JBC, Vol. 252, Issue 2, 479-482, Jan, 1977
Specificity of fructose-1, 6-P2 aldolase (muscle) and partition of the enzyme among catalytic intermediates in the steady state
I. A. Rose and E. L. O'Connell
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.
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