Yang Likui, Manithody Chandrashekhara, Rezaie Alireza R
Edward A. Doisy Department of Biochemistry and Molecular Biology, Saint Louis University School of Medicine, Saint Louis, Missouri 63104, USA.
Biochemistry. 2002 May 14;41(19):6149-57. doi: 10.1021/bi015899r.
The role of basic residues of the 70-80-loop, Arg(74), Arg(75), and Lys(78) (chymotrypsin numbering) in the catalytic function of activated protein C (APC) was investigated by expressing mutants of protein C in which these residues were replaced with Ala in three separate constructs. Following purification to homogeneity and activation by thrombin, the catalytic properties of the mutants were characterized with respect to their ability to cleave the chromogenic substrate Spectrozyme PCa, react with protein C inhibitor (PCI), and inactivate factor Va. Relative to wild-type APC, the mutants cleaved Spectrozyme PCa with identical or improved catalytic efficiencies. Similarly, PCI inhibited mutants with identical or improved second-order rate constants (k(2)) in the absence of heparin. However, the heparin-catalyzed inhibition of mutants by PCI was impaired approximately 10-fold. Analysis of k(2) values by a ternary complex model revealed that the affinities of mutants for heparin were impaired to a similar extent. Moreover, analysis of the NaCl gradient elution profiles of APC derivatives from Heparin-Sepharose supported this conclusion. An oligosaccharide containing 14 residues efficiently catalyzed the PCI inhibition of APC by a template mechanism. Further studies revealed that the ability of Arg(74) and Arg(75) mutants to inactivate factor Va was markedly impaired. We conclude that basic residues of the 70-80-loop are critical for the catalytic function of APC.
通过在三种不同构建体中表达将70 - 80环的碱性残基精氨酸（74）、精氨酸（75）和赖氨酸（78）（胰凝乳蛋白酶编号）替换为丙氨酸的蛋白C突变体，研究了这些残基在活化蛋白C（APC）催化功能中的作用。在纯化至同质并经凝血酶激活后，对突变体的催化特性进行了表征，包括它们切割生色底物Spectrozyme PCa的能力、与蛋白C抑制剂（PCI）反应的能力以及灭活因子Va的能力。相对于野生型APC，突变体切割Spectrozyme PCa的催化效率相同或有所提高。同样，在不存在肝素的情况下，PCI以相同或更高的二级反应速率常数（k(2)）抑制突变体。然而，肝素催化的PCI对突变体的抑制作用受损约10倍。通过三元复合物模型分析k(2)值表明，突变体对肝素的亲和力受损程度相似。此外，对来自肝素 - 琼脂糖的APC衍生物的NaCl梯度洗脱图谱的分析支持了这一结论。一种含有14个残基的寡糖通过模板机制有效地催化了PCI对APC的抑制作用。进一步的研究表明，精氨酸（74）和精氨酸（75）突变体灭活因子Va的能力明显受损。我们得出结论，70 - 80环的碱性残基对APC的催化功能至关重要。
