Abstract
The kinetic and equilibrium isotope effects on the fructose- 1,6-bisphosphate aldolase reaction have been determined using the rabbit muscle enzyme, The natural C-13 abundance for both atoms participating in the bond splitting were measured in position C-1 of dihydroxyacetone phosphate and glyceraldehyde 3-P after irreversible conversion to glycerol-3-P and 3- phosphoglycerate, respectively, and chemical degradation, The carbon isotope effects were determined comparing the C-13 content of the corresponding positions after partial and complete turnover, and after complete equilibration of the reactants. (13)(V-max/K-m) on C-3 was 1.016 +/- 0.007 and 0.997 +/- 0.009 on position C-4, and the equilibrium isotope effects K-12/K-13 on these positions were 1.0036 +/- 0.0002 and 1.0049 +/- 0.0001. The observed kinetic isotope effect on C-3 is discussed to originate from the formation of the enamine, which comes to equilibrium before the rate determining release of glyceraldehyde 3-P from the ternary complex. The equilibrium isotope effect is seen as the reason for an earlier-found relative C-13 enrichment in position C-3 and C-4 of glucose and for varying enrichments in C-13 of carbohydrates from different compartments of cells. The kinetic isotope effect is suggested to cause C-13 discriminations in the C-3 pool in context with the hexose formation in competition with other dihydroxyacetone phosphate turnover reactions.
