Steady-state analysis of kinetic isotope effects in enzymic reactions

The decrease in the rate of bond cleavage resulting from the presence of heavy isotopes is expressed to a greater or lesser extent as an isotope effect on steady-state kinetic parameters of enzyme-catalyzed reactions, depending upon complex relationships between individual rate constants. This paper describes these relationships and derives general kinetic expressions which allow the determination of the limits of the relative contribution of different reaction components to V/sub max/ and V/sub max//K/sub m/. The value of the true isotope effect on a carbon-hydrogen bond breaking step, the rate of this step, and its percentage of partial rate limitation of the overall reaction, plus the dissociation constant of the enzyme-substrate complex, can be determined from the derived expressions by comparing deuterium and tritium isotope effects on V/sub max//K/sub m/. In the absence of a measure of the true isotope effect, the lower limit of primary isotope effects of deuterium which may be interpreted as identifying the slowest or ''rate-determining'' step in an enzyme-catalyzed reaction is found to be k/sub H//k/sub D/ = 8, and not k/sub H//k/sub D/ = 2 as is currently supposed. (U.S.)