Mycobacterium tuberculosis β-ketoacyl-acyl carrier protein (ACP) reductase

Kinetic and chemical mechanisms

Rafael G. Silva, Luiz Pedro S De Carvalho, John S. Blanchard, Diógenes S. Santos, Luiz A. Basso

Research output: Contribution to journalArticle

15 Citations (Scopus)

Abstract

β-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 β-ketoacyl-ACP to generate β-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 C4-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 D2OV/K and an increase in D2OV when [4S-2H]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.

Original languageEnglish (US)
Pages (from-to)13064-13073
Number of pages10
JournalBiochemistry
Volume45
Issue number43
DOIs
StatePublished - Oct 31 2006

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3-Oxoacyl-(Acyl-Carrier-Protein) Reductase
Mycobacterium tuberculosis
NADP
Isotopes
Kinetics
Deuterium
Acyl Carrier Protein
Protons
Complement C4
Catalysis
Elongation
Hydrogen
Bacteria
Fatty Acids
Nucleotides
Activation energy
Observation
Equipment and Supplies
Temperature

ASJC Scopus subject areas

  • Biochemistry

Cite this

Mycobacterium tuberculosis β-ketoacyl-acyl carrier protein (ACP) reductase : Kinetic and chemical mechanisms. / Silva, Rafael G.; De Carvalho, Luiz Pedro S; Blanchard, John S.; Santos, Diógenes S.; Basso, Luiz A.

In: Biochemistry, Vol. 45, No. 43, 31.10.2006, p. 13064-13073.

Research output: Contribution to journalArticle

Silva, Rafael G. ; De Carvalho, Luiz Pedro S ; Blanchard, John S. ; Santos, Diógenes S. ; Basso, Luiz A. / Mycobacterium tuberculosis β-ketoacyl-acyl carrier protein (ACP) reductase : Kinetic and chemical mechanisms. In: Biochemistry. 2006 ; Vol. 45, No. 43. pp. 13064-13073.
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