Diversity of enzymatic specificities and genome structures of vertebrate pepsinogens.

以多种寡肽为底物，研究了人胃蛋白酶A和猴凝乳酶的蛋白水解特异性。人胃蛋白酶A对P'1的疏水/芳香残基有严格的偏好，而猴凝乳酶对带电残基和疏水/芳香残基有多样化的偏好。比较哺乳动物胃蛋白酶A和凝乳酶之间形成S'1亚位的残基，发现存在保守残基，包括Tyr^<189>, Ile^<213>和Ile^<300>，以及在c端附近的289-298环区域的群特异性残基。基团特异性残基包括胃蛋白酶A中的疏水残基（Met^<289>, Leu/Ile/Val^<291>和Leu^<298>）和凝乳酶中的电荷或极性残基（Asp/Glu^<289>和Gln/His/Lys^<298>）。由于环中的残基似乎与各自类型酶的独特特异性有关，因此进行了位点定向诱变，用凝乳酶特异性残基取代胃蛋白酶a特异性残基，反之亦然。研究了一种用于GST融合蛋白表达的酵母表达载体。胃蛋白酶- a突变体的特异性可以成功地改变为类似凝乳酶的偏好，而凝乳酶突变体的特异性可以改变为类似胃蛋白酶的特异性，这证实了S'1环上的残基对于酶的独特蛋白水解特性是必不可少的。在胃蛋白酶- a突变体中，对P'1带电残基的偏好增加可能是由于氢键相互作用的增加。在凝血酶突变体中，相反的情况是可能的。在P'1带电荷残基的多肽催化效率的变化主要是由kcat值而不是Km值决定的。

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.