Steady-state and stopped-flow kinetic measurements of the primary deuterium isotope effect in the reaction catalyzed by p-cresol methylhydroxylase.
Steady-state kinetic studies for the reaction of the flavocytochrome p-cresol methylhydroxylase with the reducing substrates (S) p-cresol, 4-ethylphenol, and their corresponding alpha-deuteriated analogues are presented. The results from these experiments and those from studies involving various reoxidizing substrates support the proposed apparent ping-pong mechanism. With phenazine methosulfate (PMS) as the reoxidant for studies at pH 7.6 and 6 or 25 degrees C, the isotope effects on kcat are lower than the intrinsic isotope effect. The values for D(kcat/KS) are equal to the intrinsic effect for p-cresol at 25 degrees C and for 4-ethylphenol at both 6 and 25 degrees C. However, the value for this steady-state parameter at 6 degrees C for p-cresol is lower than the intrinsic effect. The values for D(kcat/KPMS) are nearly equal to 1.0 under all conditions. In contrast, the steady-state kinetic analysis for the isolated flavoprotein subunit of p-cresol methylhydroxylase involving p-cresol and PMS as substrates indicates that a random-binding mechanism is operating. Additionally, several of the steady-state parameters yield values for the apparent intrinsic isotope effect for the flavoprotein. The results of stopped-flow kinetic studies are also reported. At pH 7.6 the intrinsic isotope effect (Dk2) for the reduction of the enzyme by 4-ethylphenol is 4.8-5.0 at 25 degrees C and 4.0 at 6 degrees C. This technique yields a value for Dk2 of 7.05 at 6 degrees C and pH 7.6 for p-cresol.(ABSTRACT TRUNCATED AT 250 WORDS)
D001665:Binding Sites; D003903:Deuterium / Q000493:pharmacokinetics* / Q000494:pharmacology; D005420:Flavoproteins / Q000378:metabolism; D008433:Mathematics; D006899:Mixed Function Oxygenases / Q000493:pharmacokinetics* / Q000494:pharmacology; D010636:Phenols / Q000378:metabolism; D011487:Protein Conformation; D011549:Pseudomonas / Q000201:enzymology; D013050:Spectrometry, Fluorescence / Q000379:methods
D005420:Flavoproteins; D010636:Phenols; C042291:4-ethylphenol; D003903:Deuterium; D006899:Mixed Function Oxygenases; C019947:p-cresol oxidoreductase