OXIDATION/REDUCTION-ELECTRON TRANSFER PROTEINS AND BLOOD CLOTTING ENZYMES

这个子项目是许多利用 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 该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）风味酶或醌酶内的氧化还原和电子转移到其氧化还原伙伴和（2）凝血酶的变构调节，凝血酶是参与凝血级联反应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质，amicyanin，与一个含蒽醌的酶，甲胺脱氢酶和细胞色素的相互作用。已经制备了几种amicyanin突变体，它们对电子转移的速率和机制表现出一系列影响。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产生的微妙变化以及它们如何影响这一基本过程。在相关领域，正在研究两种形式的细菌黄素酶肌氨酸氧化酶，一种是仅含有FAD的简单单体形式，另一种是含有FAD、FMN和NAD的复杂异源四聚体形式。目前正在研究两种酶的几种突变体与各种外源配体复合的结构，以确定底物氧化的催化机制和控制电子转移的因素，质子和后一种酶中催化位点之间的不稳定中间体。凝血酶在纤维蛋白原裂解后的血液凝固中起促凝作用，并且在蛋白C裂解中也起抗凝作用。这些作用的调节是由一价阳离子，特别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几个残基，强烈影响这一钠调节。这些突变蛋白在这些不同阳离子的存在和不存在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中的每一个在变构调节中发挥的作用以及它们之间的相互作用。

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.