Kinetic and mechanistic analysis of the E. coli panE-encoded ketopantoate reductase.
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.
Zheng
R
R
Department of Biochemistry, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, New York 10461, USA.
Blanchard
J S
JS
D000429:Alcohol Oxidoreductases / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D003903:Deuterium; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D010957:Plasmids / Q000737:chemistry / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D012997:Solvents; D013237:Stereoisomerism; D013816:Thermodynamics
D011994:Recombinant Proteins; D012997:Solvents; D003903:Deuterium; D000429:Alcohol Oxidoreductases; C021934:2-dehydropantoate 2-reductase