Activation of nitrite reductase from Escherichia coli K12 by oxidized nicotinamide-adenine dinucleotide.
Nitrite reductase from Escherichia coli K12 requires the presence of NAD+, one of the products of the reduction of NO2-by NADH, for full activity. The effect is observed with both crude extracts and purified enzyme. NAD+ also acts as a product inhibitor at high concentrations, and plots of initial rate against NAD+ concentration are bell-shaped. The maximum occurs at about 1 mM-NAD+, but increases with increasing NADH concentration. In the presence of 1 mM-NAD+ and saturating NO2-(2mM) the Michaelis constant for NADH is about 16 micron. The Michaelis constant for NO2-is about 5 micron and is largely independent of the NAD+ concentration. Similar but more pronounced effects of NAD+ are observed with hydroxylamine as electron acceptor instead of NO2-. The maximum rate of NADH oxidation by hydroxylamine is about 5.4 times greater than the maximum rate of NADH oxidation by NO2- when assayed with the same volume of the same preparation of purified enzyme. The Michaelis constant for hydroxylamine is 5.3 mM, however, about 1000 times higher than for NO2-. These results are consistent with a mechanism in which the same enzyme-hydroxylamine complex occurs as an intermediate in both reactions.
D004789:Enzyme Activation; D004926:Escherichia coli / Q000201:enzymology*; D006898:Hydroxylamines / Q000378:metabolism; D007700:Kinetics; D009243:NAD / Q000378:metabolism*; D009247:NADH, NADPH Oxidoreductases / Q000378:metabolism*; D009572:Nitrite Reductases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D009573:Nitrites / Q000378:metabolism; D010084:Oxidation-Reduction
D006898:Hydroxylamines; D009573:Nitrites; D009243:NAD; D009247:NADH, NADPH Oxidoreductases; D009572:Nitrite Reductases