title: Site-directed mutagenesis of ricin A-chain and implications for the mechanism of action.
abstract: Ricin A-chain is an N-glycosidase that attacks ribosomal RNA at a highly conserved adenine residue. The enzyme is representative of a large family of medically significant proteins used in the design of anticancer agents and in the treatment of HIV infection. The x-ray structure has been used as a guide to create several active site mutations by directed mutagenesis of the cloned gene. Glu177 is a key catalytic residue, and conversion to Gln reduces activity 180-fold. Asn209 is shown to participate in substrate binding by kinetic analysis. Conversion to Ser increases Km sixfold but has no effect on kcat. Conversion of Tyr80 and Tyr123 to Phe decreases activity by 15- and 7-fold respectively. A mechanism of action is proposed that involves binding of the substrate adenine in a syn configuration that resembles the transition state; the putative oxycarbonium ion is probably stabilized by interaction with Glu177.
authors.affiliation: Clayton Foundation Biochemical Institute, Department of Chemistry and Biochemistry, University of Texas, Austin 78712.
authors.forename: M P
authors.initials: MP
authors.lastname: Ready
authors.forename: Y
authors.initials: Y
authors.lastname: Kim
authors.forename: J D
authors.initials: JD
authors.lastname: Robertus
mesh_terms: D001483:Base Sequence; D001665:Binding Sites; D003001:Cloning, Molecular; D004252:DNA Mutational Analysis; D006026:Glycoside Hydrolases / Q000502:physiology*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D012276:Ricin / Q000235:genetics* / Q000494:pharmacology; D013329:Structure-Activity Relationship
chemical_list: D012276:Ricin; D006026:Glycoside Hydrolases