title: A histidine residue in p-hydroxybenzoate hydroxylase essential for binding of reduced nicotinamide adenine dinucleotide phosphate.
abstract: Chemical modification with diethylpyrocarbonate (ethoxyformic anhydride) was examined to demonstrate the existence of an essential histidine residue at the NADPH-binding site of p-hydroxybenzoate hydroxylase (EC 1.14.13.2) from Pseudomonas desmolytica. Among some ligands, NADPH was noticeable in protecting the enzyme from the modification-caused inactivation. Although several amino acid residues were modified during the inactivation process, inhibition of the enzyme could be correlated with modification of a single histidine residue which was masked by addition of NADPH. The pK of the essential histidine residue was estimated to be 6.5-6.7. The Kd (Km) for NADPH of the inactivated enzyme was shown to have been increased greatly, although the Kd for substrate (p-hydroxybenzoate) was not changed.
mesh_terms: D015109:4-Hydroxybenzoate-3-Monooxygenase / Q000378:metabolism*; D004047:Diethyl Pyrocarbonate / Q000494:pharmacology*; D004229:Dithiothreitol / Q000494:pharmacology; D005561:Formates / Q000494:pharmacology*; D006639:Histidine; D006898:Hydroxylamines / Q000494:pharmacology; D007700:Kinetics; D006899:Mixed Function Oxygenases / Q000378:metabolism*; D009249:NADP / Q000378:metabolism*; D011549:Pseudomonas / Q000201:enzymology*
chemical_list: D005561:Formates; D006898:Hydroxylamines; D006639:Histidine; D009249:NADP; D006899:Mixed Function Oxygenases; D015109:4-Hydroxybenzoate-3-Monooxygenase; D004047:Diethyl Pyrocarbonate; D004229:Dithiothreitol