Kinetics of the diacetyl and 2,3-pentanedione reduction by diacetyl reductase (alpha-diketone reductase (NAD)) from Staphylococcus aureus.

The kinetic mechanism of diacetyl and 2,3-pentanedione reduction by diacetyl reductase from Staphylococcus aureus was investigated. The shape of the primary double reciprocal plots, the product inhibition pattern, and the features of the inhibition by a substrate analogue (acetone) show that diacetyl is reduced via an Ordered Bi-Bi mechanism, and 2,3-pentanedione by an Ordered Bi-Bi or Theorell-Chance mechanism. NADH is the leading substrate in both reactions. Affinity constants for the coenzyme and the substrates and inhibition constants for NAD, acetoin, and acetone were also calculated. This enzyme has a high affinity for NADH; Km (31-50 microM) and Ks (20-27 microM) for this compound are around one-tenth of the NADH intracellular concentration. Therefore, it must operate in vivo saturated with the coenzyme. This condition is not adequate to play the role, formerly proposed for diacetyl reductases, of regulating the equilibrium between oxidized and reduced forms of pyridine-nucleotides.