro and in vivo (1-3). These results suggest that mycobacteria, engulfed within the macrophage phagolysosome-like compartment, cannot obtain sufficient L-leucine from the surrounding environment. These findings also validate the L-leucine biosynthetic pathway as a target for inhibitor design and drug development to treat Tuberculosis. In spite of its importance, the L-leucine biosynthetic pathway is among the least studied amino acid biosynthetic pathways. The L-leucine branch of the branched-chain amino acid pathway (Scheme 1) starts with the AcCoA-dependent carboxymethylation of α–KIV catalyzed by α–isopropylmalate synthase (EC 2.3.3.13). This enzyme is subject to feedback inhibition by L-leucine in many organisms and the M. tuberculosis IPMS has recently been shown to exhibit slow-onset, feedback inhibition by L-leucine (4). α–Isopropylmalate is subsequently converted to β-isopropylmalate by α–isopropylmalate isomerase (EC 4.2.1.33) and then oxidatively decarboxylated to α–ketoisocaproate by β-isopropylmalate dehydrogenase (EC 1.1.1.85). α–Ketoisocaproate is converted directly to L-leucine by the action of a branched-chain amino acid transaminase (5). All of the genes involved in L-leucine biosynthesis have defined orthologues in the M. tuberculosis genome (6, 7). Scheme 1 α–Isopropylmalate synthase (IPMS) catalyzes a Claisen-type condensation between α–KIV and AcCoA. The most well-characterized members of this family are malate synthase (MS), a component of the glyoxylate pathway, citrate synthase (CS), a component of the Krebs cycle, and most recently homocitrate synthase (HCS), a component of the yeast α–aminoadipate pathway (8). Despite the similarities in their respective substrates and the chemical reactions catalyzed, MS and CS perform the condensation reaction using quite different catalytic mechanisms. MS from both yeast and M. tuberculosis use a Mg2+ ion (9, 10) to bind and polarize the carbonyl group of glyoxylate, while CS does not use a divalent metal, but rather a pair of histidine residues to polarize the carbonyl group of oxaloacetate. In addition, CS from different organisms can display quite different pH dependences, with the Thermoplasma acidophilum CS reaction being independent of pH and the pig heart CS reaction being dependent on two ionizable enzyme groups (11). IPMS from Salmonella enterica serovar typhimurium (12-15), Alcaligenes eutrophus H16 (16-18), Neurospora (19), and Saccharomyces (20) have been partially characterized. The common features of most α–IPM synthases are a requirement for monovalent cations for maximal activity, feedback inhibition by L-leucine, and narrow substrate specificity for analogs of α–KIV. In contrast, there are many differences revealed by these studies including the effects of divalent metals, inhibition by some α-keto acids, feedback regulation, and allosteric properties. Chanchaem and Palittapongarnpim have cloned, expressed and purified active MtIPMS, but no rigorous kinetic studies were performed (21). Recently, Koon et al. solved the three-dimensional crystal structure of IPMS from M. tuberculosis with Zn2+ and α–KIV bound at the active site (22). The insertion of two residues of one monomer into the active site of the other in the dimeric enzyme suggests how regulation by L-leucine may be achieved (22). Our own studies have revealed an absolute dependence for a monovalent cation for activity and potassium appears to be the physiologically relevant monovalent ion. Additionally, the monovalent cation is required for the binding of, and activation of catalysis by, a divalent metal that is used to coordinate the carbonyl and carboxyl oxygens of α-KIV (25). This paper describes the detailed biochemical characterization of the recombinant α-isopropylmalate synthase from M. tuberculosis H37Rv. Using a combination of steady-state kinetics, primary deuterium and solvent kinetic isotope effects, isotopic labeling, and 1H-NMR spectroscopy, the following questions were addressed. (i) What is the substrate specificity for α–keto acids and acyl-CoA analogs? (ii) What is the kinetic mechanism of MtIPMS? (iii) Does acid/base chemistry play an important role in catalysis? (iv) Does the enzyme hydrolyze acyl-CoA analogs? (v) What is the rate-limiting step in the reaction? (vi) What is the chemical mechanism used for intermediate cleavage?