A novel subtilase from common bean leaves.
We describe the isolation of a protease from common bean leaves grown in the field. On the basis of its biochemical properties it was classified as serine proteinase belonging to the subtilisin clan. Isoelectric focusing resulted in a single band at pH 4.6, and SDS-PAGE in a single band corresponding to M(r) 72 kDa. The proteinase activity is maximal at pH 9.9 and shows high stability in the alkaline region. The relative activities of the proteinase for eight different synthetic substrates were determined. The requirement for Arg in the P1 position appeared obligatory. k(cat)/K(m) values indicate that, for highest catalytic efficiency, a basic amino acid is also required in the P2 position, presenting a motif typical of the cleavage site for the kexin family of subtilases. The sequence of the 17 N-terminal amino acids of this proteinase shows similarity to those of other plant subtilases, sharing the highest number of identical amino acids with proteinase C1 from soybean seedling cotyledons and a cucumisin-like proteinase from white gourd (Benincasa hispida).
Popovic
Tatjana
T
Department of Biochemistry and Molecular Biology, Jozef Stefan Institute, Jamova 39, 1000 Ljubljana, Slovenia. tatjana.popovic@ijs.si
Puizdar
Vida
V
Brzin
Joze
J
D000595:Amino Acid Sequence; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases / Q000302:isolation & purification / Q000378:metabolism*; D004795:Enzyme Stability; D006863:Hydrogen-Ion Concentration; D007525:Isoelectric Focusing; D027805:Phaseolus / Q000201:enzymology*; D018515:Plant Leaves / Q000201:enzymology*; D011480:Protease Inhibitors / Q000494:pharmacology; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
D011480:Protease Inhibitors; D010450:Endopeptidases