PMID:10736170
| Citation |
Zheng, R and Blanchard, JS (2000) Kinetic and mechanistic analysis of the E. coli panE-encoded ketopantoate reductase. Biochemistry 39:3708-17 |
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| Abstract |
Ketopantoate reductase (EC 1.1.1.169) catalyzes the NADPH-dependent reduction of alpha-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 (1)H 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, DeltaG, of -14 kcal/mol suggest that ketopantoate reductase reaction is very favorable in the physiologically important direction of pantoate formation. |
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| Keywords |
Alcohol Oxidoreductases/biosynthesis; Alcohol Oxidoreductases/chemistry; Alcohol Oxidoreductases/genetics; Alcohol Oxidoreductases/metabolism; Amino Acid Sequence; Deuterium; Escherichia coli/enzymology; Escherichia coli/genetics; Hydrogen-Ion Concentration; Kinetics; Molecular Sequence Data; Plasmids/chemistry; Plasmids/metabolism; Recombinant Proteins/biosynthesis; Recombinant Proteins/chemistry; Recombinant Proteins/isolation & purification; Solvents; Stereoisomerism; Thermodynamics |
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