Involvement of a protein with molybdenum cofactor in the in vitro activation of nitrate reductase from a chlA mutant of Escherichia coli K12.
Chlorate-resistant mutants are pleiotropically defective in molybdoenzyme activities. The inactive derivative of the molybdoenzyme, respiratory nitrate reductase (nitrite: (acceptor) oxidoreductase, EC 1.7.99.4), which is present in cell-free extracts of chlA mutants can be activated by addition of purified protein PA, the presumed active product of the chlA+ locus, but the activity of the purified protein PA is low, since comparatively large amounts of protein PA are required for the activation. Addition of 10 mM tungstate to the growth medium of a chlBchlC double mutant leads to inactivation of both the molybdenum cofactor and protein PA. Protein PA prepared from such cells was unable to potentiate the in vitro activation of nitrate reductase present in the soluble fraction of a chlA mutant. Quantitation of inactive protein PA was determined immunologically using protein PA-specific antiserum. When a heat-treated extract of a wild-type strain was added to purified protein PA or to the supernatant fraction of a chlBchlC double mutant grown with tungstate, a large stimulation in the ability of these preparations to activate chlA nitrate reductase was found. We equate the activator of protein PA with molybdenum cofactor because: (1) both are absent from heated extracts of tungstate-grown chlBchlC double mutant and cofactor defective chlA and chlE mutants; (2) both are present in heated extracts of wild-type strain; and (3) they behave identically on molecular-sieve columns.
D003067:Coenzymes; D004789:Enzyme Activation; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D005838:Genotype; D007700:Kinetics; D008667:Metalloproteins / Q000378:metabolism*; D000090006:Molybdenum Cofactors; D009154:Mutation; D009492:Neurospora crassa / Q000378:metabolism; D050901:Nitrate Reductase; D009565:Nitrate Reductases / Q000378:metabolism*; D011621:Pteridines / Q000378:metabolism*
D003067:Coenzymes; D008667:Metalloproteins; D000090006:Molybdenum Cofactors; D050901:Nitrate Reductase; D009565:Nitrate Reductases; D011621:Pteridines; C022261:molybdopterin