Studies on the interaction of aldolase with substrate analogues.

Interactions of specific and nonspecific agents at the dihydroxyacetone-P and glyceraldehyde-3-P sites of rabbit muscle aldolase are shown by several methods: quenching of protein fluorescence, protection against the destructive action of trypsin, the inhibition of initial rates and of equilibrium exchange rates, and the inactivation caused by NaBH4. It is concluded from inactivation and labeling studies that glycolaldehyde-P can form a Schiff’s base at the dihydroxyacetone-P site. D-Glyceraldehyde-3-P and erythrose-4-P, like dihydroxyacetone-P, cause fluorescence quenching at low concentration, whereas L-glyceraldehyde-3-P, which is a good acceptor for dihydroxyacetone-P, does not. The competitive inhibition shown by small anions such as Cl-, Pi, and ethyl-P is first order for these anions with fructose-l-P as thle substrate but approximately second order with fructose-1,6-di-P as substrate. This suggests that binding of fructose-l-P to the enzyme is prevented by an anion bound to the dihydroxyacetone-P site, but not by one at the aldehyde-P site, whereas fructose-di-P binding is prevented by anion interaction with either site. 0 II Acetol-P (CH8-C-CH20P03=) is a substrate for the proton exchange reaction of aldolase. It has a much higher Km than dihydroxyacetone-P, the maximum velocity of exchange is about 17% as great, and its rate of condensation with glyceraldehyde-3-P only about 1% that found with dihydroxyacetone-P. L-Glyceraldehyde-3-P is a competitive inhibitor of the proton exchange reaction of acetol-P but does not react with the dihydroxyacetone-P site as shown by lack of fluorescence quenching. This suggests that, although the enzyme can bind aldehyde, the complex is inappropriate for productive reaction with dihydroxyacetone-P. This is consistent with earlier studies requiring that the condensation is an ordered reaction with dihydroxyacetone-P reacting before aldehyde. The inactivation of aldolase as a result of reaction with L-