抄録
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 β-endorphin (β-EP) most effectively, with a Km value of 0.36 μM and a kcat value of 750 min−1. β-EP consists of 31 residues and was predominantly cleaved by the enzyme at Lys19–Asn20. Kinetic analyses using a series of β-EP derivatives with N and/or C-terminal truncations and with amino acid substitutions revealed that three hydrophobic residues (Leu14, Val15, and Leu17) and the region 22–26 in β-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 β-EP, suggesting that the substrate binding pocket of pitrilysin spans its catalytic site.
