Covalent Nucleophilic Catalysis: Structures of Enzyme Intermediates in Covalent Enzyme Catalysis

Abstract Enzyme catalysis relies on several factors, such as proper fit of the substrate to the enzyme active site and interactions between substrate and residues that will aid in the chemical reaction. The overall effect is to lower the energy barrier for the reaction. One factor that is observed in some reactions is the formation of an intermediate covalent adduct to a residue in the active site in what is considered the first half of the reaction. This intermediate then goes on to react with another substrate to finish the overall reaction. Sometimes these covalent intermediates can be captured and characterised structurally. The chemical structure of an intermediate can define how a reaction proceeds, how the enzyme responds to binding of substrate, which residues of the active site are involved in the chemical transformation and how the substrate undergoes the rearrangements needed to produce the products. Capture of intermediates is not straightforward and requires different strategies, such as trapping with altered reaction conditions, use of low temperature and the use of mutants that prevent the second half of the reaction to proceed. A number of residues are able to form adducts to a ligand, depending on the nucleophilicity of the atom which undergoes the addition to a substrate (or part of a substrate). Key Concepts Covalent intermediates can lower the energy barrier in an enzyme‐catalysed reaction. Catalysis by an enzyme allows a reaction to occur under conditions that would be unfavourable or exceedingly slow in the absence of the enzyme. Strategies to accomplish this feat depend on the structure and physical properties of the protein to promote precise orientation of a substrate relative to the catalytic residues in an active site and stabilising unstable species with polarising bonds and forming covalent adducts. Structures of covalent intermediates help to define the mechanism by which an enzyme achieves its catalytic power. The structure of covalent intermediates can be trapped by a number of strategies, such as altering reaction conditions such as pH, by drastically lowering the temperature in order to prevent the second half of the reaction to occur or using mutants that prevent the second half of the reaction to occur. The most common covalent intermediates involve acylation, phosphorylation, glycosylation and Schiff base formation. Formation of a covalent adduct can lower the energy barrier of a reaction.