title: Role of S'1 loop residues in the substrate specificities of pepsin A and chymosin.
abstract: Proteolytic specificities of human pepsin A and monkey chymosin were investigated with a variety of oligopeptides as substrates. Human pepsin A had a strict preference for hydrophobic/aromatic residues at P'1, while monkey chymosin showed a diversified preferences accommodating charged residues as well as hydrophobic/aromatic ones. A comparison of residues forming the S'1 subsite between mammalian pepsins A and chymosins demonstrated the presence of conservative residues including Tyr(189), Ile(213), and Ile(300) and group-specific residues in the 289-299 loop region near the C terminus. The group-specific residues consisted of hydrophobic residues in pepsin A (Met(289), Leu/Ile/Val(291), and Leu(298)) and charged or polar residues in chymosins (Asp/Glu(289) and Gln/His/Lys(298)). Because the residues in the loop appeared to be involved in the unique specificities of respective types of enzymes, site-directed mutagenesis was undertaken to replace pepsin-A-specific residues by chymosin-specific ones and vice versa. A yeast expression vector for glutathione-S-transferase fusion protein was newly developed for expression of mutant proteins. The specificities of pepsin-A mutants could be successfully altered to the chymosin-like preference and those of chymosin mutants, to pepsin-like specificities, confirming residues in the S'1 loop to be essential for unique proteolytic properties of the enzymes. An increase in preference for charged residues at P'1 in pepsin-A mutants might have been due to an increase in the hydrogen-bonding interactions. In chymosin mutants, the reverse is possible. The changes in the catalytic efficiency for peptides having charged residues at P'1 were dominated by k(cat) rather than K(m) values.
authors.affiliation: Center for Human Evolution Modeling Research, Primate Research Institute, Kyoto University, Inuyama 484-8506, Japan. kageyama@ pri.kyoto-u.ac.jp
authors.forename: Takashi
authors.initials: T
authors.lastname: Kageyama
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D002427:Cebidae; D012085:Chymosin / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics; D006801:Humans; D006868:Hydrolysis; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009842:Oligopeptides / Q000378:metabolism; D010434:Pepsin A / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics; D017433:Protein Structure, Secondary / Q000235:genetics; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000235:genetics; D013379:Substrate Specificity / Q000235:genetics
chemical_list: D009842:Oligopeptides; D011994:Recombinant Proteins; D010434:Pepsin A; D012085:Chymosin