Prediction of protein cleavage sites by the barley cysteine endoproteases EP-A and EP-B based on the kinetics of synthetic peptide hydrolysis

Hordeins, the natural substrates of barley (Hordeum vulgare) cysteine endop roteases (EPs), were isolated as protein bodies and degraded by purified EP -B from green barley malt. Cleavage specificity was determined by synthesiz ing internally quenched, fluorogenic tetrapeptide substrates of the general formula 2-aminobenzoyl-P-2-P-1-P-1'-P-2' 1-tyrosine(NO2)-aspartate. The ba rley EPs preferred neutral amino acids with large aliphatic and nonpolar (l eucine, valine, isoleucine, and methionine) or aromatic (phenylalanine, tyr osine, and tryptophan) side chains at P-2, and showed less specificity at P -1, although asparagine, aspartate, valine, and isoleucine were particularl y unfavorable. Peptides with proline at P-1 or P-1' were extremely poor sub strates. Cleavage sites with EP-A and EP-B preferred substrate sequences ar e found in hordeins, their natural substrates. The substrate specificity of EP-B with synthetic peptides was used successfully to predict the cleavage sites in the C-terminal extension of barley beta-amylase. When all of the primary cleavage sites in C hordein, which occur mainly in the N- and C-ter minal domains, were removed by site-directed mutagenesis, the resulting pro tein was degraded 112 times more slowly than wild-type C hordein. We sugges t that removal of the C hordein terminal domains is necessary for unfolding of the beta-reverse turn helix of the central repeat domain, which then be comes more susceptible to proteolytic attack by EP-B.