Chemical modification of phenol hydroxylase by ethoxyformic anhydride.
Phenol hydroxylase was inactivated by ethoxyformic anhydride. Part of the inactivation was related to modification of histidyl residues. The remaining part of the inactivation is proposed to be due to the modification of a lysyl residue which, we suggest, is identical with the one previously described, being essential for the binding of NADPH [Neujahr, H. Y. and Kjellén, K. G. (1980) Biochemistry 19, 4967-4972]. The overall inactivation reaction is biphasic and follows pseudo-first-order kinetics. Numerical analysis of kinetic data was applied to discriminate between simultaneous reactions at different sites. It is proposed that phenol hydroxylase contains two essential histidyl residues, located in or near the NADPH-binding sites. Ethoxyformylation of the lysyl residue(s) caused tightening of the binding of phenol and perturbation of the FAD spectrum of phenol hydroxylase, similar to that caused by phenolic effectors.
D004047:Diethyl Pyrocarbonate / Q000494:pharmacology*; D005561:Formates / Q000494:pharmacology*; D006639:Histidine; D007700:Kinetics; D008239:Lysine; D008433:Mathematics; D006899:Mixed Function Oxygenases / Q000037:antagonists & inhibitors*; D011732:Pyridoxal Phosphate / Q000494:pharmacology; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D013053:Spectrophotometry
D005561:Formates; D006639:Histidine; D011732:Pyridoxal Phosphate; D006899:Mixed Function Oxygenases; C022336:phenol 2-monooxygenase; D008239:Lysine; D004047:Diethyl Pyrocarbonate