Amino acid sequence and some properties of phytolacain G, a cysteine protease from growing fruit of pokeweed, Phytolacca americana

A protease, phytolacain G, has been found to appear on CM-Sepharose ion-exc hange chromatography of greenish small-size fruits of pokeweed, Phytolacca americana L, from ca. 2 weeks after flowering, and increases during fruit e nlargement. Reddish ripe fruit of the pokeweed contained both phytolacain G and R. The molecular mass of phytolacain G was estimated to be 25.5 kDa by SDS-PAGE. Its amino acid sequence was reconstructed by automated sequence analysis of the peptides obtained after cleavage with Achromobacter proteas e I, chymotrypsin, and cyanogen bromide. The enzyme is composed of 216 amin o acid residues, of which it shares 152 identical amino acid residues (70%) with phytolacain R, 126 (58%) with melain G, 108 (50%) with papain, 106 (4 9%) with actinidain, and 96 (44%) with stem bromelain. The amino acid resid ues forming the substrate binding S-2 pocket of papain, Tyr67, Pro68, Trp69 , Val133, and Phe207, were predicted to be replaced by Trp, Met, His, Ala, and Ser in phytolacain G, respectively. As a consequence of these substitut ions, the S-2 pocket is expected to be less hydrophobic in phytolacain G th an in papain. (C) 2000 Elsevier Science B.V. All rights reserved.