[4] Site-directed mutagenesis: A tool for studying enzyme catalysis

Enzymes catalyze reactions, by binding and orienting substrates in an active site, formed with many amino acid residues, that participate in the chemical transformation. Three-dimensional structures, determined by X-ray crystallography, show that enzymes bind substrates with a combination of ionic and hydrogen bonds and Van Der Waals interactions. The specificity of enzymes is due to multiple interactions that define the size and shape of the active site. The enzyme catalyzes the reaction, by affecting solvation and the electrostatic environment and by facilitating proton transfers and covalent chemistry, at the reaction center. Understanding the principles of enzyme action should allow designing specific catalysts. This chapter discusses the purposes for using site-directed mutagenesis, some of the precautions for such studies, and an overall approach that is designed to provide maximal information. Some studies are selected to discuss what can be learned about the marvels of enzymes and to show why some conclusions are uncertain.