Diversity of enzymatic specificities and genome structures of vertebrate pepsinogens.

以不同的寡肽为底物，研究了人胃蛋白酶A和猴凝乳酶的蛋白降解特性。人胃蛋白酶A对P‘1位疏水/芳香族残基有严格的偏好，而猴凝乳酶对带电残基和疏水/芳香族残基表现出多样化的偏好。哺乳动物胃蛋白酶A和凝乳酶形成S‘1亚基的残基比较表明，在C末端附近的第289-298环区存在酪氨酸、Ile^&lt；213和Ile^&lt；300&gt；等保守残基和组特异性残基。组特异性残基包括胃蛋白酶A中的疏水残基(Met^&lt；289&gt；，Leu/Ile/Val^&lt；291&gt；和Leu^&lt；298&gt；)，以及凝乳酶中的带电或极性残基(Asp/Glu^&lt；289&gt；和Gln/His/Lys^&lt；298&gt；)。由于环中的残基似乎与不同类型酶的独特特异性有关，因此采用了定点突变，将胃蛋白酶-A特异性残基替换为凝乳酶特异性残基，反之亦然。为了表达突变蛋白，新研制了GST融合蛋白酵母表达载体。胃蛋白酶-A突变体的特异性可以成功地改变为类凝乳酶的偏好，而凝乳酶突变体的特异性可以成功地改变为类胃蛋白酶的特异性，证实了S 1环中的残基对于酶的独特的蛋白降解特性是必不可少的。胃蛋白酶-A突变体对P‘1位带电残基的偏好增加可能是由于氢键相互作用增加所致。在凝乳酶突变体中，相反的情况是可能的。带电残基位于P‘1的多肽的催化效率变化主要由kcat决定，而不是Km值。

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.