Kinetic and chemical mechanism of the dihydrofolate reductase from Mycobacterium tuberculosis

Clarissa M. Czekster, An Vandemeulebroucke, John S. Blanchard

Research output: Contribution to journalArticle

11 Citations (Scopus)

Abstract

Dihydrofolate reductase from Mycobacterium tuberculosis (MtDHFR) catalyzes the NAD(P)-dependent reduction of dihydrofolate, yielding NAD(P)+ and tetrahydrofolate, the primary one-carbon unit carrier in biology. Tetrahydrofolate needs to be recycled so that reactions involved in dTMP synthesis and purine metabolism are maintained. In this work, we report the kinetic characterization of the MtDHFR. This enzyme has a sequential steady-state random kinetic mechanism, probably with a preferred pathway with NADPH binding first. A pKa value for an enzymic acid of approximately 7.0 was identified from the pH dependence of V, and the analysis of the primary kinetic isotope effects revealed that the hydride transfer step is at least partly rate-limiting throughout the pH range analyzed. Additionally, solvent and multiple kinetic isotope effects were determined and analyzed, and equilibrium isotope effects were measured on the equilibrium constant. D2OV and D2OV/K[4R-4-2H]NADH were slightly inverse at pH 6.0, and inverse values for D2OV[4R-4-2H]NADH and D 2OV/K[4R-4-2H]NADH suggested that a pre-equilibrium protonation is occurring before the hydride transfer step, indicating a stepwise mechanism for proton and hydride transfer. The same value was obtained for DkH at pH 5.5 and 7.5, reaffirming the rate-limiting nature of the hydride transfer step. A chemical mechanism is proposed on the basis of the results obtained here.

Original languageEnglish (US)
Pages (from-to)367-375
Number of pages9
JournalBiochemistry
Volume50
Issue number3
DOIs
StatePublished - Jan 25 2011

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Tetrahydrofolate Dehydrogenase
Mycobacterium tuberculosis
NAD
Hydrides
Kinetics
Isotopes
Protonation
Equilibrium constants
NADP
Metabolism
Protons
Carbon
Acids
Enzymes

ASJC Scopus subject areas

  • Biochemistry

Cite this

Kinetic and chemical mechanism of the dihydrofolate reductase from Mycobacterium tuberculosis. / Czekster, Clarissa M.; Vandemeulebroucke, An; Blanchard, John S.

In: Biochemistry, Vol. 50, No. 3, 25.01.2011, p. 367-375.

Research output: Contribution to journalArticle

Czekster, Clarissa M. ; Vandemeulebroucke, An ; Blanchard, John S. / Kinetic and chemical mechanism of the dihydrofolate reductase from Mycobacterium tuberculosis. In: Biochemistry. 2011 ; Vol. 50, No. 3. pp. 367-375.
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