Neutral imidazole is the electrophile in the reaction catalyzed by triosephosphate isomerase: structural origins and catalytic implications

Abstract

To illuminate the role of histidine-95 in the catalytic reaction mediated by triosephosphate isomerase, 13C and 15N 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. 15N 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 pKa of histidine-95 is below 4.5. This abnormally low pKa 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. 15N 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.(ABSTRACT TRUNCATED AT 250 WORDS)

Optimal alignment for enzymatic proton transfer: Structure of the Michaelis complex of triosephosphate isomerase at 1.2-Å resolution

“…As for all other TIM structures, the hydrogen bonding context of His-95 suggests that it is neutral, and protonated at the (amino) N position. Previous NMR studies demonstrated that the imidazole group is neutral at physiological pH values (13). In the Michaelis complex structure, the imino N␦ forms a hydrogen bond with an amidic NH moiety from the protein backbone; N forms a bifurcated hydrogen bond to O2 (2.6-2.7 Å) and O1 (3.0 Å) of the substrate (Fig.…”

“…This electrophile has a crucial kinetic role in facilitating proton transfer, as evidenced by the disastrous effects on k cat when it is replaced by other functionalities (16). Short hydrogen bonds involving His-95 and analog compounds have been characterized with diffraction (9), IR (16), and NMR experiments (13,17), as well as with computational tools (19). The structures reported herein, at nearatomic resolution, allowed us to define the hydrogen bonding parameters in the presence of the actual substrate for the first time.…”

“…Spectroscopic and mutation studies have focused on the polarization of the substrate by catalytic residues as well as on the stabilization of the enendiol(ate) intermediate (10)(11)(12)(13)(14)(15)(16)(17)(18). Computational effort has focused on the clarification of steps between the deprotonation at C1 of DHAP by glutamic acid 165 and protonation of C2 to form GAP (19)(20)(21)(22), wherein the substrate's oxygens also participate in proton transfer reactions, as well as the contribution of proton tunneling in the reaction mechanism (23).…”

An aspartate and a water molecule mediate efficient acid-base catalysis in a tailored antibody pocket

“…The extraordinary efficiency of TIM (23) and KSI (24) (k cat /K m Ϸ10 8 M Ϫ1 s Ϫ1 ), for instance, arises from synergistic action of a carboxylate base, which initiates proton abstraction from carbon, and a general acid, either a histidine (25) or a tyrosine (26), that stabilizes the resulting anionic intermediate via hydrogen bonding.…”

pH Titration studies of an SH2 domain‐phosphopeptide complex: Unusual histidine and phosphate pKa values

“…Examples include changes in 31P chemical shifts to study phosphate pKas (Vogel, 1989), "&l, and ISNf2 chemical shifts of histidine imidazole groups (Bachovchin, 1986;van Dijk et al, 1990van Dijk et al, . 1992Lodi & Knowles, 1991;Annand et al, 1993;Pelton et al, 1993;Tsilikonas et al, 1996) and I3C side chain carboxyl chemical shifts for aspartic and glutamic acid residues (Jeng & Dyson, 1996;Qin et al, 1996).…”