Interaction between rabbit muscle aldolase and dihydroxyacetone phosphate.

It is generally accepted that the mechanism of enzyme activity includes a combination of enzyme and substrate. This concept forms the basis for the conventional kinetic analyses of enzymatic reactions (1). Direct evidence for the existence of enzyme-substrate combinations is as yet meager. The binding of pyridine nucleotide coenzymes as substrates to various dehydrogenases has been shown to result in changes in absorption and fluorescence (2). In several other cases, the enzyme has been demonstrated to be a participant in the overall reaction, accepting, for example, an acyl group in one step of the reactions of chymotrypsin (3) and trios, phosphate dehydrogenase (4)) or undergoing oxidation in the reactions of perosidase (5). Equilibrium dialysis and ultracentrifugation (6) also have been used to measure the binding of enzyme and substrate; such techniques have shown specific binding of substrate, as well as nonspecific absorption. Rabbit muscle aldolase is a well known representative of a group of enzymes that catalyze aldol reactions with various substrates (7). Similar reactions catalyzed by the transferring enzymes, transaldolase (8) and transketolase (9, lo), recently have been shown to involve the intermediate formation of a derivative of the enzyme with the fragment to be transferred. Particularly in the case of transaldolase, the overall reaction bears a great similarity to the reaction of aldolase, but it remains to be determined whether the two processes involve similar mechanisms. In a previous report, it was shown that the combination of dihydrosyacetone phosphate and aldolase absorbs ultraviolet light, and it was suggested that the absorbing material is the catalytically active enzyme-substrate complex (11). The measurements reported in this paper indicate that enzymatically altered, active aldolase (12) produces spectral changes with dihydrosyacetone phosphate similar to those caused by native aldolase, whereas inactivated enzyme produces no change. The increases in optical density measured as a function of concentration have been used to calculate the dissociation constant of aldolase and dihydroxyacetone phosphate in the absence of a second substrate.