The rate of oxidation of ferricyanide of the aldolase-dihydroxyacetone phosphate complex was measured under different conditions. The following conclusions are drawn. 1. In the cleavage of fructose diphosphate, catalysed by native aldolase, the steady-state concentration of the enzyme-dihydroxyacetone phosphate carbanion intermediate represents less than 6% of the total enzyme-substrate intermediates. 2. Fructose diphosphate and dihydroxyacetone phosphate compete for the four catalytic sites on aldolase, the binding of fructose diphosphate being about twice as tight. 3. The equilibrium concentration of the carbanion intermediate formed by reaction of carboxypeptidase-treated aldolase with dihydroxyacetone phosphate is independent of pH between 5.0 and 9.0. The rates of fromation of the carbanion intermediate and of the reverse reaction are, however, concomitantly increased by increasing pH between 5.0 and 6.5.
