Dynamic interactions of enzymes involved in triosephosphate metabolism.
A steady-state kinetic analysis of the coupled reactions catalysed by the three-enzyme system, aldolase, glyceraldehyde-3-phosphate dehydrogenase and triosephosphate isomerase, was performed. The kinetic parameters of the progress curves of end-product formation calculated for noninteracting enzymes were compared with those measured in the two-enzyme and three-enzyme systems. Changes in the fluorescence anisotropy of labelled dehydrogenase upon addition of aldolase and/or isomerase were also measured. Glyceraldehyde-3-phosphate oxidation catalysed by glyceraldehyde-3-phosphate dehydrogenase in the presence of isomerase (which ensures rapid equilibration of the triosephosphates) follows single first-order kinetics. The rate constant depends simply on the concentration of the dehydrogenase, indicating no kinetically significant isomerase-dehydrogenase interaction. Fluorescence anisotropy measurements also fail to reveal complex formation between the two enzymes. The steady-state velocity of 3-phosphoglycerate formation from fructose 1, 6-bisphosphate in the reactions catalysed by aldolase and dehydrogenase is not increased twofold on addition of the isomerase, even though a 1:2 stoichiometry of fructose 1,6-bisphosphate/glyceraldehyde 3-phosphate is expected. In fact, by increasing the concentration of the isomerase, the steady-state velocity actually decreases. This effect of the isomerase may be a kinetic consequence of an aldolase-isomerase interaction, which results in a decrease of aldolase activity. Furthermore, the fluorescence anisotropy of labelled dehydrogenase, measured at different aldolase concentrations, is significantly lower when the sample contains isomerase. The decrease in the steady-state velocity of the consecutive reactions caused by the elevation of isomerase concentration could be negated by increasing the dehydrogenase concentrations in the three-enzyme system. All of these observations fit the assumption that the amount of aldolase-dehydrogenase complex is reduced due to competition of isomerase with dehydrogenase. The alternate binding of dehydrogenase and isomerase to aldolase may regulate the flux rate of glycolysis.
D000818:Animals; D002238:Carbohydrate Epimerases / Q000378:metabolism*; D005454:Fluorescence Polarization; D005634:Fructose-Bisphosphate Aldolase / Q000378:metabolism*; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000378:metabolism*; D007700:Kinetics; D009132:Muscles / Q000201:enzymology; D011817:Rabbits; D014305:Triose-Phosphate Isomerase / Q000378:metabolism*
D005987:Glyceraldehyde-3-Phosphate Dehydrogenases; D005634:Fructose-Bisphosphate Aldolase; D002238:Carbohydrate Epimerases; D014305:Triose-Phosphate Isomerase