Kinetic and Mechanistic Analysis of theE. colipanE-Encoded Ketopantoate Reductase

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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 Vfor and V/KNADPH, respectively, and 2.1 and 1.3 on Vrev and V/KHP, respectively, suggest that hydride transfer is not rate-limiting in catalysis. Solvent kinetic isotope effects of 1.3 on both Vfor and V/KKP, and 1.4 and 1.5 on Vrev and V/KHP, respectively, support this conclusion. The apparent equilibrium constant, Keq‘, 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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