IDENTIFICATION OF AMINO-ACID-RESIDUES OF ABRIN-A A-CHAIN IS ESSENTIALFOR CATALYSIS AND REASSOCIATION WITH ABRIN-A B-CHAIN BY SITE-DIRECTEDMUTAGENESIS

Abrin is a toxic protein consisting of two subunits, an enzymatic A ch ain (ABRaA) and a lectin-active B chain (ABRaB), linked by a disulfide bond, Site-directed mutagenesis was performed using PCR to study how the conserved amino acid residues, Tyr74, Tyr113 Glu164 and Trp198, ar ound the active site of ABRaA are involved in enzyme catalysis, enzyme -substrate recognition and reassociation of ABRaA with ABRaB, The prot ein biosynthesis inhibitory activities of Y74F, Y113F and W198F were d ecreased moderately to that of wild type reABRaA, while that of E164Q decreased dramatically, Kinetic analysis showed that the k(cat) of Y74 F, Y113F and W198F resembled that of wild type, while the K-m increase d significantly, W198F did not reassociate with ABRaB to form heterodi mes, while Y74F, Y113F and E164Q did, SDS-PAGE analysis of ABRaA treat ed with trypsin showed that reABRaA, Y74F, Y113F and E164Q survived di gestion, whereas W198F was not protected from digestion, CD spectra re vealed that W198F showed significant conformational changes, These obs ervations suggest that E164 is directly involved in catalysis, and Tyr 74, Tyr113 and Trp198 in substrate binding, while Trp198 also plays an important role in maintaining the conformation of ABRaA required for its reassociation with ABRaB.