Department of Molecular and Cellular Hemostasis, Sanquin Research, Amsterdam, The Netherlands.
Department of Biomolecular Mass Spectrometry and Proteomics, Utrecht Institute for Pharmaceutical Sciences (UIPS), Utrecht University, Utrecht, The Netherlands.
J Thromb Haemost. 2021 Jun;19(6):1447-1459. doi: 10.1111/jth.15288. Epub 2021 Apr 5.
Activated factor IX (FIXa) is an inefficient enzyme that needs activated factor VIII (FVIII) for full activity. Recently, we identified a network of FVIII-driven changes in FIXa employing hydrogen-deuterium eXchange mass spectrometry (HDX-MS). Some changes also occurred in active-site inhibited FIXa, but others were not cofactor-driven, in particular those within the 220-loop (in chymotrypsin numbering).
The aim of this work is to better understand the zymogen-to-enzyme transition in FIX, with specific focus on substrate-driven changes at the catalytic site.
Footprinting mass spectrometry by HDX and Tandem-Mass Tags (TMT) labelling were used to explore changes occurring upon the conversion from FIX into FIXa. Mutagenesis and kinetic studies served to assess the role of the 220-loop.
HDX-MS displayed remarkably few differences between FIX and FIXa. In comparison with FIX, FIXa did exhibit decreased deuterium uptake at the N-terminus region. This was more prominent when the FIXa active site was occupied by an irreversible inhibitor. TMT-labelling showed that the N-terminus is largely protected from labelling, and that inhibitor binding increases protection to a minor extent. Occupation of the active site also reduced deuterium uptake within the 220-loop backbone. Mutagenesis within the 220-loop revealed that a putative H-bond network contributes to FIXa activity. TMT labeling of the N-terminus suggested that these 220-loop variants are more zymogen-like than wild-type FIXa.
In the absence of cofactor and substrate, FIXa is predominantly zymogen-like. Stabilization in its enzyme-like form involves, apart from FVIII-binding, also interplay between the 220-loop, N-terminus, and the substrate binding site.
激活的凝血因子 IX（FIXa）是一种效率较低的酶，需要激活的凝血因子 VIII（FVIII）才能充分发挥活性。最近，我们采用氢氘交换质谱（HDX-MS）技术鉴定了 FVIII 驱动的 FIXa 变化网络。一些变化也发生在活性位点受抑制的 FIXa 中，但也有一些变化不是辅因子驱动的，特别是在 220 环内（按糜蛋白酶编号）。
本研究旨在更好地了解 FIX 中的酶原向酶的转变，特别是关注催化部位的底物驱动变化。
采用 HDX 和串联质量标签（TMT）标记的足迹质谱法研究从 FIX 转化为 FIXa 过程中发生的变化。通过突变和动力学研究来评估 220 环的作用。
HDX-MS 显示 FIX 和 FIXa 之间的差异非常小。与 FIX 相比，FIXa 在 N 端区域的氘摄取减少。当 FIXa 的活性位点被不可逆抑制剂占据时，这种情况更为明显。TMT 标记显示 N 端大部分受到保护，而抑制剂结合在较小程度上增加了保护。活性位点的占据也降低了 220 环骨架内的氘摄取。220 环内的突变显示，一个假定的氢键网络有助于 FIXa 的活性。N 端的 TMT 标记表明这些 220 环变体比野生型 FIXa 更具有酶原样特征。
在没有辅因子和底物的情况下，FIXa 主要表现为酶原样。除了 FVIII 结合外，其酶样形式的稳定还涉及 220 环、N 端和底物结合位点之间的相互作用。
