The stereospecificity of the reduction of nitrate by reduced nicotinamide-adenine dinucleotides catalysed by Candida utilis preparations.
The reduction of nitrate by reduced nicotinamide-adenine dinucleotides, catalysed by extract of Candida utilis, exhibits an apparent high degree of stereospecificity for the 'B' methylene hydrogen atom of NADPH and mixed stereospecificity for the methylene hydrogen atoms of NADH. Purified nitrate reductase, on the other hand, exhibits 'A' stereospecificity for NADH and NADPH. The apparent switch of stereospecificity from the 'B' to the 'A' side of NADPH, which occurs after purification of the enzyme, is partly explained by the fact that in crude extracts nitrate is reduced completely to ammonia. Nitrite does not accumulate but is reduced to ammonia by nitrite dehydrogenase, which is 'B'-specific, so that up to 75% of hydrogen removed from NADPH during the reduction of nitrate could occur from the 'B' side. A further increase in the removal of hydrogen from the 'B' side of NADPH could be the kinetic isotope effect that is observed when ['A'-3H]NADPH is the reductant, the H--C bond being cleaved 2.3 times faster than the 3H--C bond. The mixed stereospecificity observed with NADH has been traced to an uncharacterized enzyme that catalyses a 'B'-specific exchange between NAD+ and NADH. This reaction is discussed in relation to the possibility that it may explain other cases of apparent mixed stereospecificity that have been reported.
D002175:Candida / Q000201:enzymology; D007700:Kinetics; D009243:NAD / Q000502:physiology*; D009249:NADP / Q000502:physiology*; D009565:Nitrate Reductases / Q000378:metabolism*; D009572:Nitrite Reductases / Q000378:metabolism; D010084:Oxidation-Reduction; D013329:Structure-Activity Relationship
D009243:NAD; D009249:NADP; D009565:Nitrate Reductases; D009572:Nitrite Reductases