Analysis of active site residues of the antiviral protein from summer leaves from Phytolacca americana by site-directed mutagenesis

The summer leaf isoform of the pokeweed (Phytolacca americana) antiviral pr otein, PAP II, was produced in high yields from inclusion bodies in recombi nant E. coli. On the basis of its sequence similarity with the spring leaf isoform (PAP I) and with the A chain of ricin, a three-dimensional model of the protein was constructed as an aid in the design of active-site mutants . PAP II variants mutated in residues Asp 88 (D88N), Tyr 117 (Y117S), Glu 1 72 (E172Q), Arg 175 (R175H) and a combination of Asp 88 and Arg 175 (D88N/R 175H) were produced in E. coil and assayed for their ability to inhibit pro tein synthesis in a rabbit reticulocyte lysate, All of these mutations had effects deleterious to the enzymatic activity of PAP II. The results were i nterpreted in the light of three reaction mechanisms proposed for ribosome- inactivating proteins (RIPs). We conclude that none of the proposed mechani sms is entirely consistent with the data presented here. (C) 1998 Academic Press.