Evolution of the Chemical Step in Enzyme Catalysis

Abstract

Enzymes have evolved to increase chemical reaction rates, some by factors exceeding the trillions, thus enabling the remarkable success of life on Earth. A typical enzymatic process includes substrate binding, a chemical step involving covalent bond rearrangements, and product release. A distinct energy threshold must be overcome for each of these steps to proceed. Past studies of enzyme evolution have focused on how the overall catalytic process or specific steps such as binding respond to selective pressures, but researchers have not deliberately monitored the evolution of the chemical step per se until now. To study the chemical step, we measured the temperature dependence of intrinsic kinetic isotope effects of dihydrofolate reductase from primitive to evolved variants. We found a progressive decrease in the temperature dependence of intrinsic kinetic isotope effects with evolution, indicating gradual narrowing of the thermally averaged donor–acceptor distance for hydride transfer in step with an increased catalytic efficiency. The important role played by residues that are remote from the active site in optimizing the chemical step of this complex, multistep enzymatic pathway is particularly notable.