Studies on the Enzymatic Reduction of C-Nitroso Compounds

III. The Kinetic Analysis of C-Nitrosoreductase Reaction Catalyzed by the Cytoplasmic Enzyme from Porcine Liver

Abstract

The reaction kinetics of the major cytoplasmic NADH-nitrosoreductase, which is the identical enzyme to alcohol dehydrogenase [EC 1. 1. 1. 1], was studied with a partially purified preparation from porcine liver.
On the basis of the data obtained, the following scheme is proposed as the mechanism of this enzyme reaction:
E+S^K₁_??_ES E+n^K₂_??_EN ES+N^K₂_??_ESN
EN+S^K₁_??_ESN ESN^K₁_??_E'+P₁ E'+N^K₂_??_E+p₁+p₂,
where E and E' are the enzyme unit (one subunit of alcohol dehydrogenase) and an intermediate form of the enzyme unit-substrate compound which appears by two-electron reduction of the enzyme unit-substrate compound, respectively, S is p-nitrosophenol (p-NSP), N is NADH, and P₁ and P₂ are NAD⁺ and p-aminophenol (p-AmP), respectively. In this case, it is assumed that k₁_??_k₂.
Para-aminophenol, the reaction product, showed an inhibition competitive to p-NSP at fixed concentrations of NADH and also showed a mixed-type inhibition to NADH at fixed concentrations of p-NSP. NAD⁺ inhibited the reaction in a competitive manner to NADH at fixed concentrations of p-NSP and in a non-competitive manner to p-NSP at fixed concentrations of NADH. These results can also be accounted for by the proposed mechanism.