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Substrate Specificity of Barley Cysteine Endoproteases EP-A and EP-B1

Anne Davy, Ib Svendsen, Susanne O. Sørensen2, Mikael Blom Sørensen, Jacques Rouster, Morten Meldal, David J. Simpson*, and Verena Cameron-Mills

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

The cysteine endoproteases (EP)-A and EP-B were purified from green barley (Hordeum vulgare L.) malt, and their identity was confirmed by N-terminal amino acid sequencing. EP-B cleavage sites in recombinant type-C hordein were determined by N-terminal amino acid sequencing of the cleavage products, and were used to design internally quenched, fluorogenic peptide substrates. Tetrapeptide substrates of the general formula 2-aminobenzoyl-P2-P1-P1'-P2'-tyrosine(NO2)-aspartic acid, in which cleavage occurs between P1 and P1', showed that the cysteine EPs preferred phenylalanine, leucine, or valine at P2. Arginine was preferred to glutamine at P1, whereas proline at P2, P1, or P1' greatly reduced substrate kinetic specificity. Enzyme cleavage of C hordein was mainly determined by the primary sequence at the cleavage site, because elongation of substrates, based on the C hordein sequence, did not make them more suitable substrates. Site-directed mutagenesis of C hordein, in which serine or proline replaced leucine, destroyed primary cleavage sites. EP-A and EP-B were both more active than papain, mostly because of their much lower Km values.

1   This work was supported by the Danish Academy of Technical Sciences (A.D. received a studentship). This is Adapting Barley for Industrial Needs publication no. 159.
2   Present address: Danisco Biotechnology, Langebrogade 1, DK-1001 Copenhagen K, Denmark.
*   Corresponding author; e-mail simpson{at}biobase.dk; fax 45-3327-4766.

Plant Physiol. (1998) 117: 255-261
Copyright Clearance Center:   0032-0889/98/117/0255/07
© 1998 American Society of Plant Physiologists




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