Mechanistic studies of the flavoprotein tryptophan 2-monooxygenase. 2. pH and kinetic isotope effects

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 pK_a 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 pK_a 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 pK_a value. The temperature dependence of this pK_a 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/K_trp) 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/K_met 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/K_O2 value with methionine increases severalfold with a pK_a value of 6.8; this is assigned to the reduced FAD. There is no solvent isotope effect on the V/K_O2 value with tryptophan, consistent with rate-limiting electron transfer in the reaction with oxygen. With all three substrates, the V_max value decreases 20-50-fold when a single residue is protonated. This pK_a 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 V_max value with tryptophan is 2.5. This is consistent with slow proton transfer being coupled to product release.