Identification of amino acid residues of abrin-a A chain is essential for catalysis and reassociation with abrin-a B chain by site-directed mutagenesis.
Abrin is a toxic protein consisting of two subunits, an enzymatic A chain (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, around the active site of ABRaA are involved in enzyme catalysis, enzyme-substrate recognition and reassociation of ABRaA with ABRaB. The protein biosynthesis inhibitory activities of Y74F, Y113F and W198F were decreased moderately to that of wild type reABRaA, while that of E164Q decreased dramatically. Kinetic analysis showed that the kat of Y74F, Y113F and W198F resembled that of wild type, while the Km increased significantly. W198F did not reassociate with ABRaB to form heterodimers, while Y74F, Y113F and E164Q did. SDS-PAGE analysis of ABRaA treated with trypsin showed that reABRaA, Y74F, Y113F and E164Q survived digestion, whereas W198F was not protected from digestion. CD spectra revealed that W198F showed significant conformational changes. These observations suggest that E164 is directly involved in catalysis, and Tyr74, 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.
Chen
J K
JK
Institute of Biochemistry, College of Medicine, National Taiwan University, Taipei, Republic of China.
Hung
C H
CH
Liaw
Y C
YC
Lin
J Y
JY
D000036:Abrin / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D000596:Amino Acids / Q000737:chemistry; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D002384:Catalysis; D002942:Circular Dichroism; D017931:DNA Primers / Q000235:genetics; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D016133:Polymerase Chain Reaction; D011487:Protein Conformation; D015202:Protein Engineering; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D014357:Trypsin
D000596:Amino Acids; D017931:DNA Primers; D011994:Recombinant Proteins; D000036:Abrin; D014357:Trypsin