Department of Biochemistry, University of Oulu, PO Box 3000, 90014 University of Oulu, Oulu, Finland.
J Mol Biol. 2010 Mar 5;396(4):883-92. doi: 10.1016/j.jmb.2009.12.014. Epub 2009 Dec 21.
Protein disulfide isomerase (PDI) plays a central role in disulfide bond formation in the endoplasmic reticulum. It is implicated both in disulfide bond formation and in disulfide bond reduction and isomerization. To be an efficient catalyst of all three reactions requires complex mechanisms. These include mechanisms to modulate the pK(a) values of the active-site cysteines of PDI. Here, we examined the role of arginine 120 in modulating the pK(a) values of these cysteines. We find that arginine 120 plays a significant role in modulating the pK(a) of the C-terminal active-site cysteine in the a domain of PDI and plays a role in determining the reactivity of the N-terminal active-site cysteine but not via direct modulation of its pK(a). Mutation of arginine 120 and the corresponding residue, arginine 461, in the a' domain severely reduces the ability of PDI to catalyze disulfide bond formation and reduction but enhances the ability to catalyze disulfide bond isomerization due to the formation of more stable PDI-substrate mixed disulfides. These results suggest that the modulation of pK(a) of the C-terminal active cysteine by the movement of the side chain of these arginine residues into the active-site locales has evolved to allow PDI to efficiently catalyze both oxidation and isomerization reactions.
蛋白质二硫键异构酶（PDI）在内质网中二硫键形成中发挥核心作用。它既参与二硫键的形成，也参与二硫键的还原和异构化。作为所有这三种反应的有效催化剂，需要复杂的机制。这些机制包括调节 PDI 活性位点半胱氨酸的 pK(a) 值的机制。在这里，我们研究了精氨酸 120 在调节这些半胱氨酸的 pK(a) 值中的作用。我们发现，精氨酸 120 在调节 PDI 的 a 结构域中 C 末端活性位点半胱氨酸的 pK(a) 值方面发挥重要作用，并在决定 N 末端活性位点半胱氨酸的反应性方面发挥作用，但不是通过直接调节其 pK(a) 值。a' 结构域中精氨酸 120 和相应的精氨酸 461 的突变严重降低了 PDI 催化二硫键形成和还原的能力，但由于形成了更稳定的 PDI-底物混合二硫键，增强了催化二硫键异构化的能力。这些结果表明，这些精氨酸残基侧链进入活性部位位置对 C 末端活性半胱氨酸的 pK(a) 值的调节已经进化，以使 PDI 能够有效地催化氧化和异构化反应。
