Unexpected crucial role of residue 225 in serine proteases
Unexpected crucial role of residue 225 in serine proteases
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DOI：
10.1073/pnas.96.5.1852
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发表时间：
1999-03-02
影响因子：
11.1
通讯作者：
Di Cera, E
中科院分区：
文献类型：
--
作者：
Guinto, ER;Caccia, S;Di Cera, E
在近 300 个可用序列中，超过 95% 的胰凝乳蛋白酶家族丝氨酸蛋白酶中的残基 225 是 Pro 或 Tyr。含有 Y225 的蛋白酶（如一些凝血因子和补体因子）几乎只存在于脊椎动物中，而含有 P225 的蛋白酶（如降解酶）则存在于细菌和人类中。凝血酶中 Y225 的饱和诱变表明，残基 225 对配体识别的影响高达 60,000 倍。除了 Tyr 和 Phe 之外，所有残基都具有与 Pro 相当或大大降低的催化活性。催化活性差异很大的三个突变体（Y225F、Y225P 和 Y225I）的晶体结构表明，尽管残基 225 不与底物接触，但它极大地影响了主要特异性位点周围的水通道的形状。获得的凝血酶活性谱还表明，脊椎动物中记录的 Pro 向 Tyr 或 Phe 的转化是通过 Ser 和 aas 发生的，催化活性显着增加（高达 50 倍）。事实上，Ser 和 Phe 存在于 4% 的丝氨酸蛋白酶中，它们与 Pro 和 Tyr 一起几乎构成了 225 位残基的全部分布。225 位残基在丝氨酸蛋白酶中意想不到的关键作用解释了该位置残基的进化选择，并表明蛋白酶活性和特异性的结构决定因素比目前认为的更为复杂。这些发现对于合理设计具有增强催化特性的酶具有广泛的意义。
Residue 225 in serine proteases of the chymotrypsin family is Pro or Tyr in more than 95% of nearly 300 available sequences. Proteases with Y225 (like some blood coagulation and complement factors) are almost exclusively found in vertebrates, whereas proteases with P225 (like degradative enzymes) are present from bacteria to human. Saturation mutagenesis of Y225 in thrombin shows that residue 225 affects ligand recognition up to 60,000-fold. With the exception of Tyr and Phe, all residues are associated with comparable or greatly reduced catalytic activity relative to Pro. The crystal structures of three mutants that differ widely in catalytic activity (Y225F, Y225P, and Y225I) show that although residue 225 makes no contact with substrate, it drastically influences the shape of the water channel around the primary specificity site. The activity profiles obtained for thrombin also suggest that the conversion of Pro to Tyr or Phe documented in the vertebrates occurred through Ser and aas driven by a significant gain (up to 50-fold) in catalytic activity. In fact, Ser and Phe are documented in 4% of serine proteases, which together with Pro and Tyr account for almost the entire distribution of residues at position 225. The unexpected crucial role of residue 225 in serine proteases explains the evolutionary selection of residues at this position and shows that the structural determinants of protease activity and specificity are more complex than currently believed. These findings have broad implications in the rational design of enzymes with enhanced catalytic properties.