title: Extensive comparison of the substrate preferences of two subtilisins as determined with peptide substrates which are based on the principle of intramolecular quenching.
abstract: Subtilisins are serine endopeptidases with an extended binding cleft comprising at least eight binding subsites. Interestingly, subsites distant from the scissile bond play a dominant role in determining the specificity of the enzymes. The development of internally quenched fluorogenic substrates, which allow polypeptides of more than 11 amino acids to be inserted between the donor and the acceptor, has rendered it possible to perform a highly systematic mapping of the individual subsites of the active sites of subtilisin BPN' from Bacillus amyloliquefaciens and Savinase from Bacillus lentus. For each enzyme, the eight positions S5-S'3 were characterized by determination of kcat/KM values for the hydrolysis of substrates in which the amino acids were systematically varied. The results emphasize that in both subtilisin BPN' and Savinase interactions between substrate and S4 and S1 are very important. However, it is apparent that interactions between other subsites and the substrate exert a significant influence on the substrate preference. The results are rationalized on the basis of the structural data available for the two enzymes.
authors.affiliation: Department of Chemistry, Carlsberg Laboratory, Copenhagen Valby, Denmark.
authors.forename: H
authors.initials: H
authors.lastname: Grøn
authors.forename: M
authors.initials: M
authors.lastname: Meldal
authors.forename: K
authors.initials: K
authors.lastname: Breddam
mesh_terms: D000595:Amino Acid Sequence; D001407:Bacillus / Q000201:enzymology; D001665:Binding Sites; D005456:Fluorescent Dyes; D007700:Kinetics; D008969:Molecular Sequence Data; D010449:Peptide Mapping; D010455:Peptides / Q000737:chemistry / Q000378:metabolism*; D012697:Serine Endopeptidases / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D013381:Subtilisins / Q000378:metabolism*; D013816:Thermodynamics
chemical_list: D005456:Fluorescent Dyes; D010455:Peptides; D012697:Serine Endopeptidases; D013381:Subtilisins; C032923:microbial serine proteinases