Abstract
Conformational changes are crucial for the catalytic action of many enzymes. A prototypical and well-studied example is loop opening and closure in triosephosphate isomerase (TIM), which is thought to determine the rate of catalytic turnover in many circumstances. Specifically, TIM loop 6 "grips" the phosphodianion of the substrate and, together with a change in loop 7, sets up the TIM active site for efficient catalysis. Crystal structures of TIM typically show an open or a closed conformation of loop 6, with the tip of the loop moving ∼7 Å between conformations. Many studies have interpreted this motion as a two-state, rigid-body transition. Here, we use extensive molecular dynamics simulations, with both conventional and enhanced sampling techniques, to analyze loop motion in apo and substrate-bound TIM in detail, using five crystal structures of the dimeric TIM from Saccharomyces cerevisiae. We find that loop 6 is highly flexible and samples multiple conformational states. Empirical valence bond simulations of the first reaction step show that slight displacements away from the fully closed-loop conformation can be sufficient to abolish most of the catalytic activity; full closure is required for efficient reaction. The conformational change of the loops in TIM is thus not a simple "open and shut" case and is crucial for its catalytic action. Our detailed analysis of loop motion in a highly efficient enzyme highlights the complexity of loop conformational changes and their role in biological catalysis.
Highlights
The simulation time in the present study is substantially longer than in previous molecular dynamics (MD) studies of Triosephosphate isomerase (TIM) loop dynamics,[7,8,39,40,102] and it is plausible that similar observations of loop opening would have been made in those studies had the simulation time been extended
We have previously modeled the deprotonation of substrates dihydroxyacetone phosphate (DHAP) and GAP by TIM in the closed state of the enzyme,[26] using the empirical valence bond (EVB) approach and the same PDB structure (PDB ID: 1NEY)[16,35] that was used in this work
Understanding how TIM is regulated is of interest from a biocatalysis perspective, as the TIM barrel is one of the most versatile and evolvable protein scaffolds.[11,15,24,32]
Summary
Triosephosphate isomerase (TIM) is an enzyme that catalyzes a simple reversible isomerization reaction, namely the isomerization of dihydroxyacetone phosphate (DHAP) and (R)-glyceraldehyde-3-phosphate (GAP, Figure 1A).[1−5] It does so with tremendous catalytic proficiency, such that the turnover of this enzyme has been argued to be limited by diffusion.[2,3] As a result, TIM has often been described as a “catalytically perfect” enzyme[3] and has been the subject of extensive experimental and computational studies, as a model system for understanding the factors that drive enzyme catalysis (see refs 2 and 4−27 as just a few examples).Structurally, TIM is usually a homodimer and gives its name to the archetypal TIM barrel fold, which consists of eight αhelices and eight parallel β-sheets alternating along the protein backbone (Figure 1B). We note that exceptions exist: In some organisms, TIM is a tetramer, and the change in oligomerization state can be functionally important.[28−30] TIM barrels, which are by far one of the most commonly occurring protein folds,[31] provide a versatile[11] and highly evolvable[11,15,24,32] scaffold and have been argued to have facilitated the early evolution of protein-mediated metabolism.[24] In addition, TIM barrel enzymes are excellent model systems for understanding enzymatic thermal adaptation.[33,34] A defining feature of reaction in TIM is the large motion (up to 7 Å) of a phosphate gripper loop, loop 6 (residues Pro166-Ala[176] for yeast TIM from Saccharomyces cerevisiae, yTIM, PDB ID: 1NEY),[16,35] which interacts first with loop 5 in the unliganded form of TIM, and subsequently with loop 7 of TIM and with the phosphodianion of the bound substrate (Figure 1C). This dianion is, in turn, anchored to the enzyme through (up to) four hydrogen bonds with the protein backbone (Figure 1D).[16]
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