The mechanistic pathway of a mutant triosephosphate isomerase.

The glycolytic enzyme triosephosphate isomerase catalyzes the interconversion of dihydroxyacetone phosphate and D-glyceraJdehyde 3-phosphate, following the pathway iHustrated in SCHEME 1.1,2 From a variety of kinetic,3 stereochemical,4 and chemical modification studies,s.6 it is known that an enzymic base abstracts the I-pro-R proton of dihydroxyacetone phosphate to yield the cis-enediol (or enedioiate) intermediate. which then collapses as a proton is delivered to carbon-2 in the formation of D-glyceraJdehyde 3-phosphate. When stereospecificaHy labeled 1[(R)_3H] dihydroxyacetone phosphate is used as substrate, some 3% to 6% of the tritium label ends up at carbon 2 of the product D-glyceraldehyde 3-phosphate. This small but significant level of proton transfer from carbon-l to carbon-2 strongly suggests that there is a single base at the active site of the isomerase. The rate of exchange of the proton in B-H (SCHEME 1, at the enediol stage of the reaction) with the solvent is