The study of two enzymes using unnatural amino acid mutagenesis: p21(Ras) and chloramphenicol acetyl transferase

Unnatural amino acid mutagenesis allows the site-specific incorporation of amino acids with steric and electronic properties tailored to answer specific questions about protein structure and function. This work describes the use of unnatural amino acid mutagenesis to study the mechanism of p21;Initial work on p21;The p21;In studies on the active site of CAT, it was possible to distinguish the action of Thr174 as a hydrogen bond donor rather than an acceptor and to show that the interaction of Thr174 with waters in the substrate-binding pocket is solely responsible for transmitting effects between the substrate binding sites. Ser148 was shown to make a contact to acetyl coenzyme A in the binary complex that must be broken in the transition state. Finally, mutations at His195 support its role as an active site base and a mutation of the backbone between His195 and Ala196 support a structural role for Arg18.