Functional modification of an arginine residue on salicylate hydroxylase.
Salicylate hydroxylase from Pseudomonas putida (EC 1.14.13.1, salicylate, NADH:oxygen oxidoreductase) is an FAD-containing monooxygenase, which catalyzes decarboxylative hydroxylation of salicylate to produce catechol in the presence of NADH and O2. By chemical treatment of the enzyme with dicarbonyl reagents, such as glyoxal, the original oxygenase activity was converted to the salicylate-dependent NADH-dehydrogenase activity with free FAD as electron acceptor. One of twenty arginine residues of this enzyme is concerned with this alteration of activity, as shown by the result of its modification at pH 6.9. This result is further supported by the isolation of one arginine-modified enzyme by chromatographic methods on DEAE-Sephadex, A-50 columns. It exhibits the dehydrogenase activity predominantly. This modified enzyme is spectrophotometrically and electrophoretically characterized by a minute conformational change around the active site, and kinetically by a 7-fold increase in an apparent Km for NADH and a decrease of more than 5-fold in an apparent Km for FAD as electron acceptor, with an apparent Vmax of 22 s-1 for the dehydrogenase activity. Flow kinetics also showed a marked decrease in the rate for oxygenation of the reduced enzyme-salicylate complex from 21 s-1 (native enzyme) to 3.3 s-1 (modified enzyme). These facts suggest that one arginine residue of the enzyme is responsible for the NADH binding site, and chemical modification of one arginine residue of the enzyme induces some conformational change around the active site to alter the catalytic activity from oxygenation to dehydrogenation.
Suzuki
K
K
Department of Chemistry, Faculty of Science, Kanazawa University, Japan.
Ohnishi
K
K
D001120:Arginine / Q000737:chemistry*; D002942:Circular Dichroism; D004591:Electrophoresis, Polyacrylamide Gel; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism; D006037:Glyoxal / Q000737:chemistry; D007700:Kinetics; D006899:Mixed Function Oxygenases / Q000737:chemistry* / Q000378:metabolism; D009243:NAD / Q000378:metabolism; D009245:NADH Dehydrogenase / Q000378:metabolism; D010084:Oxidation-Reduction; D011549:Pseudomonas / Q000201:enzymology; D013379:Substrate Specificity
D009243:NAD; D005182:Flavin-Adenine Dinucleotide; D006037:Glyoxal; D001120:Arginine; D006899:Mixed Function Oxygenases; C022619:salicylate 1-monooxygenase; D009245:NADH Dehydrogenase