STRUCTURAL STUDIES OF REDOX ENZYMES AND MEDICALLY IMPORTANT SERINE PROTEASES

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 这是一项为期两年的APS束流时间方案，用于生物大分子结构研究。它集中在哺乳动物的丝氨酸蛋白酶凝血酶，两个黄素蛋白氧化还原系统，以及铜还蛋白的电子转移(ET)突变体，该突变体接受奎诺酶的电子。 人凝血酶催化纤维蛋白原蛋白分解转化为纤维蛋白，纤维蛋白是血液凝块的主要成分。这些蛋白质的几种突变形式以及各种酶-底物和酶-抑制物复合体的结构将被确定，以阐明它们的作用模式。这些研究将扩展到鼠酶，它在几个方面与人类的酶不同。 从嗜麦芽假单胞菌中分离到的氧化还原黄素蛋白异四聚体肌氨酸氧化酶(TSOX)含有FAD、FMN并结合四氢叶酸(THF)。TSOX催化肌氨酸(N-甲基甘氨酸)氧化成亚胺中间体和还原的FAD；在第二步，亚胺中间体与四氢呋喃结合形成5，10-亚甲基-四氢呋喃。两个催化中心相隔约35A，FAD被ET重新氧化成FMN，FMN再还原分子氧生成过氧化氢。 第二种氧化还原黄酶是nikD，它催化肌腱链霉菌生物合成尼可霉素抗生素的早期步骤。它是一种含有FAD的45 kDa的单体，其结构有两种不同的构象。它的突变体的结构将被研究。 氨蓝蛋白是一种大小为9 kDa的蓝铜蛋白，接受脱硝副球藻中甲胺脱氢酶(MADH)的电子。几个氨基蓝蛋白突变体的氧化还原性质与野生型蛋白有很大的不同；这些突变体的原子分辨结构是已知的。这些突变体与MADH的ET络合物的晶体将被研究。

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 is a 2-year proposal for APS beam time for structural studies of biological macromolecules. It focuses on the mammalian serine protease thrombin, on two flavoprotein redox systems and on electron transfer (ET) mutants of a cupredoxin that accepts electrons from a quinoenzyme. Human thrombin catalyzes the proteolytic conversion of fibrinogen to fibrin, the major component of a blood clot. The structures of several mutant forms of these proteins, as well as of various enzyme-substrate and enzyme-inhibitor complexes will be determined in order to elucidate their modes of action. These studies will be extended to the murine enzyme which differs in several respects from the human enzyme. One of the redox flavoproteins, heterotetrameric sarcosine oxidase (TSOX), isolated from Pseudomonas maltophilia, contains FAD, FMN and binds tetrahydrofolate (THF). TSOX catalyzes the oxidation of sarcosine (N-methyl glycine) to an imine intermediate and reduced FAD; in a second step the imine intermediate combines with THF to form 5,10-methylene-THF. The two catalytic sites are separated by about 35 A. The FAD is reoxidized by ET to FMN and the latter then reduces molecular oxygen to yield peroxide. A second redox flavoenzyme is nikD, which catalyzes an early step in the biosynthesis of nikkomycin antibiotics in Streptomyces tendae. It is a monomer of 45 kDa containing FAD; its structure is known in two distinct conformation. Structures of its mutants will be studied. Amicyanin is a blue copper protein of 9 kDa that accepts electrons from methylamine dehydrogenase (MADH) in Paracoccus denitrificans. The redox properties of several amicyanin mutants show significant differences from the wild type protein; atomic resolution structures of these mutants are known. Crystals of ET complexes of these mutants with MADH will be studied.