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GrainGenes Reference Report: PPS-122-137

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Reference	PPS-122-137
Title	Prediction of protein cleavage sites by the barley cysteine endoproteases EP-A and EP-B based on the kinetics of synthetic peptide hydrolysis
Journal	Plant Physiology
Year	2000
Volume	122
Pages	137-145
Author	Davy A Sorensen MB Svendsen I Cameron-Mills V Simpson DJ
Abstract	Summary: Hordeins, the natural substrates of barley (Hordeum vulgare) cysteine endoproteases (EPs), were isolated as protein bodies and deraded 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 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
Keyword	[ Hide all but 1 of 39 ] amino acid amino acid sequence attack barley beta amylase beta-amylase c-hordein chemical structure cleavage cleavage sites cysteine cysteine proteinases domain endoproteases extension helix hordein hordeum vulgare hydrolysis kinetics malt methionine molecular conformation mutagenesis n p1 p2 peptide prediction proline protein body proteolysis sequence site-directed mutagenesis specificity substrate substrate-specificity synthetic peptides wild-type

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