Kageyama Takashi
Center for Human Evolution Modeling Research, Primate Research Institute, Kyoto University, Inuyama 484-8506, Japan. kageyama@ pri.kyoto-u.ac.jp
Biochemistry. 2004 Dec 7;43(48):15122-30. doi: 10.1021/bi048440g.
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.
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位的疏水/芳香族残基有严格偏好，而猴凝乳酶则表现出多样化的偏好，既能容纳带电荷的残基，也能容纳疏水/芳香族残基。对哺乳动物胃蛋白酶A和凝乳酶之间形成S'1亚位点的残基进行比较，发现存在保守残基，包括Tyr(189)、Ile(213)和Ile(300)，以及C末端附近289 - 299环区域的基团特异性残基。基团特异性残基在胃蛋白酶A中由疏水残基（Met(289)、Leu/Ile/Val(291)和Leu(298)）组成，在凝乳酶中由带电荷或极性残基（Asp/Glu(289)和Gln/His/Lys(298)）组成。由于环中的残基似乎参与了各类型酶的独特特异性，因此进行了定点诱变，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。新开发了一种用于谷胱甘肽 - S - 转移酶融合蛋白的酵母表达载体，用于表达突变蛋白。胃蛋白酶A突变体的特异性能够成功改变为凝乳酶样偏好，而凝乳酶突变体的特异性则改变为胃蛋白酶样特异性，证实S'1环中的残基对于酶的独特蛋白水解特性至关重要。胃蛋白酶A突变体对P'1位带电荷残基偏好的增加可能是由于氢键相互作用的增加。在凝乳酶突变体中，情况可能相反。对P'1位带有电荷残基的肽的催化效率变化主要由k(cat)而非K(m)值决定。
