Steady-state and stopped-flow kinetic measurements of the primary deuterium isotope effect in the reaction catalyzed by p-cresol methylhydroxylase

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Abstract
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.degree.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.degree.C and for 4-ethylphenol at both 6 and 25.degree.C. However, the value for this steady-state parameter at 6.degree.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.degree.C and 4.0 at 6.degree.C. This technique yields a value for Dk2 of 7.05 at 6.degree.C and pH 7.6 for p-cresol. The combined results from the stopped-flow and steady-state kinetic experiments at pH 7.6 and 6.degree.C for p-cresol also allow the calculation of several important kinetic parameters for this enzyme. These calculations are viewed with caution, since some discrepancies develop when a comparison of the data from the two kinetic methods is made for both p-cresol and 4-ethylphenol. Surprisingly, the stopped-flow method could not be used to measure an isotope effect for the isolated flavoprotein subunit.