p-Cresol methylhydroxylase. Assay and general properties

Area of Science:

• Biochemistry
• Enzymology
• Microbial Metabolism

Background:

• p-Cresol methylhydroxylase (PCMH) from Pseudomonas putida is an anaerobic dehydrogenase.
• PCMH catalyzes the oxidation of p-cresol to p-hydroxybenzyl alcohol and p-hydroxybenzaldehyde.
• The enzyme's flavoprotein and cytochrome c subunits can reversibly dissociate and recombine, retaining activity.

Purpose of the Study:

• To investigate the kinetic behavior and mechanism of p-cresol methylhydroxylase.
• To explore the implications of subunit dissociation and recombination on enzyme activity.
• To examine substrate interactions, electron transfer, and the influence of pH and ionic strength.

Main Methods:

• Bisubstrate kinetic analysis using double-reciprocal plots.
• Reductive titration with dithionite and substrates.
• Spectroscopic analysis of enzyme oxidation states.
• Enzyme activity assays under varying pH and ionic strength.

Main Results:

• Unresolved enzyme exhibits parallel-line kinetics, while the flavoprotein subunit shows converging lines.
• Reductive titration reveals 3-electron uptake and intermediate flavin radical formation.
• Substrate titration indicates sequential reduction of cytochrome and flavin subunits, suggesting intermolecular electron transfer.
• pH and ionic strength affect enzyme activity, specificity, and substrate/acceptor interactions.

Conclusions:

• The distinct kinetic behaviors suggest mechanistic differences between the resolved and unresolved enzyme.
• Intermolecular electron transfer likely plays a role in the enzyme's function.
• Environmental factors like pH and ionic strength significantly modulate PCMH activity and specificity.