title: Cleavage of various peptides with pitrilysin from Escherichia coli: kinetic analyses using beta-endorphin and its derivatives.
abstract: Pitrilysin from Escherichia coli was overproduced, purified, and analyzed for enzymatic activity using 14 peptides as a substrate. Pitrilysin cleaved all the peptides, except for two of the smallest, at a limited number of sites, but showed little amino acid specificity. It cleaved beta-endorphin (beta-EP) most effectively, with a K(m) value of 0.36 microM and a k(cat) value of 750 min(-1). beta-EP consists of 31 residues and was predominantly cleaved by the enzyme at Lys(19)-Asn(20). Kinetic analyses using a series of beta-EP derivatives with N and/or C-terminal truncations and with amino acid substitutions revealed that three hydrophobic residues (Leu(14), Val(15), and Leu(17)) and the region 22-26 in beta-EP are responsible for high-affinity recognition by the enzyme. These two regions are located on the N- and C-terminal sides of the cleavage site in beta-EP, suggesting that the substrate binding pocket of pitrilysin spans its catalytic site.
authors.affiliation: Department of Material and Life Science, Graduate School of Engineering, Osaka University, Suita, Osaka 565-0871, Japan.
authors.forename: Joel
authors.initials: J
authors.lastname: Cornista
authors.forename: Satoshi
authors.initials: S
authors.lastname: Ikeuchi
authors.forename: Mitsuru
authors.initials: M
authors.lastname: Haruki
authors.forename: Atsuko
authors.initials: A
authors.lastname: Kohara
authors.forename: Kazufumi
authors.initials: K
authors.lastname: Takano
authors.forename: Masaaki
authors.initials: M
authors.lastname: Morikawa
authors.forename: Shigenori
authors.initials: S
authors.lastname: Kanaya
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics; D001426:Bacterial Proteins / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology*; D006868:Hydrolysis; D008666:Metalloendopeptidases / Q000378:metabolism*; D008969:Molecular Sequence Data; D010455:Peptides / Q000378:metabolism; D011498:Protein Precursors / Q000378:metabolism*; D013379:Substrate Specificity / Q000502:physiology; D001615:beta-Endorphin / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D010455:Peptides; D011498:Protein Precursors; D001615:beta-Endorphin; D008666:Metalloendopeptidases; C046583:pitrilysin