Specificity of fructose-1, 6-P2 aldolase (muscle) and partition of the enzyme among catalytic intermediates in the steady state.
Others have concluded that beta-fructose 1, 6-bisphosphate is a substrate for muscle aldolase on the basis of rapid kinetic measurements. In view of new data showing excellent aldol cleavage of an analog of the keto form and a very high rate of spontaneous ring opening, Midelfort et al. (Midelfort, C. F., Gupta, R., and Rose, I.A. (1976) Biochemistry 15, 2178-2185) have suggested that the beta form may be used only after spontaneous conversion to the keto form in solution, followed by reaction of the keto form with the aldolase. In order to determine whether beta-fructose-1, 6-P2 is itself a substrate a steady state approach was devised in which the beta form is specifically produced by phosphofructokinase for use by the aldolase present. Since aldolase binds very tightly to bisphosphates similar in size to beta-fructose-1, 6-P2, it was expected that if a spontaneous ring opening were essential for catalysis, it would be possible to decrease the rate of production of triose-Ps by using very high concentrations of aldolase. If the beta form were itself a substrate, the rate would reach a constant value with increasing aldolase, limited by the phosphofructokinase rate. It was found that under conditions where only approximately 2 per cent of the total fructose-1, 6-P2 present in the steady state would be free, the turnover of the complexed fructose-1, 6-P2 was about 20-fold greater than that in which spontaneous ring opening is a required step. Using similar methods, the turnover of enzyme-bound glyceraldehyde-P and dihydroxyacetone-P were determined. It was concluded that at saturation both the beta and acyclic forms have about the same rates as substrates.
D000818:Animals; D005634:Fructose-Bisphosphate Aldolase / Q000378:metabolism*; D007700:Kinetics; D009132:Muscles / Q000201:enzymology*; D014305:Triose-Phosphate Isomerase / Q000378:metabolism
D005634:Fructose-Bisphosphate Aldolase; D014305:Triose-Phosphate Isomerase