A Proteinase from Germinated Barley : II. Hydrolytic Specificity of a 30 Kilodalton Cysteine Proteinase From Green Malt.

The hydrolytic specificity of a 30 kilodalton cysteine proteinase purified from germinated barley (Hordeum vulgare L. cv Morex) was investigated using high performance liquid chromatography to characterize its hydrolysis of two small barley seed proteins, the alpha- and beta-hordothionins. The reduced and pyridylethylated thionins were rapidly cleaved, resulting in the production of a limited number of peptides. Peptide bonds Gly9-Arg10, Cys 16-Arg17, Cys25-Ala26, and Thr34-Ser35 were most susceptible to hydrolysis, the peptide bonds Arg5-Ser6, Arg19-Gly20 in both thionins and Lys38-Cys39 in beta-hordothionin and Cys29-Arg30 of alpha-hordothionin being broken at much slower rates. The hydrolysis patterns were highly reproducible from assay to assay and with various enzyme preparations. The specificity was apparently defined by the amino acids in the P(2) position, not those immediately adjacent to the susceptible bonds. The P(2) amino acid residues of the released peptides were always either leucine, valine, tyrosine, or pyridylethylcysteine. From these observations and from the rates of release of the various peptides, it appears that the barley 30 kilodalton endoproteinase has an S2 subsite that preferentially binds the leucine side chain: i.e. for hydrolyzing the peptide bond P(1)-P(1)' in the general sequence NH(2)-P(2)-P(1)-P(1)'-COOH, the enzyme is selective for leucine and, to a lesser extent, valine and tyrosine at position P(2). The barley proteinase thus resembles two other cysteine proteinases, papain and Streptococcal proteinase, in its specificity.