Energetically unfavorable protein angles: Exploration of a conserved dihedral angle in triosephosphate isomerase.

Over the past 3.5 billion years of evolution, enzymes have adopted a myriad of conformations to suit life on earth. However, torsional angles of proteins have settled into limited zones of energetically favorable dihedrals observed in Ramachandran plots. Areas outside said zones are believed to be disallowed to all amino acids, except glycine, due to steric hindrance. Triosephosphate isomerase (TIM), a homodimer with a catalytic rate approaching the diffusion limit, contains an active site lysine residue (K13) with dihedrals within the fourth quadrant (Φ = +51/Ψ = -143). Both the amino acid and the dihedral angles are conserved across all species of TIM and known crystal structures regardless of ligand. Only crystal structures of the engineered monomeric version (1MSS) show accepted ß-sheet dihedral values of Φ = -135/Ψ = +170 but experiments show a 1000-fold loss in activity. Based on these results, we hypothesized that adopting the unfavorable torsion angle for K13 contributes to catalysis. Using both, computational and experimental approaches, four residues that interact with K13 (N11, M14, E97, and Q64) were mutated to alanine. In silico molecular dynamics (MD) simulations were performed using 2JK2 unliganded human TIM as a starting structure. Ramachandran plots, containing K13 dihedral values reveal full or partial loss of disallowed zone angles. N11A showed no detectable catalytic activity and lost the unfavorable K13 dihedral angles across four separate force fields during simulation while all other mutants plus wild type retained activity and retained the conserved K13 dihedral angles.

Evaluating the catalytic importance of a conserved Glu97 residue in triosephosphate isomerase.

Investigating enzyme activity is central to our understanding of biological function, and the design of biocatalysts continues to find applications in synthesis. While a role for active site residues can be proposed based on structure and mechanism, our understanding of the catalytic importance for residues surrounding the active site is less well understood. In triosephosphate isomerase (TIM), Glu97 is situated adjacent to the active site and is found in essentially all sequences. Prior studies reported mutation of Glu97 to Asp and Gln in TIM from Plasmodium falciparum (PfTIM) led to a 100- and 4000-fold decrease in activity, respectively, while the E97D mutation in TIM from Gallus gallus (cTIM) had no effect on activity. To investigate further the question of how mutations in essentially superimposable structures give different effects, we mutated E97 in TIM from Trypanosoma brucei brucei (TbbTIM), Saccharomyces cerevisiae (yTIM), and human (hTIM). The E97D, E97A, and E97Q mutations led to a ∼three-tenfold decrease in activity, a modest effect compared to the 102-103-fold effect in PfTIM. CD and fluorescence studies showed the overall structures for the mutants were essentially unchanged. Structural analysis shows that several residues surrounding E97 differ between PfTIM and TIM from the other organisms, and rearrangements or mispositioning of residues in PfTIM may lead to the different rate effects. The results illustrate the interplay of active site and surrounding residues in affecting catalysis and highlight that understanding of the role of residues surrounding the active site may aid in the incorporation of favorable or avoidance of unfavorable interactions when designing enzymes.