A study of p-hydroxybenzoate hydroxylase from Pseudomonas fluorescens: chemical modification of histidine residues.
The flavoprotein p-hydroxybenzoate hydroxylase from Pseudomonas fluorescens is inactivated by diethyl pyrocarbonate. Below pH 7, diethyl pyrocarbonate reacts specifically with histidine residues. The inactivation reaction is biphasic and follows pseudo-first-order kinetics. Four of the nine histidine residues of the enzyme are modified. During the first phase of the reaction, one histidine residue is modified and leads to a loss of about 30% of the activity. Modification of the additional three histidine residues during the second phase leads to complete loss of activity. Two of the latter histidine residues are essential for activity and are involved in the binding of reduced nicotinamide adenine dinucleotide phosphate (NADPH). The activity can be restored almost quantitatively upon treatment of modified enzyme with hydroxylamine. The modified enzyme is still capable of binding NADPH. The dissociation constant of the enzyme-NADPH complex is larger by a factor of 10 for the modified enzyme as compared to that for the native enzyme. The modification does not affect the affinity of the enzyme for the substrate, although effectors protect two histidine residues from chemical modification by diethyl pyrocarbonate. The rate of inactivation of the enzyme is pH dependent and increases with increasing pH values. From the pH dependence of the rate constant, it is calculated that two cooperative histidine residues participate in the reaction with diethyl pyrocarbonate. Both histidine residues possess a pKa' value of 6.2. At pH greater than 7, other reactions take place which are completely abolished in the presence of an effector (substrate) of the enzyme.
D015109:4-Hydroxybenzoate-3-Monooxygenase / Q000378:metabolism*; D000244:Adenosine Diphosphate / Q000378:metabolism; D006639:Histidine; D007700:Kinetics; D008433:Mathematics; D006899:Mixed Function Oxygenases / Q000378:metabolism*; D009249:NADP / Q000378:metabolism; D011551:Pseudomonas fluorescens / Q000201:enzymology*
D006639:Histidine; D009249:NADP; D000244:Adenosine Diphosphate; D006899:Mixed Function Oxygenases; D015109:4-Hydroxybenzoate-3-Monooxygenase