Liberation of the triosephosphate isomerase reaction intermediate and its trapping by isomerase, yeast aldolase, and methylglyoxal synthase

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Abstract
When a mixture of triosephosphate isomerase (rabbit muscle) and dihydroxyacetone phosphate (DHAP) is quenched with acid, a compound is liberated, presumed to be the cis-enediol 3-phosphate, that decomposes to Pi and methylglyoxal. The decomposition can be prevented by rapid neutralization if a catalytic amount of fresh isomerase is present. Varying the time between acidification and rescue gave a half-life of the liberated compound of .apprx. 12-17 ms. Varying the concentration of enzyme used for rescue gave a minimum 2nd-order rate constant for trapping of 109 M-1 s-1. These results add further evidence favoring a stepwise mechanism for the aldose-ketose isomerase reactions in which a chemically defined enzyme-bound intermediate is formed. The high rate of trapping over a wide pH range indicates that the enediol phosphate, not the enediolate phosphate, is the intermediate. One property of the enzyme is to stabilize the intermediate with respect to its fragmentation in solution by > 1000-fold. Yeast aldolase is also able to rescue all of the isomerase intermediate, though higher concentrations of enzyme are required. Although different enantiotopic protons of DHAP are abstracted by isomerase and aldolase, both enzymes use the same enediol phosphate intermediate. Methylglyoxal synthase at a 50-fold greater concentration was unable to compete with triosephosphate isomerase for cis-enediol phosphate. Either the synthetase has a low V/K for the cis isomer or it uses the trans-enediol phosphate form specifically. A new strategy for the chemical and enzymological characterization of enzyme reaction intermediates is provided here based on the liberation of the intermediate from the reaction equilibrium and its recovery by fresh enzyme or another enzyme species.