OXIDATION/REDUCTION-ELECTRON TRANSFER PROTEINS AND BLOOD CLOTTING ENZYMES

本子项目是利用由NIH/NCRR资助的中心赠款提供的资源的众多研究子项目之一。子项目和研究者（PI）可能已经从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。列出的机构是中心的，不一定是研究者的机构。这个子项目涉及基础生物化学和医学的两个重要领域的结构生物学，(1)风味酶或喹啉酶的氧化还原和电子转移到它们的氧化还原伙伴，以及(2)凝血酶的变构调节，凝血酶是参与血液凝固级联的丝氨酸蛋白酶家族的关键成员。氧化还原研究的一个方面涉及铜蛋白，氨基青色素，与含有色氨酸的酶，甲胺脱氢酶和细胞色素的相互作用。已经制备了几种变异的氨基花青素，它们对电子转移的速率和机制表现出一系列的影响。在各种条件下对这些突变体的原子分辨率研究开始揭示这些突变所产生的细微变化以及它们如何影响这一基本过程。在相关领域，细菌黄酶肌氨酸氧化酶的两种形式正在研究中，一种是仅含有FAD的简单单体形式，另一种是含有FAD、FMN和NAD的复杂异四聚体形式，这两种酶的几种突变体与各种外源配体的复合物，目前正在研究中，以确定底物氧化的催化机制和控制电子转移的因素。后一种酶催化位点之间的质子和不稳定中间体。凝血酶在纤维蛋白原裂解的凝血过程中发挥促凝作用，在蛋白c的裂解过程中也发挥抗凝作用。这些作用的调节是由单价阳离子（尤其是钠）的结合水平变构介导的。丙氨酸扫描诱变已经确定了十多个强烈影响钠调节的残基。这些突变蛋白在存在和缺乏这些不同阳离子的情况下的结构研究目前正在进行中。这项工作旨在确定这些残基在变构调节和它们之间的相互作用中所起的作用。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. This subproject involves the structural biology of two important areas of basic biochemistry and medicine, (1) oxidation-reduction within flavor- or quino-enzymes and electron transfer to their redox partners and (2) the allosteric regulation of thrombin, a key member of the family of serine proteases involved in the blood clotting cascade. One aspect of the redox studies involves the interactions of a copper protein, amicyanin, with a tryptophylquinone-containing enzyme, methylamine dehydrogenase and a cytochrome. Several mutants of amicyanin have been prepared that display a range of effects on the rates and mechanism of electron transfer. Atomic resolution studies of these mutants under a variety of conditions are beginning to reveal the subtle changes produced by these mutations and how they effect this fundamental process. In a related area, two forms of the bacterial flavoenzyme sarcosine oxidase are under investigation, a simple monomeric form containing FAD only and a complex heterotetrameric form containing FAD, FMN and NAD Structures of several mutants of both enzyme, in complex with a variety of exogenous ligands, are currently being investigated to define the catalytic mechanism of substrate oxidation and the factors controlling the transfer of electrons, protons and labile intermediates between catalytic sites in the latter enzyme. Thrombin plays both a procoagulant role in blood clotting upon cleavage of fibrinogen and also an anticoagulant role in the cleavage of protein C. Regulation of these roles is allosterically mediated by the binding level of monovalent cations, particularly sodium. Alanine-scanning mutagenesis has identified over a dozen residues that strongly affect this sodium regulation. Structural studies of these mutant protein in the presence and absence of these various cations are currently underway. This work is designed to identify the rolls played by each of these residues in the allosteric regulation and the interplay between them.