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 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-P(2)-P(1)-P(1)'-P(2)' 1-tyrosine(NO(2))-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 P(2), and showed less specificity at P(1), although asparagine, aspartate, valine, and isoleucine were particularly unfavorable. Peptides with proline at P(1) or P(1)' 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 beta-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 beta-reverse turn helix of the central repeat domain, which then becomes more susceptible to proteolytic attack by EP-B.
Davy
A
A
Carlsberg Research Laboratory, Carlsberg Laboratory, Gamle Carlsbergvej 10, DK-2500 Valby, Denmark.
SŁrensen
M B
MB
Svendsen
I
I
Cameron-Mills
V
V
Simpson
D J
DJ
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000737:chemistry; D003546:Cysteine Endopeptidases / Q000737:chemistry*; D005983:Glutens; D001467:Hordeum / Q000737:chemistry*; D006868:Hydrolysis; D008666:Metalloendopeptidases / Q000737:chemistry*; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry; D010940:Plant Proteins / Q000737:chemistry*; D001614:beta-Amylase / Q000737:chemistry
D000596:Amino Acids; D010446:Peptide Fragments; D010940:Plant Proteins; D005983:Glutens; D001614:beta-Amylase; D003546:Cysteine Endopeptidases; C028307:endopeptidase B; D008666:Metalloendopeptidases; C022510:thimet oligopeptidase