Plant Physiol, January 2000, Vol. 122, pp. 137-146

Prediction of Protein Cleavage Sites by the Barley Cysteine Endoproteases EP-A and EP-B Based on the Kinetics of Synthetic Peptide Hydrolysis¹

Carlsberg Research Laboratory (A.D., M.B.S., V.C.-M.), Department of Chemistry (I.S.), and Department of Physiology (D.J.S.), Carlsberg Laboratory, Gamle Carlsbergvej 10, DK-2500 Valby, Denmark.

Hordeins, the natural substrates of barley (Hordeum vulgare) cysteine endoproteases (EPs), were isolated as protein bodies and degraded by purified EP-B from green barley malt. Cleavage specificity was determined by synthesizing internally quenched, fluorogenic tetrapeptide substrates of the general formula 2-aminobenzoyl-P₂-P₁-P₁'-P₂' 1-tyrosine(NO₂)-aspartate. The barley EPs preferred neutral amino acids with large aliphatic and nonpolar (leucine, valine, isoleucine, and methionine) or aromatic (phenylalanine, tyrosine, and tryptophan) side chains at P₂, and showed less specificity at P₁, although asparagine, aspartate, valine, and isoleucine were particularly unfavorable. Peptides with proline at P₁ or P₁' were extremely poor substrates. Cleavage sites with EP-A and EP-B preferred substrate sequences are 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 -amylase. When all of the primary cleavage sites in C hordein, which occur mainly in the N- and C-terminal domains, were removed by site-directed mutagenesis, the resulting protein was degraded 112 times more slowly than wild-type C hordein. We suggest that removal of the C hordein terminal domains is necessary for unfolding of the -reverse turn helix of the central repeat domain, which then becomes more susceptible to proteolytic attack by EP-B.