Mycobacterium tuberculosis beta-ketoacyl-ACP reductase: alpha-secondary kinetic isotope effects and kinetic and equilibrium mechanisms of substrate binding.
Beta-ketoacyl-ACP reductase catalyzes the NADPH-dependent reduction of beta-ketoacyl-acyl carrier protein to generate beta-hydroxyacyl-acyl carrier protein and NADP+, the second step of the fatty acid elongation system type II of bacteria, plants, and apicomplexan organisms. Here, a modified and more efficient purification protocol is reported for recombinant Mycobacterium tuberculosis beta-ketoacyl-ACP reductase (MabA). The increase in alpha-secondary deuterium kinetic isotope effect values measured at pH 10 as compared to those obtained at pH 7 points to isotope- and pH-sensitive steps occurring concomitantly. Equilibrium and kinetic fluorescence studies demonstrate positive cooperativity in binding of NADPH to MabA, with two forms of free enzyme in solution. Equilibrium dialysis shows no cooperativity in acetoacetyl-CoA binding to the enzyme. Moreover, modest affinity loss occurs when the substrates bind to the monomer as compared to the dimer of MabA. A mechanism of substrate binding to MabA is proposed on the basis of the experimental data.
D064431:3-Oxoacyl-(Acyl-Carrier-Protein) Reductase; D000214:Acyl Coenzyme A / Q000737:chemistry / Q000378:metabolism; D000429:Alcohol Oxidoreductases / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D001426:Bacterial Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D002384:Catalysis; D003903:Deuterium / Q000737:chemistry / Q000378:metabolism; D019281:Dimerization; D006859:Hydrogen / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D008956:Models, Chemical; D009169:Mycobacterium tuberculosis / Q000201:enzymology*; D009171:Mycolic Acids / Q000138:chemical synthesis / Q000378:metabolism; D009249:NADP / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D011522:Protons; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013379:Substrate Specificity
D000214:Acyl Coenzyme A; D001426:Bacterial Proteins; D009171:Mycolic Acids; D011522:Protons; D011994:Recombinant Proteins; C010667:acetoacetyl CoA; D009249:NADP; D006859:Hydrogen; D003903:Deuterium; D000429:Alcohol Oxidoreductases; D064431:3-Oxoacyl-(Acyl-Carrier-Protein) Reductase