p-Cresol methylhydroxylase. Assay and general properties.
p-Cresol methylhydroxylase from Pseudomonas putida, an anaerobic dehydrogenase that catalyses the oxidation of p-cresol to p-hydroxybenzyl alcohol and then to p-hydroxybenzaldehyde, is an enzyme of great interest in several respects. One of these is the fact that its flavoprotein and cytochrome c subunits may be reversibly dissociated with ease, with full regeneration of the activity and its native properties on recombining the components. Bisubstrate kinetic analysis of the unresolved enzyme gives parallel-line kinetics in double-reciprocal plots, whereas the reaction of the separated flavoprotein subunit with substrates is described by converging lines. The mechanistic implications of these behaviours are discussed. Reductive titration with dithionite results in the uptake of 3 electrons by the enzyme, with the intermediate formation of the anionic flavin radical [McIntire, Edmondson, Hopper & Singer (1981) Biochemistry 20, 3068-3075]. Reductive titration with substrates resulted initially only in reduction of the cytochrome subunit, followed by formation of the anionic radical and finally the fully reduced enzyme. These observations suggest rapid intermolecular electron transfer between p-cresol methylhydroxylase molecules. This paper also examines the effect of pH and ionic strength on the activity and specificity of the enzyme with respect to substrates and natural, as well as artificial, electron acceptors. The absorption coefficients of the enzyme and of its subunits in various oxidation states are also presented.
McIntire
W
W
Hopper
D J
DJ
Singer
T P
TP
D001592:Benzyl Alcohols / Q000378:metabolism; D003408:Cresols / Q000378:metabolism; D003574:Cytochrome c Group / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008773:Methylphenazonium Methosulfate / Q000378:metabolism; D006899:Mixed Function Oxygenases / Q000378:metabolism*; D009994:Osmolar Concentration; D011549:Pseudomonas / Q000201:enzymology; D013053:Spectrophotometry; D013379:Substrate Specificity
D001592:Benzyl Alcohols; D003408:Cresols; D003574:Cytochrome c Group; C018966:4-hydroxybenzyl alcohol; C032538:4-cresol; D008773:Methylphenazonium Methosulfate; D006899:Mixed Function Oxygenases; C019947:p-cresol oxidoreductase