title: Chemical modification of arginine residues in p-hydroxybenzoate hydroxylase from Pseudomonas fluorescens: a kinetic and fluorescence study.
abstract: The flavoprotein p-hydroxybenzoate hydroxylase from Pseudomonas fluorescens was modified by several arginine-specific reagents. Modifications by 2,3-butanedione led to the loss of activity of the enzyme, but the binding of p-hydroxybenzoate and NADPH to the enzyme was little or not at all affected. However the formation of the enzyme-substrate complex of the modified enzyme was accompanied by an increase of the fluorescence of protein-bound FAD, in contrast to that of native enzyme which leads to quenching of the fluorescence. Enzyme modified by phenylglyoxal did not bind p-hydroxybenzoate nor NADPH. Quantification and protection experiments showed that two arginine residues are essential and a model is described which accounts for the results. Modification by 4-hydroxy-3-nitrophenylglyoxal reduced the affinity of the enzyme for the substrate and NADPH. The ligands offered no protection against inactivation. From this it is concluded that one arginine residue is essential at some stage of the catalysis. This residue is not associated with the substrate- or NADPH-binding site of the enzyme. Time-resolved fluorescence studies showed that the average fluorescence lifetime and the mobility of protein-bound FAD are affected by modification of the enzyme.
mesh_terms: D015109:4-Hydroxybenzoate-3-Monooxygenase / Q000378:metabolism*; D001120:Arginine / Q000378:metabolism*; D001665:Binding Sites; D003931:Diacetyl / Q000494:pharmacology; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism; D005453:Fluorescence; D007700:Kinetics; D008433:Mathematics; D006899:Mixed Function Oxygenases / Q000378:metabolism*; D009249:NADP / Q000378:metabolism; D010658:Phenylglyoxal / Q000031:analogs & derivatives / Q000494:pharmacology; D011551:Pseudomonas fluorescens / Q000201:enzymology*
chemical_list: D005182:Flavin-Adenine Dinucleotide; D009249:NADP; C027540:4-hydroxy-3-nitrophenylglyoxal; D001120:Arginine; D006899:Mixed Function Oxygenases; D015109:4-Hydroxybenzoate-3-Monooxygenase; D003931:Diacetyl; D010658:Phenylglyoxal