Ketopantoate reductase (EC 126.96.36.199) catalyzes the NADPH-dependent reduction of α-ketopantoate to D-(-)-pantoate in the biosynthesis of pantothenate. The pH dependence of V and V/K for the E. coli enzyme suggests the involvement of a general acid/base in the catalytic mechanism. To identify residues involved in catalysis and substrate binding, we mutated the following six strictly conserved residues to Ala: Lys72, Lys176, Glu210, Glu240, Asp248, and Glu256. Of these, the K176A and E256A mutant enzymes showed 233- and 42-fold decreases in Vmax, and 336- and 63-fold increases in the Km value of ketopantoate, respectively, while the other mutants exhibited WT kinetic properties. The Vmax for the K176A and E256A mutant enzymes was markedly increased, up to 25% and 75% of the wild-type level, by exogenously added primary amines and formate, respectively. The rescue efficiencies for the K176A and E256A mutant enzymes were dependent on the molecular volume of rescue agents, as anticipated for a finite active site volume. The protonated form of the amine is responsible for recovery of activity, suggesting that Lys176 functions as a general acid in catalysis of ketopantoate reduction. The rescue efficiencies for the K176A mutant by primary amines were independent of the pKa value of the rescue agents (Brønsted coefficient, α = -0.004 ± 0.008). Insensitivity to acid strength suggests that the chemical reaction is not rate-limiting, consistent with (a) the catalytic efficiency of the wild-type enzyme (kcat/Km = 2 × 106 M-1 s-1) and (b) the small primary deuterium kinetic isotope effects, DV = 1.3 and DV/K = 1.5, observed for the wild-type enzyme. Larger primary deuterium isotope effects on V and V/K were observed for the K176A mutant (DV = 3.0, DV/K = 3.7) but decreased nearly to WT values as the concentration of ethylamine was increased. The nearly WT activity of the E256A mutant in the presence of formate argues for an important role for this residue in substrate binding. The double mutant (K176A/E256A) has no detectable ketopantoate reductase activity. These results indicate that Lys176 and Glu256 of the E. coli ketopantoate reductase are active site residues, and we propose specific roles for each in binding ketopantoate and catalysis.