Neutron structures of Leishmania mexicana triosephosphate isomerase in complex with reaction-intermediate mimics shed light on the proton-shuttling steps.

Vinardas Kelpšas,Matthew P Blakeley,Nicolas Coquelle,Claes Von Wachenfeldt,Octav Caldararu,Esko Oksanen,Rikkert K Wierenga,Ulf Ryde

doi:10.1107/s2052252521004619

Vinardas Kelpšas, Matthew P Blakeley + Show 6 more

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https://doi.org/10.1107/s2052252521004619

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Abstract

Triosephosphate isomerase (TIM) is a key enzyme in glycolysis that catalyses the interconversion of glyceraldehyde 3-phosphate and dihydroxy-acetone phosphate. This simple reaction involves the shuttling of protons mediated by protolysable side chains. The catalytic power of TIM is thought to stem from its ability to facilitate the deprotonation of a carbon next to a carbonyl group to generate an enediolate intermediate. The enediolate intermediate is believed to be mimicked by the inhibitor 2-phosphoglycolate (PGA) and the subsequent enediol intermediate by phosphoglycolohydroxamate (PGH). Here, neutron structures of Leishmania mexicana TIM have been determined with both inhibitors, and joint neutron/X-ray refinement followed by quantum refinement has been performed. The structures show that in the PGA complex the postulated general base Glu167 is protonated, while in the PGH complex it remains deprotonated. The deuteron is clearly localized on Glu167 in the PGA-TIM structure, suggesting an asymmetric hydrogen bond instead of a low-barrier hydrogen bond. The full picture of the active-site protonation states allowed an investigation of the reaction mechanism using density-functional theory calculations.

Highlights

Triosephosphate isomerase (TIM; EC 5.3.1.1) plays a central role in glycolysis by interconverting dihydroxyacetone phosphate (DHAP) and glyceraldehyde 3-phosphate (GAP)
The freeenergy profile reveals that the TIM-catalyzed reaction is 109 times faster compared with general base catalysis in solution (Albery & Knowles, 1976). kcat for the DHAP-to-GAP reaction is 860 s–1, while kcat for the reverse reaction is approximately ten times larger (Zhai et al, 2015)
The X-ray refinement of perdeuterated LmTIM in complex with the inhibitor PGA (PDB entry 7abx) at 1.19 Aresolution at 100 K shows a structure of the active site that is similar to that in the 0.82 Aresolution structure of the PGH complex at

Summary

Introduction

Triosephosphate isomerase (TIM; EC 5.3.1.1) plays a central role in glycolysis by interconverting dihydroxyacetone phosphate (DHAP) and glyceraldehyde 3-phosphate (GAP). Both substrates are produced in the reaction catalyzed by fructose1,6-bisphosphate aldolase (EC 4.1.2.13). The kinetic mechanism of TIM has been very well studied, and TIM is often called a catalytically perfect enzyme because of the high rate and high catalytic efficiency of the GAP-toDHAP reaction (Zhai et al, 2015). The kcat/Km value of Saccharomyces cerevisiae TIM is 8.9 Â 106 M–1 s–1 for the GAP-to-DHAP reaction, which is close to the diffusioncontrolled rate limit (Albery & Knowles, 1977). The freeenergy profile reveals that the TIM-catalyzed reaction is 109 times faster compared with general base catalysis in solution (Albery & Knowles, 1976). The freeenergy profile reveals that the TIM-catalyzed reaction is 109 times faster compared with general base catalysis in solution (Albery & Knowles, 1976). kcat for the DHAP-to-GAP reaction is 860 s–1, while kcat for the reverse reaction is approximately ten times larger (Zhai et al, 2015)

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