Diversity of enzymatic specificities and genome structures of vertebrate pepsinogens.

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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 whilst monkey chymosin showed a diversified preferences accommodating charged residues as well as hydrophobic/aromatic ones. 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 289-298 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>). Since 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 GST fusion protein was newly developed for expression of mutant proteins. The specificities of pepsin-A mutants could be successfully altered to 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. Increase in preference for charged residues at P'1 in pepsin-A mutants might have been due to 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 kcat rather than Km values.