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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.

alpha-dicarbonyl reductase from pigeon liver catalyzes diacetyl reduction with NADPH via an ordered Bi-Bi mechanism in which the coenzyme is the leading substrate, as deduced from the inhibition pattern by products and by acetone. The activation energy of the reaction has been calculated as 16.6 kcal/mol, delta H and delta F as 15.6 and 15.3 kcal/mol, respectively, and delta S as 1 cal/mol per k. Kinetic constants obtained for substrates (KmNADPH = 15 microM, KsNADPH = 10 microM; Kmdiacetyl = 0.5 mM, Ksdiacetyl = 0.35 mM) and products (KiNADP 50 microM; Kiacetoin = 100 mM) are about 10 times lower than those reported for this enzyme in the reduction of diacetyl with NADH. This confirms that NADPH is its physiological coenzyme.