Ribulose-1,5-bisphosphate carboxylase: primary deuterium kinetic isotope effect using [3-2H]ribulose 1,5-bisphosphate

The primary deuterium kinetic isotope effect for the reaction of [3-2H]ribulose 1,5-bisphosphate with CO2 in the reaction catalyzed by ribulose-1,5-bisphosphate carboxylase has been determined. By use of highly purified substrates containing less than 0.13% of the C-3 epimer xylulose 1,5-bisphosphate (this material is known to be a potent competitive inhibitor), the kinetic isotope effect has been shown to be 1.2 +/- 0.2 (Vmax) and 1.3 +/- 0.3 (Vmax/Km). These values are clearly too small to be intrinsic isotope effects for the rate-limiting removal of the C-3 proton from ribulose 1,5-bisphosphate. The results confirm the conclusions from the tritium experiments reported in the previous two papers and emphasize the fine balance between the forward and reverse reactions of the enediol intermediate.