Energetics of triosephosphate isomerase: the fate of the 1(R)-³H label of tritiated dihydroxyacetone phosphate in the isomerase reaction

Abstract

The isomerization of specifically tritium-labeled [ 1 (R)-3H]dihydroxyacetone phosphate to D-glyceraldehyde 3-phosphate, catalyzed by the enzyme triosephosphate isomerase, has been studied. The distribution of the 3H label amongst the three possible sites (in [ 1 (R)-3Hldihydroxyace-tone phosphate, in ~-[2-~H]glyceraldehyde 3-phosphate, and in the solvent) has been followed as a function of the extent of the reaction. It is shown that the extent of transfer of the 3H label from the substrate dihydroxyacetone phosphate to Dglyceraldehyde 3-phosphate is between 3 and 6% (depending upon the extent of the reaction). The enzymic base responsible Tiosephosphate isomerase catalyzes the interconversion of the two triose phosphates, dihydroxyacetone phosphate and glyceraldehyde 3-phosphate. The crystalline enzyme from chicken muscle is a dimer of identical subunits each of molecular weight about 26 000 (Putman et a]., 1972). It has no cofactor or specific metal ion requirements. As an isomerase, with a single substrate and a single product, it is particularly amenable to mechanistic study, and the equilibrium constant for the reaction is close enough to unity to allow the reaction to be run in either direction, utilizing the appropriate dehydrogenases as coupling enzymes (a-glycerophosphate dehydrogenase to remove dihydroxyacetone phosphate, or glyceraldehyde-phosphate dehydrogenase to remove glyceraldehyde phosphate). In the presence of excess of the appropriate dehydrogenase and cofactor, the reaction can be studied under conditions such that the isomerase step is rate limiting (Plaut and Knowles, 1972).Aside from these attractive features, this system presents a rare opportunity for crystallographic and spectroscopic study. With most enzymes that use two or more substrates in either direction, even if one of these is water, it is currently impossible to study productive enzyme-substrate complexes directly by crystallographic methods. approaches to the problem of the structure of enzyme-substrate complexes have had to rely on the structures of enzyme-inhibitor complexes, or "dead-end" binary complexes or abortive ternary complexes \lor two substrate systems). From such data, one has to try to deduce the structure of enzyme with real substrate bound (see, e.g., Blake et al., 1967; Steitz et al., 1969). For an isomerase, however, it is possible to find the structure both of native enzyme and of an enzyme-substrate complex. Progress with each for proton abstraction from substrate is, therefore, in almost complete isotopic equilibrium with the solvent. The remaining substrate after partial reaction increases in specific radioactivity as the reaction proceeds, showing that the preferential reaction of ' H substrate is more important than the washing out of 3H label at the stage of the exchanging intermediate. Quantitatively, these results provide the data for the first steps in the analysis described in the previous paper (Albery, W. J., and Knowles, J. R. (1976), Biochemistry, preceding paper in this issue). of these probl...

Substrate product equilibrium on a reversible enzyme, triosephosphate isomerase

“…As much as 50% intramolecular transfer of proton from GAP to DHAP and Ϸ18% from DHAP to GAP were documented. The later is considerably higher than the Ͻ6% detected by radioactivity and mass spectral analysisbased assays (3,25). Notably, the same study also found that partitioning of the endiol(ate) depends on the substrate identity; the reaction of the two substrates is not likely to share a common, single intermediate.…”

“…The precise path of the transferred proton and the identity of the reaction intermediates have been, by and large, deduced from the fate of isotopic labels (1,2). The existence of a stable enediol(ate) intermediate has been inferred from the loss of an isotope label from the substrate during the reaction (1,3); this reaction intermediate must exist with a sufficient lifetime to allow partial equilibration of the catalytic base with the surrounding solvent. Moreover, when the reaction takes place in tritiated water the solvent's tritium is incorporated into both the substrate and product, evidence that the hydrogen of the newly formed carbonhydrogen bond is mostly derived from the solvent (4,5) and that exchange occurs from an enzyme bound intermediate.…”

Common Enzymological Experiments Allow Free Energy Profile Determination

“…A well known example is that of Knowles et al . where the FEP for TIM was partially defined using various isotopic measurements (1, 2, 33, 60–62). Other notable examples include work by Knowles et al .…”

Segmental motion in catalysis: investigation of a hydrogen bond critical for loop closure in the reaction of triosephosphate isomerase

“…Phosphoglycolohydroxamate [bis(cyclohexylammonium) salt] was synthesized by Dr. J. G. Belasco as described in Belasco and Knowles (1980). [ 1 (R)-3H] Dihydroxyacetone phosphate (9 Ci/mol) was prepared by Dr. R. T. Raines according to the method of Herlihy et al (1976). Phospho-[l-I4C]glycerate (14.1 Ci/mol) was prepared by Dr. D. L. Pompliano according to the method of Guilford et al (1987).…”