CARBANION INTERMEDIATES IN REACTION OF YEAST AND MUSCLE ALDOLASE

Fructose 1,6-diphosphate aldolase from rabbit muscle is a class I aldolase, i.e., it forms a Schiff base ezymesubstrate complex involving an active-center lysyl residue. Its mechanism of action involves a tetranitromethane-reactive intermediate, most likely a carbanion (Christen, P., and Riordan, J. F. (1968), Biochemistry 7, 1531). The carbanion reactivity of tetranitromethane has now been confirmed by titration studies of a series of carbon acids. Yeast aldolase is a class II aldolase, i.e., it requires a metal ion for catalysis and does not form a Schiff base complex. The present investigation demonstrates that this enzyme also forms a tetranitromethanereactive carbanion intermediate. Kinetic data reveal that the reactive carbanion occurs during catalysis and is located on the dihydroxyacetone phosphate moiety of the yeast aldolase-substrate complex. The activating effect of potassium ions on the tetranitromethane reaction closely parallels their known effects on cleavage activity of this enzyme suggesting that potassium ions enhance the rate of carbanion formation. EDTA abolishes the tetranitromethane reaction, consistent with the view that the metal ion of the yeast enzyme serves a function analogous to that of the lysyl residue of the muscle enzyme. The tetranitromethane reaction traps similar fractional amounts of the total intermediary dihydroxyacetone phosphate carbanion of both the yeast and muscle enzymes. Even though these two enzymes employ different catalytically functional residues their reaction mechanisms are apparently very similar. © 1969, American Chemical Society. All rights reserved.