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

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Abstract
The primary deuterium kinetic isotope effect for the reaction of [3-2H]ribulose 1,5-biphosphatase with CO2 in the reaction catalyzed by [Rhodospirillum rubrum] ribulose-1,5-biphosphate carboxylase was determined. By use of highly purified substrates containing < 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 was 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-biphosphate. The results confirm the conclusions from the 3H experiments reported in the previous 2 papers and emphasize the fine balance between the forward and reverse reactions of the enediol intermediate.