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 methionin e 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 pK(a) value equal to that of the amino grou p 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 en zyme with a pK(a) value of 6 must be deprotonated for tight binding of amide inhibitors and protonated for tight binding of acids, establish ing this as the intrinsic pK, value. The temperature dependence of thi s pK(a) value is consistent with involvement of a histidinyl residue. Deprotonation of the residue with a pK(a) value of 10 decreases bindin g 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 7-1 5 with this substrate. The (D)(V/K)(ala) value is pH dependent, increa sing 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 pK(a) values of 5.1 and 10. The increase in both the isotope effect and the V/K-met value at high pH is consis tent 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 int rinsic isotope effect with this substrate. The beta-secondary isotope effect with [beta,beta,beta-H-2(3)]alanine is 0.965 +/- 0.041, consist ent with a carbanion mechanism. The proposed role of the residue with a pK(a) value of 6 is to remove the substrate alpha-proton to form the carbanion. The V/K-O2 values for phenylalanine and tryptophan are ess entially insensitive to pH between pH 5 and pH 10. The V/K-O2 value wi th 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 th e 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 assi gned to a conformational change which precedes product release. The so lvent isotope effect on the V-max value with tryptophan is 2.5. This i s consistent with slow proton transfer being coupled to product releas e.