Chemical modification of tyrosine-38 in p-hydroxybenzoate hydroxylase from Pseudomonas fluorescens by 5'-p-fluorosulfonylbenzoyladenosine: a probe for the elucidation of the NADPH binding site? Involvement in catalysis, assignment in sequence and fitting to the tertiary structure.
p-Hydroxybenzoate hydroxylase from Pseudomonas fluorescens was covalently modified by the nucleotide analog 5'-(p-fluorosulfonylbenzoyl)-adenosine in the presence of 20% dimethylsulfoxide. The inactivation reaction is pH-dependent and does not obey pseudo-first-order kinetics, due to spontaneous hydrolysis of the reagent. The kinetic data further indicate that a weak, reversible enzyme-inhibitor complex is an intermediate in the inactivation reaction and that only one amino acid residue is responsible for the loss of activity. The inactivation is strongly inhibited by NADPH and 2',5'ADP. Steady-state kinetics and 2',5'ADP bioaffinity chromatography of the modified enzyme suggest that the essential residue is not directly involved in NADPH binding. Sequence studies show that Tyr-38 is the main residue protected from modification in the presence of NADPH. From crystallographic studies it is known that the hydroxyl group of Tyr-38 is 1.84 nm away from the active site. Model-building studies using computer graphics show that this distance can be accommodated when FSO2BzAdo binds in an extended conformation with the sulfonylbenzoyl portion in an orientation different from the nicotin-amide ring of NADPH.
D015109:4-Hydroxybenzoate-3-Monooxygenase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D000241:Adenosine / Q000031:analogs & derivatives* / Q000494:pharmacology; D001665:Binding Sites; D002384:Catalysis; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007700:Kinetics; D008433:Mathematics; D006899:Mixed Function Oxygenases / Q000378:metabolism*; D009249:NADP / Q000378:metabolism*; D011487:Protein Conformation; D011551:Pseudomonas fluorescens / Q000201:enzymology*; D014443:Tyrosine / Q000378:metabolism
D014443:Tyrosine; D009249:NADP; C010959:5'-(4-fluorosulfonylbenzoyl)adenosine; D006899:Mixed Function Oxygenases; D015109:4-Hydroxybenzoate-3-Monooxygenase; D000241:Adenosine