Abstract
Enzyme motions on a broad range of time scales can play an important role in various intra- and intermolecular events, including substrate binding, catalysis of the chemical conversion, and product release. The relationship between protein motions and catalytic activity is of contemporary interest in enzymology. To understand the factors influencing the rates of enzyme-catalyzed reactions, the dynamics of the protein-solvent-ligand complex must be considered. The current review presents two case studies of enzymes—dihydrofolate reductase (DHFR) and thymidylate synthase (TSase)—and discusses the role of protein motions in their catalyzed reactions. Specifically, we will discuss the utility of kinetic isotope effects (KIEs) and their temperature dependence as tools in probing such phenomena.
Highlights
Enzymes are involved in a well-orchestrated series of metabolic reactions that facilitate many processes essential to life
The current review describes selected experimental approaches that explore the relationship between protein dynamics and the bond activations of two enzymes, dihydrofolate reductase (DHFR) and thymidylate synthase (TSase)
H-transfer steps in different reaction mechanisms, and extend our understanding of enzyme catalysis. Those studies used a combination of different experimental techniques, including temperature dependence of intrinsic kinetic isotope effects (KIEs), steady state kinetics, site-directed mutagenesis, NMR and X-ray crystallography to study two different model enzymatic systems, TSase and DHFR
Summary
Thymidylate synthase (EC 2.1.1.45, TSase), a pyrimidine-metabolizing enzyme, is one of the most highly-conserved proteins found in nature. This is not surprising as it is involved in the last step of de novo synthesis of a precursor of DNA, 2′-deoxythymidine-5′-monophosphate (dTMP), using (6R)-N5,N10-methylene-5,6,7,8-tetrahydrofolate (CH2H4folate) as a cofactor and 2′-deoxyuridine-5′monophosphate (dUMP) as the substrate [66]. The mechanism of TSase (Scheme 1) involves several chemical bond activations including two H transfers: a rate limiting hydride transfer (step 5) and a much faster proton transfer (step 4) Adapted from Ref [11] with copyright permission from the American Chemical Society
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