Mycobacterium tuberculosis beta-ketoacyl-acyl carrier protein (ACP) reductase: kinetic and chemical mechanisms.
Beta-ketoacyl-acyl carrier protein (ACP) reductase from Mycobacterium tuberculosis (MabA) is responsible for the second step of the type-II fatty acid elongation system of bacteria, plants, and apicomplexan organisms, catalyzing the NADPH-dependent reduction of beta-ketoacyl-ACP to generate beta-hydroxyacyl-ACP and NADP(+). In the present work, the mabA-encoded MabA has been cloned, expressed, and purified to homogeneity. Initial velocity studies, product inhibition, and primary deuterium kinetic isotope effects suggested a steady-state random bi-bi kinetic mechanism for the MabA-catalyzed reaction. The magnitudes of the primary deuterium kinetic isotope effect indicated that the C(4)-proS hydrogen is transferred from the pyridine nucleotide and that this transfer contributes modestly to the rate-limiting step of the reaction. The pH-rate profiles demonstrated groups with pK values of 6.9 and 8.0, important for binding of NADPH, and with pK values of 8.8 and 9.6, important for binding of AcAcCoA and for catalysis, respectively. Temperature studies were employed to determine the activation energy of the reaction. Solvent kinetic isotope effects and proton inventory analysis established that a single proton is transferred in a partially rate-limiting step and that the mechanism of carbonyl reduction is probably concerted. The observation of an inverse (D)2(O)V/K and an increase in (D)2(O)V when [4S-(2)H]NADPH was the varied substrate obscured the distinction between stepwise and concerted mechanisms; however, the latter was further supported by the pH dependence of the primary deuterium kinetic isotope effect. Kinetic and chemical mechanisms for the MabA-catalyzed reaction are proposed on the basis of the experimental data.
Silva
Rafael G
RG
Centro de Pesquisas em Biologia Molecular e Funcional, Faculdade de Biociências e Faculdade de Farmacia, Instituto de Pesquisas Biomédicas, PUCRS, 6681/92-A Avenida Ipiranga, 90619-900, Porto Alegre, RS, Brazil.
de Carvalho
Luiz Pedro S
LP
Blanchard
John S
JS
Santos
Diógenes S
DS
Basso
Luiz A
LA
D064431:3-Oxoacyl-(Acyl-Carrier-Protein) Reductase; D000429:Alcohol Oxidoreductases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D015394:Molecular Structure; D009169:Mycobacterium tuberculosis / Q000201:enzymology*; D010084:Oxidation-Reduction; D013379:Substrate Specificity; D013696:Temperature
D000429:Alcohol Oxidoreductases; D064431:3-Oxoacyl-(Acyl-Carrier-Protein) Reductase