The mechanism of action of aldolases.

In previous work from this laboratory, we have demonstrated the formation of a Schiff base intermediale between fructose 1,6-diphosphate aldolase and one of its substrates, dihydroxyacetone phosphate (l-3). Upon reduction of the intermediate with sodium borohydride, it is converted to a stable secondary amine derivative which has been isolated from the protein hydrolysates and identified as NG-P-glyceryllysine. The formation of this stable derivative has provided a means of labeling the active site of fructose diphonphate aldolase. Since fructose diphosphate aldolase is composed of three peptide chains of approximately equal molecular weight, one of which appears to differ from the other two in its carboxyl-terminal sequence (448), it became of interest to establish the number of active sites in the enzyme protein. Our first evidence (1) suggested that only one was present, but later studies, based on the labeling of the protein with 14C-dihydroxyacetone phosphate, indicated t,hat 2 moles of substrate could combine (3). This was in contrast to results obtained in equilibrium dialysis (9), which were consistent with only a single active site in the pr0tein.l During the course of an investigation of the structure of the active site of fructose diphosphate aldolase, we have obtained further evidence for the presence of two combining sites per enzyme molecule; these results are reported here.