REACTION OF AN ALDOLASE-SUBSTRATE INTERMEDIATE WITH TETRANITROMETHANE

The suitability of applying selective reagents to the detection and identification of enzyme-substrate intermediates has been examined using tetranitromethane (TNM) and aldolase of rabbit muscle. The rate of production of nitroform (є350 14,400) from TNM on addition to aldolase is markedly enhanced by the presence of substrates. Kinetic studies have demonstrated that this phenomenon is due to a reaction of TNM with an aldolase-substrate complex. The rate of nitroform production is directly proportional to the concentration of active enzyme. The relationship between the rate of increase in A350 and substrate concentration reflects apparent Michaelis-Menten kinetics. The substrate concentration resulting in half-maximal rate of nitroform production (Km′) is virtu ally identical with the values of Km for fructose 1,6-diphosphate and fructose 1-phosphate determined enzymatically. Phosphate competitively inhibits the reaction (Ki′ = Ki). The consumption of substrate as a function of time indicates that TNM reacts with the substrate moiety of the enzyme-substrate complex. Comparison of the kinetics of nitroform production by native and carboxypeptidase-treated aldolase with different substrates indicates that the dihydroxyacetone phosphate-aldolase carbanion is the most likely TNM-reactive species. The carbanion specificity of TNM has been demonstrated also with a series of model systems. The potential usefulness of TNM in elucidating enzymatic mechanisms involving carbanion intermediates is discussed. © 1968, American Chemical Society. All rights reserved.