The steady-state kinetics of the NADH-dependent nitrite reductase from Escherichia coli K 12. Nitrite and hydroxylamine reduction.
The reduction of both NO2- and hydroxylamine by the NADH-dependent nitrite reductase of Escherichia coli K 12 (EC 1.6.6.4) appears to follow Michaelis-Menten kinetics over a wide range of NADH concentrations. Substrate inhibition can, however, be detected at low concentrations of the product NAD+. In addition, NAD+ displays mixed product inhibition with respect to NADH and mixed or uncompetitive inhibition with respect to hydroxylamine. These inhibition characteristics are consistent with a mechanism in which hydroxylamine binds during catalysis to a different enzyme form from that generated when NAD+ is released. The apparent maximum velocity with NADH as varied substrate increases as the NAD+ concentration increases from 0.05 to 0.7 mM with 1 mM-NO2- or 100 mM-hydroxylamine as oxidized substrate. This increase is more marked for hydroxylamine reduction than for NO2- reduction. Models incorporating only one binding site for NAD can account for the variation in the Michaelis-Menten parameters for both NADH and hydroxylamine with [NAD+] for hydroxylamine reduction. According to these models, activation of the reaction occurs by reversal of an over-reduction of the enzyme by NADH. If the observed activation of the enzyme by NAD+ derives both from activation of the generation of the enzyme-hydroxylamine complex from the enzyme-NO2- complex during NO2- reduction and from activation of the reduction of the enzyme-hydroxylamine complex to form NH4+, then the variation of Vapp. for NO2- or hydroxylamine with [NAD+] is consistent with the occurrence of the same enzyme-hydroxylamine complex as an intermediate in both reactions.
D004926:Escherichia coli / Q000201:enzymology*; D019811:Hydroxylamine; D006898:Hydroxylamines / Q000378:metabolism*; D007700:Kinetics; D008956:Models, Chemical; D009243:NAD / Q000494:pharmacology; D009244:NAD+ Nucleosidase / Q000494:pharmacology; D009247:NADH, NADPH Oxidoreductases / Q000378:metabolism*; D050898:Nitrite Reductase (NAD(P)H); D009572:Nitrite Reductases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D009573:Nitrites / Q000378:metabolism; D010084:Oxidation-Reduction
D006898:Hydroxylamines; D009573:Nitrites; D009243:NAD; D019811:Hydroxylamine; D009247:NADH, NADPH Oxidoreductases; D009572:Nitrite Reductases; D050898:Nitrite Reductase (NAD(P)H); D009244:NAD+ Nucleosidase