title: Mechanistic studies of the flavoprotein tryptophan 2-monooxygenase. 2. pH and kinetic isotope effects.
abstract: 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 pKa 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 pKa 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 pKa 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 pKa 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.(ABSTRACT TRUNCATED AT 250 WORDS)
authors.affiliation: Department of Biochemistry and Biophysics, Texas A&M University, College Station 77843-2128.
authors.forename: J J
authors.initials: JJ
authors.lastname: Emanuele
authors.forename: P F
authors.initials: PF
authors.lastname: Fitzpatrick
mesh_terms: D006863:Hydrogen-Ion Concentration; D007554:Isotopes; D007700:Kinetics; D006899:Mixed Function Oxygenases / Q000037:antagonists & inhibitors / Q000737:chemistry*; D012997:Solvents
chemical_list: D007554:Isotopes; D012997:Solvents; D006899:Mixed Function Oxygenases; C023204:tryptophan 2-monooxygenase