Kinetic and mechanistic analysis of the E. coli panE-encoded ketopantoate reductase

Renjian Zheng, John S. Blanchard

Research output: Contribution to journalArticle

37 Citations (Scopus)

Abstract

Ketopantoate reductase (EC 1.1.1.169) catalyzes the NADPH-dependent reduction of α-ketopantoate to form D-(-)-pantoate in the pantothenate/coenzyme A biosynthetic pathway. The enzyme encoded by the pane gene from E. coli K12 was overexpressed and purified to homogeneity. The native enzyme exists in solution as a monomer with a molecular mass of 34 000 Da. The steady-state initial velocity and product inhibition patterns are consistent with an ordered sequential kinetic mechanism in which NADPH binding is followed by ketopantoate binding, and pantoate release precedes NADP+ release. The pH dependence of the kinetic parameters V and V/K for substrates in both the forward and reverse reactions suggests the involvement of a single general acid/base in the catalytic mechanism. An enzyme group exhibiting a pK value of 8.4 ± 0.2 functions as a general acid in the direction of the ketopantoate reduction, while an enzyme group exhibiting a pK value of 7.8 ± 0.2 serves as a general base in the direction of pantoate oxidation. The stereospecific transfer of the pro-S hydrogen atom of NADPH to the C-2 position of ketopantoate was demonstrated by 1H NMR spectroscopy. Primary deuterium kinetic isotope effects of 1.3 and 1.5 on V(for) and V/K(NADPH), respectively, and 2.1 and 1.3 on V(rev) and V/K(HP), respectively, suggest that hydride transfer is not rate-limiting in catalysis. Solvent kinetic isotope effects of 1.3 on both V(for) and V/K(KP), and 1.4 and 1.5 on V(rev) and V/K(HP), respectively, support this conclusion. The apparent equilibrium constant, K(eq)', of 676 at pH 7.5 and the standard free energy change, ΔG, of -14 kcal/mol suggest that ketopantoate reductase reaction is very favorable in the physiologically important direction of pantoate formation.

Original languageEnglish (US)
Pages (from-to)3708-3717
Number of pages10
JournalBiochemistry
Volume39
Issue number13
DOIs
StatePublished - Apr 4 2000

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NADP
Escherichia coli
Kinetics
Enzymes
Isotopes
Escherichia coli K12
Acids
Deuterium
Biosynthetic Pathways
Equilibrium constants
Molecular mass
Coenzyme A
Catalysis
Kinetic parameters
Catalyst supports
Hydrides
Nuclear magnetic resonance spectroscopy
Free energy
Hydrogen
Magnetic Resonance Spectroscopy

ASJC Scopus subject areas

  • Biochemistry

Cite this

Kinetic and mechanistic analysis of the E. coli panE-encoded ketopantoate reductase. / Zheng, Renjian; Blanchard, John S.

In: Biochemistry, Vol. 39, No. 13, 04.04.2000, p. 3708-3717.

Research output: Contribution to journalArticle

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abstract = "Ketopantoate reductase (EC 1.1.1.169) catalyzes the NADPH-dependent reduction of α-ketopantoate to form D-(-)-pantoate in the pantothenate/coenzyme A biosynthetic pathway. The enzyme encoded by the pane gene from E. coli K12 was overexpressed and purified to homogeneity. The native enzyme exists in solution as a monomer with a molecular mass of 34 000 Da. The steady-state initial velocity and product inhibition patterns are consistent with an ordered sequential kinetic mechanism in which NADPH binding is followed by ketopantoate binding, and pantoate release precedes NADP+ release. The pH dependence of the kinetic parameters V and V/K for substrates in both the forward and reverse reactions suggests the involvement of a single general acid/base in the catalytic mechanism. An enzyme group exhibiting a pK value of 8.4 ± 0.2 functions as a general acid in the direction of the ketopantoate reduction, while an enzyme group exhibiting a pK value of 7.8 ± 0.2 serves as a general base in the direction of pantoate oxidation. The stereospecific transfer of the pro-S hydrogen atom of NADPH to the C-2 position of ketopantoate was demonstrated by 1H NMR spectroscopy. Primary deuterium kinetic isotope effects of 1.3 and 1.5 on V(for) and V/K(NADPH), respectively, and 2.1 and 1.3 on V(rev) and V/K(HP), respectively, suggest that hydride transfer is not rate-limiting in catalysis. Solvent kinetic isotope effects of 1.3 on both V(for) and V/K(KP), and 1.4 and 1.5 on V(rev) and V/K(HP), respectively, support this conclusion. The apparent equilibrium constant, K(eq)', of 676 at pH 7.5 and the standard free energy change, ΔG, of -14 kcal/mol suggest that ketopantoate reductase reaction is very favorable in the physiologically important direction of pantoate formation.",
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