Affiliation:
1. Program for Apoptosis & Cell Death Research, The Burnham Institute, 10901 North Torrey Pines Road, La Jolla, CA 92037, U.S.A.
2. Carlsberg Laboratory, Center for Solid Phase Organic Combinatorial Chemistry, Department of Chemistry, Gamle Carlsberg Vej 10, DK-2500 Copenhagen, Denmark
Abstract
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.
Subject
Cell Biology,Molecular Biology,Biochemistry
Cited by
188 articles.
订阅此论文施引文献
订阅此论文施引文献,注册后可以免费订阅5篇论文的施引文献,订阅后可以查看论文全部施引文献