pH and kinetic isotope effects on steady-state kinetic parameters have been determined for the flavoprotein tryptophan 2-monooxygenase with tryptophan, phenylalanine, 2-hydrazino-3-propanoic acid, and methionine as substrates. The V/K values of the amino acid substrates show that a residue with an apparent pKa value of 5 must be unprotonated for activity, a residue with a p value equal to that of the amino group of the substrate must be protonated, and deprotonation of a residue with pKa value of 10 increases the V/K value. A group in the free enzyme with a value of 6 must be deprotonated for tight binding of amide inhibitors and protonated for tight binding of acids, establishing this as the intrinsic pKa value. The temperature dependence of this pKa value is consistent with involvement of a histidinyl residue. Deprotonation of the residue with a value of 10 decreases binding of amide inhibitors. The D(V/Ktrp) value is less than 1.7 between pH 5 and 10, consistent with a forward commitment to catalysis of 7-15 with this substrate. The D(V/K)met value is pH dependent, increasing from a minimal value of 1.8 at pH 8.3 to a limiting value of 5.3 at both high and low pH, with p values of 5.1 and 10. The increase in both the isotope effect and the V/Kmet value at high pH is consistent with a conformational change to a more open active site above pH 10. The D(V/K)ala value is 5.3 at pH 8.3; this is probably the intrinsic isotope effect with this substrate. The β-secondary isotope effect with [ββ,β-2 ]alanine is 0.965 ± 0.041, consistent with a carbanion mechanism. The proposed role of the residue with a value of 6 is to remove the substrate-proton to form the carbanion. The V/Ko2 values for phenylalanine and tryptophan are essentially insensitive to pH between pH 5 and pH 10. The V/KO2 value with methionine increases severalfold with a pKa value of 6.8; this is assigned to the reduced FAD. There is no solvent isotope effect on the V/KO2 value with tryptophan, consistent with rate-limiting electron transfer in the reaction with oxygen. With all three substrates, the Vmax value decreases 20-50-fold when a single residue is protonated. This pKa value varies with the identity of the substrate; it is assigned to a conformational change which precedes product release. The solvent isotope effect on the Vmax value with tryptophan is 2.5. This is consistent with slow proton transfer being coupled to product release.
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