EFFECT OF COMMITMENTS TO CATALYSIS ON THE DEGREE OF CURVATURE IN PROTON INVENTORIES OF THE KINETIC-PARAMETERS FOR ENZYME-CATALYZED REACTIONS - APPLICATION TO TRYPTOPHAN INDOLE-LYASE

A relatively simple method for obtaining (D)k, the intrinsic solvent deuterium isotope effect for an enzyme-catalyzed reaction, is presented and discussed. Steady-state and pre-steady-state determinations of the solvent deuterium and substrate deuterium isotope effects, the later obtained in both H2O and D2O, enable the development of a catalytic mechanism. The interrelatedness of the isotope effect equations for the enzyme system allows calculation of (D)k. As a result, the variation of 1/(D)k(n), the reciprocal of the partial intrinsic solvent deuterium isotope effect, with n(D2O), the atom fraction of deuterium in mixed isotopic waters, has been determined for the tryptophan indole-lyase enzyme-catalyzed reaction, and is compared to the variation of (V/K)n/(V/K)0 with n(D2O). Comparison of the proton inventories indicates that without knowledge of the commitments to catalysis underestimates of the number of protons calculated from a fit of the kinetic parameter data to the Gross-Butler equation can occur. A series of curves is presented which demonstrate that the degree of curvature in a bowl-shaped proton inventory decreases as the commitment to catalysis increases, and that ultimately, as the relative size of the commitment continues to increase, a dome-shaped proton inventory will result.