The steady state kinetics of the NADH-dependent nitrite reductase from Escherichia coli K12. The reduction of single-electron acceptors.
The kinetic characteristics of the diaphorase activities associated with the NADH-dependent nitrite reductase (EC 1.6.6.4) from Escherichia coli have been determined. The values of the apparent maximum velocity are similar for the reduction of Fe(CN)6(3)-and mammalian cytochrome c by NADH. These reactions may therefore have the same rate-limiting step. NAD+ activates NADH-dependent reduction of cytochrome c, and the apparent maximum velocity for this substrate increases more sharply with the concentration of NAD+ than for hydroxylamine. The simplest explanation is that NAD+ activation of hydroxylamine reduction derives solely from activation of steps involved in the reduction of cytochrome c, a flavin-mediated reaction, but these steps are only partly rate-limiting for the reduction of hydroxylamine. At 0.5 mM-NAD+, the apparent maximum velocity was 2.3 times higher for 0.1 mM-cytochrome c as substrate than for 100 mM-hydroxylamine, suggesting that the rate-limiting step during hydroxylamine reduction is a step that is not involved in cytochrome c reduction. A scheme is proposed that can account for the pattern of variation with [NAD+] of the Michaelis-Menten parameters for hydroxylamine and for NADH with hydroxylamine or cytochrome c as oxidized substrate.
D003574:Cytochrome c Group / Q000378:metabolism; D008058:Dihydrolipoamide Dehydrogenase / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology*; D019811:Hydroxylamine; D006898:Hydroxylamines / Q000378:metabolism; D007700:Kinetics; D008954:Models, Biological; D009243:NAD / Q000494:pharmacology; D009247:NADH, NADPH Oxidoreductases / Q000378:metabolism*; D050898:Nitrite Reductase (NAD(P)H); D009572:Nitrite Reductases / Q000378:metabolism*; D010084:Oxidation-Reduction
D003574:Cytochrome c Group; D006898:Hydroxylamines; D009243:NAD; D019811:Hydroxylamine; D009247:NADH, NADPH Oxidoreductases; D009572:Nitrite Reductases; D050898:Nitrite Reductase (NAD(P)H); D008058:Dihydrolipoamide Dehydrogenase