Amino acid sequence and some properties of phytolacain R, a cysteine protease from full-growth fruits of pokeweed, Phytolacca americana.
A cysteine protease, phytolacain R from full-growth greenish fruits of pokeweed, Phytolacca americana L, was purified to electrophoretic homogeneity by a simple purification procedure employing CM-Sepharose ion-exchange chromatography. The enzyme was present in low content in the young fruits about 50 d after flowering but gradually accumulated in growing fruits. Its molecular mass was estimated to be ca. 23 kDa by SDS-PAGE, and its sugar content was zero. Its amino acid sequence was established by automated sequence analysis of the peptides obtained by cleavage with Achromobacter protease I, chymotrypsin, trypsin, and cyanogen bromide. The enzyme is composed of 218 amino acid residues, of which it shares 110 residues (50%) with papain, 104 (47%) with actinidain, and 87 (40%) with stem bromelain. The amino acid residues forming the substrate-binding the S2 pocket of papain, Tyr61, Tyr67, Pro68, Trp69, Val133, and Phe207, were predicted to be replaced by Gly, Trp, Met, His, Ala, and Met in phytolacain R, respectively. As a consequence of these substitutions, the S2 pocket is expected to be less hydrophobic in phytolacain R than in papain.
Yonezawa
H
H
Department of Chemistry, Faculty of Science, Kagoshima University, Korimoto, Kagoshima, 890-0065, Japan.
Uchikoba
T
T
Arima
K
K
Shimada
M
M
Kaneda
M
M
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D001665:Binding Sites; D002241:Carbohydrates / Q000032:analysis; D002918:Chymotrypsin / Q000378:metabolism; D003488:Cyanogen Bromide / Q000737:chemistry / Q000378:metabolism; D003546:Cysteine Endopeptidases / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D015853:Cysteine Proteinase Inhibitors / Q000378:metabolism; D005638:Fruit / Q000737:chemistry; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010940:Plant Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D012697:Serine Endopeptidases / Q000378:metabolism; D014357:Trypsin / Q000378:metabolism
D000596:Amino Acids; D002241:Carbohydrates; D015853:Cysteine Proteinase Inhibitors; D010940:Plant Proteins; D012697:Serine Endopeptidases; D002918:Chymotrypsin; D014357:Trypsin; C036813:lysyl endopeptidase; D003546:Cysteine Endopeptidases; C095519:phytolacain R; D003488:Cyanogen Bromide