Chemical modification of tyrosine residues in p-hydroxybenzoate hydroxylase from Pseudomonas fluorescens: assignment in sequence and catalytic involvement.
p-Hydroxybenzoate hydroxylase was modified by diethyl pyrocarbonate at pH values greater than 7 and by p-diazobenzoate. Modification of the enzyme by diethyl pyrocarbonate abolishes the affinity of the enzyme for the substrate p-hydroxybenzoate. Modification by p-diazobenzoate has the same effect on the enzyme. The enzyme is protected against these modifications by the effector p-fluorobenzoate. The data indicate that the modification of one tyrosine residue in the active center of the enzyme is responsible for the loss of enzyme activity. This tyrosine residue has been identified by sequence studies using radioactively labeled p-diazobenzoate and was found to be most probably Tyr-222. Diethyl pyrocarbonate reacts with a tyrosine residue in the active center other than Tyr-222; the former could not be identified. Sequence studies further showed that Cys-211 is also partially modified by p-diazobenzoate. In addition, the sequence of residues 343-345 was found to be Ser-Trp-Trp instead of the tentative assignment Ser-Tyr-Trp made earlier. The results are briefly discussed on the basis of the existing three-dimensional model of the enzyme.
D015109:4-Hydroxybenzoate-3-Monooxygenase / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism*; D000595:Amino Acid Sequence; D001665:Binding Sites; D003979:Diazonium Compounds / Q000494:pharmacology*; D004047:Diethyl Pyrocarbonate / Q000494:pharmacology*; D005561:Formates / Q000494:pharmacology*; D007700:Kinetics; D006899:Mixed Function Oxygenases / Q000378:metabolism*; D010446:Peptide Fragments / Q000032:analysis; D011485:Protein Binding; D011551:Pseudomonas fluorescens / Q000201:enzymology*; D014443:Tyrosine
C016363:4-diazobenzoic acid; D003979:Diazonium Compounds; D005561:Formates; D010446:Peptide Fragments; D014443:Tyrosine; D006899:Mixed Function Oxygenases; D015109:4-Hydroxybenzoate-3-Monooxygenase; D004047:Diethyl Pyrocarbonate