Origins of catalysis by computationally designed retroaldolase enzymes

Abstract

We have investigated recently reported computationally designed retroaldolase enzymes with the goal of understanding the extent and the origins of their catalytic power. Direct comparison of the designed enzymes to primary amine catalysts in solution revealed a rate acceleration of 10(5)-fold for the most active of the designed retroaldolases. Through pH-rate studies of the designed retroaldolases and evaluation of a Brønsted correlation for a series of amine catalysts, we found that lysine pK(a) values are shifted by 3-4 units in the enzymes but that the catalytic contributions from the shifted pK(a) values are estimated to be modest, about 10-fold. For the most active of the reported enzymes, we evaluated the catalytic contribution of two other design components: a motif intended to stabilize a bound water molecule and hydrophobic substrate binding interactions. Mutational analysis suggested that the bound water motif does not contribute to the rate acceleration. Comparison of the rate acceleration of the designed substrate relative to a minimal substrate suggested that hydrophobic substrate binding interactions contribute around 10(3)-fold to the enzymatic rate acceleration. Altogether, these results suggest that substrate binding interactions and shifting the pK(a) of the catalytic lysine can account for much of the enzyme's rate acceleration. Additional observations suggest that these interactions are limited in the specificity of placement of substrate and active site catalytic groups. Thus, future design efforts may benefit from a focus on achieving precision in binding interactions and placement of catalytic groups.

Fig. 1.

Major steps in amine-catalyzed retroaldol reaction. Proton transfers, binding events, and some intermediate steps are omitted for clarity.

Fig. 2.

Three elements comprise the catalytic motif used for the retroaldolases investigated in this paper (7), illustrated here with the designed model for the most active enzyme, RA61. The experimental crystal structure for RA61 did not contain ligand, and the binding orientation of the substrate is not known. (A) A hydrophobic pocket was intended to lower the pK_a of the catalytic lysine side chain. (B) Hydrogen-bonding interactions to a bound water molecule were incorporated into the design models. In RA61, the side chains modeled in contact with the bound water are Tyr78 and Ser87. (C) Hydrophobic side chains line the active site cavity, providing a binding surface for the hydrophobic substrate.

Fig. 3.

Dependence of second-order rate constant on pH. (A) Scheme for pH dependence. The deprotonated, neutral form of lysine is required for reaction, whereas protonated, charged lysine cannot form the iminium and progress to products. Thus, in this model, the concentration of active enzyme is controlled by the pK_a of the catalytic lysine. (B) pH dependence for the RA61-catalyzed reaction. The fit to a single titratable group gives a pK_a of 6.8. Plots for the other variants are included in the SI Appendix.

Fig. 4.

(A) Relationship between the pH-independent second-order rate constant of the fully deprotonated, neutral amine (k_max) and pK_a for the retroaldol reactions of 4-hydroxy-4-(6-methoxy-2-naphthyl)-2-butanone catalyzed by amines listed in Table 3. The slope obtained by linear fit is 0.54 ± 0.03, where the error is the standard error of the slope. (B) Relationship between the observed second-order rate constant at pH 7.5 (k_obs) and pK_a for the same reactions.