Electrostatic effects on substrate activation in para-hydroxybenzoate hydroxylase: studies of the mutant lysine 297 methionine.
p-Hydroxybenzoate hydroxylase (EC 1.14.13.2) is a flavoprotein monooxygenase that catalyzes the incorporation of one atom of molecular oxygen into p-hydroxybenzoate to form 3,4-dihydroxybenzoate. The enzyme activates the substrate at the 3 position to electrophilic substitution by lowering the pKa of the phenolic oxygen. The results presented here indicate that regions of positive potential in the active site facilitate this substrate activation, which is necessary for rapid hydroxylation. We have neutralized a positive point charge by mutating lysine 297 to methionine (K297M). This mutation changes an amino acid near the active site, but not directly in contact with the flavin or the substrate. A variety of transient state kinetic and static parameters have been determined with two substrates. The results indicate that the K297M mutant does not activate the substrate through phenolic ionization to the same extent as wild-type (WT) and yet remains a competent hydroxylase. However, catalysis by the mutant is slow compared to that of WT, particularly in the oxidative half-reaction. Thus, normally quite labile oxygenated flavin intermediates encountered in the hydroxylation pathway of WT p-hydroxybenzoate hydroxylase are stabilized and their decay is rate limiting in the K297M turnover. Electrostatic potential calculations offer an explanation for the lack of substrate activation. The stability of the oxidative reaction intermediates seems to be related to a lower degree of substrate activation.
Moran
G R
GR
Department of Molecular and Cellular Biology, University of New England, New South Wales, Australia.
Entsch
B
B
Palfey
B A
BA
Ballou
D P
DP
D015109:4-Hydroxybenzoate-3-Monooxygenase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D002384:Catalysis; D004563:Electrochemistry; D004789:Enzyme Activation / Q000187:drug effects; D006900:Hydroxylation; D007700:Kinetics; D008239:Lysine; D008715:Methionine; D008958:Models, Molecular; D015394:Molecular Structure; D016296:Mutagenesis; D010226:Parabens / Q000378:metabolism; D013329:Structure-Activity Relationship
D010226:Parabens; D008715:Methionine; D015109:4-Hydroxybenzoate-3-Monooxygenase; C038193:4-hydroxybenzoic acid; D008239:Lysine