Kinetic and chemical mechanisms of ketopantoate reductase and pantothenate synthetase

Abstract

Ketopantoate reductase catalyzes the NADPH-dependent reduction of alpha-ketopantoate to form D-pantoate. The steady-state mechanism is ordered sequential. The stereospecific transfer of the pro S hydrogen atom of NADPH to the C-2 position of ketopantoate was demonstrated by 1H-NMR spectroscopy. Small primary deuterium kinetic isotope effects suggest that hydride transfer is not rate-limiting in catalysis. The pH profile revealed an enzyme group functioning as a general acid in catalysis. To identify this and other residues involved in catalysis and substrate binding, we mutated six strictly conserved residues to alanine. The K176A and E256A mutant enzymes showed a significant decrease in Vmax, and a large increase in the Km value of ketopantoate. The Vmax for the K176A and E256A mutant enzymes was markedly increased by exogenously added primary amines and formate. The protonated form of the amine is responsible for the recovery of activity, suggesting that Lys176 functions as a general acid catalyst. The rescue efficiencies for the K176A mutant by amines were independent of the pKa value of the rescue agents. Insensitivity to acid strength suggests that the chemical step is not rate-limiting. The nearly WT activity of the E256A mutant in the presence of formate argues for an important role for this residue in substrate binding.;Pantothenate synthetase catalyzes the ATP-dependent condensation of D-pantoate and beta-alanine to form pantothenate. The kinetic mechanism of pantothenate synthetase is Bi Uni Uni Bi Ping Pong. The formation of pantoyl adenylate, suggested as a key intermediate by the kinetic mechanism, was confirmed by 31P NMR spectroscopy of [18O] AMP produced by 18O transfer using [carboxyl-18O] pantoate. The rates of formation of pyrophosphate and pantothenate were determined using rapid quench techniques and are similar to the overall rate. Both half reactions are partially rate-limiting and that pantoyl adenylate is a kinetically competent intermediate in the pantothenate synthetase reaction. Reverse isotope exchange of [14C] beta-alanine into pantothenate in the presence of AMP was observed, indicating the reversible formation of the pantoyl adenylate intermediate from products in catalysis.