PMID:15502359
| Citation |
Cornista, J, Ikeuchi, S, Haruki, M, Kohara, A, Takano, K, Morikawa, M and Kanaya, S (2004) Cleavage of various peptides with pitrilysin from Escherichia coli: kinetic analyses using beta-endorphin and its derivatives. Biosci. Biotechnol. Biochem. 68:2128-37 |
|---|---|
| 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. |
| Links | |
| Keywords |
Amino Acid Sequence; Amino Acid Substitution/genetics; Bacterial Proteins/metabolism; Escherichia coli/enzymology; Hydrolysis; Metalloendopeptidases/metabolism; Molecular Sequence Data; Peptides/metabolism; Protein Precursors/metabolism; Substrate Specificity/physiology; beta-Endorphin/metabolism |
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