Subsite interactions are considered to define the stringent specificity of proteases for their natural substrates. To probe this issue in the proteolytic pathways leading to apoptosis we have examined the P4, P1 and P1´ subsite preferences of human caspases 1, 3, 6, 7 and 8, using internally quenched fluorescent peptide substrates containing o-aminobenzoyl (also known as anthranilic acid) and 3-nitro-tyrosine. Previous work has demonstrated the importance of the S4 subsite in directing specificity within the caspase family. Here we demonstrate the influence of the S1 and S1´ subsites that flank the scissile peptide bond. The S1 subsite, the major specificity-determining site of the caspases, demonstrates tremendous selectivity, with a 20000-fold preference for cleaving substrates containing aspartic acid over glutamic acid at this position. Thus caspases are among the most selective of known endopeptidases. We find that the caspases show an unexpected degree of discrimination in the P1´ position, with a general preference for small amino acid residues such as alanine, glycine and serine, with glycine being the preferred substituent. Large aromatic residues are also surprisingly well-tolerated, but charged residues are prohibited. While this describes the general order of P1´ subsite preferences within the caspase family, there are some differences in individual profiles, with caspase-3 being particularly promiscuous. Overall, the subsite preferences can be used to predict natural substrates, but in certain cases the cleavage site within a presumed natural substrate cannot be predicted by looking for the preferred peptide cleavage sites. In the latter case we conclude that second-site interactions may overcome otherwise sub-optimal cleavage sequences.
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Research Article|
August 23 2000
Internally quenched fluorescent peptide substrates disclose the subsite preferences of human caspases 1, 3, 6, 7 and 8
Henning R. STENNICKE;
Henning R. STENNICKE
*Program for Apoptosis & Cell Death Research, The Burnham Institute, 10901 North Torrey Pines Road, La Jolla, CA 92037, U.S.A.
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Martin RENATUS;
Martin RENATUS
*Program for Apoptosis & Cell Death Research, The Burnham Institute, 10901 North Torrey Pines Road, La Jolla, CA 92037, U.S.A.
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Morten MELDAL;
Morten MELDAL
†Carlsberg Laboratory, Center for Solid Phase Organic Combinatorial Chemistry, Department of Chemistry, Gamle Carlsberg Vej 10, DK-2500 Copenhagen, Denmark
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Guy S. SALVESEN
Guy S. SALVESEN
1
*Program for Apoptosis & Cell Death Research, The Burnham Institute, 10901 North Torrey Pines Road, La Jolla, CA 92037, U.S.A.
1To whom correspondence should be addressed (e-mail gsalvesen@burnham.org).
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Publisher: Portland Press Ltd
Received:
May 04 2000
Revision Received:
June 16 2000
Accepted:
June 30 2000
Online ISSN: 1470-8728
Print ISSN: 0264-6021
The Biochemical Society, London © 2000
2000
Biochem J (2000) 350 (2): 563–568.
Article history
Received:
May 04 2000
Revision Received:
June 16 2000
Accepted:
June 30 2000
Citation
Henning R. STENNICKE, Martin RENATUS, Morten MELDAL, Guy S. SALVESEN; Internally quenched fluorescent peptide substrates disclose the subsite preferences of human caspases 1, 3, 6, 7 and 8. Biochem J 1 September 2000; 350 (2): 563–568. doi: https://doi.org/10.1042/bj3500563
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