Edward A. Doisy Department of Biochemistry and Molecular Biology, Saint Louis University School of Medicine, St. Louis, MO 63104, USA.
Edward A. Doisy Department of Biochemistry and Molecular Biology, Saint Louis University School of Medicine, St. Louis, MO 63104, USA.
J Thromb Haemost. 2024 Apr;22(4):1009-1015. doi: 10.1016/j.jtha.2023.12.026. Epub 2023 Dec 30.
The residue at the site of activation of protein C is Arg in all species except the ray-finned fish, where it is Trp. This feature raises the question of whether thrombin is the physiological activator of protein C across vertebrates.
To establish if thrombin can cleave at Trp residues.
The activity of wild-type thrombin and mutant D189S was tested with a library of chromogenic substrates and toward wild-type protein C and mutants carrying substitutions at the site of cleavage.
Thrombin has trypsin-like and chymotrypsin-like specificity and cleaves substrates at Arg or Trp residues. Cleavage at Arg is preferred, but cleavage at Trp is significant and comparable with that of chymotrypsin. The D189S mutant of thrombin has broad specificity and cleaves at basic and aromatic residues without significant preference. Thrombin also cleaves natural substrates at Arg or Trp residues, showing activity toward protein C across vertebrates, including the ray-finned fish. The rate of activation of protein C in the ray-finned fish is affected by the sequence preceding Trp at the scissile bond.
The results provide a possible solution for the paradoxical presence of a Trp residue at the site of cleavage of protein C in ray-finned fish and support thrombin as the physiological activator of protein C in all vertebrates. The dual trypsin-like and chymotrypsin-like specificity of thrombin suggests that the spectrum of physiological substrates of this enzyme is broader currently assumed.
除了栉齿鲈目鱼类外，所有物种蛋白 C 激活部位的残基均为精氨酸，而栉齿鲈目鱼类的残基为色氨酸。这一特征提出了这样一个问题，即在脊椎动物中，是否凝血酶是蛋白 C 的生理激活物。
确定凝血酶是否可以在色氨酸残基处切割。
使用显色底物文库和野生型蛋白 C 及其在切割部位带有取代的突变体，测试野生型凝血酶和突变体 D189S 的活性。
凝血酶具有胰蛋白酶样和糜蛋白酶样特异性，可在精氨酸或色氨酸残基处切割。精氨酸的切割是首选，但色氨酸的切割也很显著，与糜蛋白酶相当。凝血酶的 D189S 突变体具有广泛的特异性，可在碱性和芳香族残基处切割，而没有明显的偏好。凝血酶还可在精氨酸或色氨酸残基处切割天然底物，显示出在包括栉齿鲈目鱼类在内的所有脊椎动物中对蛋白 C 的活性。在栉齿鲈目鱼类中，蛋白 C 的激活速率受切割部位色氨酸前序列的影响。
这些结果为栉齿鲈目鱼类蛋白 C 切割部位存在色氨酸残基这一矛盾现象提供了一种可能的解决方案，并支持凝血酶作为所有脊椎动物中蛋白 C 的生理激活物。凝血酶的双重胰蛋白酶样和糜蛋白酶样特异性表明，该酶的生理底物谱比目前认为的更广泛。
