A high-order structure of plant storage proprotein allows its second conversion by an asparagine-specific cysteine protease, a novel proteolytic enzyme.
During seed embryogenesis, glycinin, the 11-S seed storage protein found in soybeans, undergoes post-translational proteolytic processing, in which a proprotein molecule is cleaved into an acidic and a basic subunit by a one-point cleavage that occurs at the carboxyl side of the asparaginyl residue located at the junction of the subunits. To elucidate the mechanism of this very limited proteolysis, we purified the cysteine endoprotease and used purified proglycinin produced by Escherichia coli as a substrate. This enzyme was separated by isoelectric focusing into three isomeric forms: two had a molecular mass of 33 kDa and the third, 33.8 kDa. The cysteine protease was found both in the proteinaceous vacuoles of cotyledonary tissue of immature seeds and in mature seeds, and is the first proteolytic enzyme to be classified as an asparagine-specific endoprotease. The results also indicate that the above proteolysis is largely attributable to the conformational accessibility of the enzyme to the asparaginyl residue in the cleavage site of proglycinin. The conformation of this single enzyme-accessible region on the proglycinin molecule is relatively flexible and becomes unstable under low salt conditions, or when heat is applied, causing the enzyme to lose its specificity.
Muramatsu
M
M
Genetic Engineering Laboratory, Ministry of Agriculture, Forestry and Fisheries, Tsukuba Science City, Japan.
Fukazawa
C
C
D000595:Amino Acid Sequence; D001216:Asparagine / Q000378:metabolism; D001483:Base Sequence; D003546:Cysteine Endopeptidases / Q000737:chemistry / Q000302:isolation & purification*; D005916:Globulins / Q000378:metabolism*; D008969:Molecular Sequence Data; D009154:Mutation; D014674:Plant Proteins, Dietary / Q000378:metabolism*; D011487:Protein Conformation; D011498:Protein Precursors / Q000378:metabolism*; D030262:Soybean Proteins
D005916:Globulins; D014674:Plant Proteins, Dietary; D011498:Protein Precursors; D030262:Soybean Proteins; D001216:Asparagine; C011474:glycinin; D003546:Cysteine Endopeptidases