Enhancement of Z-aspartame synthesis by rational engineering of metalloprotease

Metalloprotease PT121^Y114S, an effective catalyst for Z-aspartame synthesis under the substrate (Z-Asp:L-Phe-OMe) molar ratio of 1:5, was obtained previously. Herein, a computational strategy combining molecular dynamics simulation of the enzyme-substrate complex with binding free energy (ΔG) calculations was established to guide the further engineering of PT121^Y114S. One His224 residue proximal to the PT121^Y114S active site was selected on the basis of the difference in ΔG decomposition of PT121^Y114S toward L-Phe-NH₂ and L-Phe-OMe. Site-saturation mutagenesis of His224 resulted in the mutants H224D, H224N, and H224S, which showed great improvement in Z-aspartame synthesis under an economical substrate molar ratio approaching 1:1. Furthermore, the kinetic constants of PT121^Y114S and its mutants revealed that the affinity of mutants toward the L-Phe-OMe was significantly higher than that of PT121^Y114S. Molecular dynamic simulation revealed that the enhanced synthetic activity may be attributed to the mutation stabilizing the transient state of the enzyme-L-Phe-OMe complex.