NEUTRAL IMIDAZOLE IS THE ELECTROPHILE IN THE REACTION CATALYZED BY TRIOSEPHOSPHATE ISOMERASE - STRUCTURAL ORIGINS AND CATALYTIC IMPLICATIONS

To illuminate the role of histidine-95 in the catalytic reaction mediated by triosephosphate isomerase, C-13 and N-15 NMR titration studies have been carried out both on the wild-type enzyme and on a mutant isomerase in which the single remaining histidine (that at the active site) has been isotopically enriched in the imidazole ring. N-15 NMR has proved especially useful in the unambiguous demonstration that the imidazole ring of histidine-95 is uncharged over the entire pH range of isomerase activity, between pH 5 and pH 9.9. The results require that the first pK(a) of histidine-95 is below 4.5. This abnormally low pK(a) rules out the traditional view that the positively charged imidazolium cation of histidine-95 donates a proton to the developing charge on the substrate's carbonyl oxygen. "N NMR experiments on the enzyme in the presence of the reaction intermediate analogue phosphoglycolohydroxamate show the presence of a strong hydrogen bond between N-epsilon-2 of histidine-95 and the bound inhibitor. These findings indicate that, in the catalyzed reaction, proton abstraction from C-1 of dihydroxyacetone phosphate first yields an enediolate intermediate that is strongly hydrogen bonded to the neutral imidazole side chain of histidine-95. The imidazole proton involved in this hydrogen bond then protonates the enediolate, with the transient formation of the enediol-imidazolate ion pair. Abstraction of the hydroxyl proton on O-1 now produces the other enediolate intermediate, which collapses to give the product glyceraldehyde 3-phosphate. This initially surprising sequence is more reasonable when it is recognized that the pK(a) values of the enediol and the perturbed pK(a)2 of the imidazole ring of histidine-95 may be rather close to each other, allowing for two facile and rapid proton transfers that interconvert the two enediolates. To our knowledge, this is the first reported example of the participation of an imidazolate side chain in an enzyme-catalyzed reaction. The imidazole ring of histidine-95 lies at the amino terminus of a short a-helix that will, in accord with what is known from the behavior of substituted imidazoles in solution, lower both the first and the second pK(a) values of the side chain of histidine-95.