G. Gleixner, H. Schmidt
Feb 28, 1997
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4
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Journal
The Journal of Biological Chemistry
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 13C 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 13C content of the corresponding positions after partial and complete turnover, and after complete equilibration of the reactants. 13(Vmax/Km) on C-3 was 1.016 ± 0.007 and 0.997 ± 0.009 on position C-4, and the equilibrium isotope effects K12/K13 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 13C enrichment in position C-3 and C-4 of glucose and for varying enrichments in 13C of carbohydrates from different compartments of cells. The kinetic isotope effect is suggested to cause 13C discriminations in the C-3 pool in context with the hexose formation in competition with other dihydroxyacetone phosphate turnover reactions.