Forbidden Protein Angles: Triosephosphate Isomerase as a Model for Studying Peripheral Residue Contribution to Catalysis.

Abstract

Over the past 3.5 billion years of evolution, enzymes have adopted a seemingly infinite number 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 forbidden to all amino acids, save glycine, due to steric hindrance. Triosephosphate Isomerase (TIM), a homodimer with a catalytic rate approaching the diffusion limit, contains an active site Lysine 13 (K13) with a dihedral within the forbidden zone (phi=69/psi=−139). Both the amino acid and the dihedral angle 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 β‐sheet dihedral values of phi=−135/psi=170 but experiments show a 1,000‐fold loss in activity. Based on these results, we hypothesized that adopting the forbidden torsion angle for K13 contributes to catalysis. Using a combination of computational and experimental approaches, four residues that interact with K13 (N11, M14, E97, and Q64) were mutated to alanine from single to quadruple mutants. In Silico Molecular Dynamic (MD) simulations of 5×200ns per mutant species were made using 2JK2 unliganded Human TIM as a starting structure. Heat Maps, containing 400,000 K13 dihedral values per run reveal full or partial loss of forbidden zone angles depending on mutant combination. Initial experimental results with E97A single mutant showed a three‐fold decrease in kcat with little change in the KM value suggesting a modest catalytic role for this residue. For this mutant the heatmaps revealed the K13 angle spends less than 30% of its time in the forbidden zone compared to over 50% occupation in the wildtype suggesting the rate decrease may be due to the mutation affecting the positioning of K13. In addition to ongoing In Vivo characterization of the remaining mutants, double mutant cycles will be used to evaluate their energetic connections with K13. As modern protein design relies on naturally occurring protein folds, understanding how to recreate forbidden angles may enhance catalytic rates in unnatural enzymes and achieve chemical reactions not yet envisioned by nature nor organic chemistry.Support or Funding InformationWe would like to thank California State University Long Beach and the Carl E. Riley Endowed STEM scholarship for their generous funding.Heat map of where K13 dihedral occupies space on Ramachandran Plot over 200ns of MD simulation using Wild Type Human Triosephosphate Isomerase (RCSB:2JK2).Figure 1