Engineering a model protein cavity to catalyze the Kemp elimination

Abstract

Synthetic cavitands and protein cavities have been widely studied as models for ligand recognition. Here we investigate the Met102 → His substitution in the artificial L99A cavity in T4 lysozyme as a Kemp eliminase. The resulting enzyme had k(cat)/K(M) = 0.43 M(-1) s(-1) and a (k(cat)/K(M))/k(uncat) = 10(7) at pH 5.0. The crystal structure of this enzyme was determined at 1.30 Å, as were the structures of four complexes of substrate and product analogs. The absence of ordered waters or hydrogen bonding interactions, and the presence of a common catalytic base (His102) in an otherwise hydrophobic, buried cavity, facilitated detailed analysis of the reaction mechanism and its optimization. Subsequent substitutions increased eliminase activity by an additional four-fold. As activity-enhancing substitutions were engineered into the cavity, protein stability decreased, consistent with the stability-function trade-off hypothesis. This and related model cavities may provide templates for studying protein design principles in radically simplified environments.

Fig. 1.

The Kemp elimination.

Fig. 2.

Structures of the model cavity sites. For panels B–F, the structures of the L99A/M102H† cavity site are shown as a blue surface with bound ligands (carbons white, nitrogens blue, oxygens red, sulfur yellow). F_o - F_c density (calculated after refinement but before the introduction of the ligand) at 3σ is shown as black mesh. (A) The molecular surface of the L99A cavity (PDB ID 181L) (inner) embedded within the overall surface of the protein (outer). The surfaces of ionic residues are shown in red, polar residues in yellow and nonpolar residues in green. Several cavity lining residues are rendered as sticks (oxygens red, nitrogens dark blue, carbons pale blue). The polar atoms of Tyr88 and Ser117 are oriented away from the cavity, which is almost entirely apolar in nature. (B) L99A/M102H† complexed with 2-mercaptoethanol at 1.30 Å. The hydrogen bond observed in all the structures between Sδ of Met106 and Nε2 of His102 is shown. (C) L99A/M102H† complexed with nitrobenzene at 1.64 Å. (D) L99A/M102H† complexed with 4-nitrophenol at 1.54 Å. (E) L99A/M102H† complexed with 2-cyanophenol at 1.49 Å. (F) L99A/M102H† complexed with benzisoxazole at 1.64 Å, modeled in the catalytically competent pose showing the 3.3 Å distance between Nδ1 of His102 and the acidic carbon of the Kemp substrate. Figures rendered with PyMol.

Fig. 3.

The relationship between Kemp eliminase activity and protein stability of the lysozyme cavity mutants. The number of substitutions required to go from WT T4 lysozyme to the specific cavity mutant is represented against the change in stability (ΔΔG, kcal/mol) between that mutant and WT. The residues present in the N-terminal hexahistidine tag are not counted towards the mutation number. The Kemp eliminase activity [k_cat/K_M (M^-1 s^-1)] of each construct is represented by the size of the circles (see Table 1 and SI Appendix, Table S1). Black arrows represent mutations that were intended to increase stability, red arrows indicate mutations that were intended to increase activity, and dashed black and red lines indicate mutations of both kinds were included in that step.

Fig. 4.

Sacrificing an ancestral active site to engineer a new one. (A) The active site of T4 lysozyme (Left) provides an enzymatic function at the cost of reducing the stability of the protein structure (Right) The axes on the charts are purely qualitative to illustrate the stability-function trade-off. (B) Stability-restoring substitutions (such as E11F, D20N,T21C, and T142C) were engineered into the muramyl peptide site of lysozyme, eliminating the enzyme's activity as a hydrolase. (C) The restored stability of the lysozyme protein can now be traded for a new function, in this case Kemp eliminase activity with the introduction of the M102H mutation.