Distal mutations enhance catalysis in designed enzymes by facilitating substrate binding and product release.

ABSTRACT: The role of amino-acid residues distant from an enzyme's active site in facilitating the complete catalytic cycle-including substrate binding, chemical transformation, and product release-remains poorly understood. Here, we investigate how distal mutations promote the catalytic cycle by engineering mutants of three de novo Kemp eliminases containing either active-site or distal mutations identified through directed evolution. Kinetic analyses, X-ray crystallography, and molecular dynamics simulations reveal that while active-site mutations create preorganized catalytic sites for efficient chemical transformation, distal mutations enhance catalysis by facilitating substrate binding and product release through tuning structural dynamics to widen the active-site entrance and reorganize surfac

SUBMITTER: Zarifi N 

PROVIDER: S-EPMC12484545 | biostudies-literature | 2025 Sep

REPOSITORIES: biostudies-literature