MECHANISTIC STUDIES OF ALKYL HYDROPEROXIDE REDUCTASE

沙门氏菌的烷基过氧化氢还原酶(AhpR)酶系 鼠伤寒沙门氏菌用于保护这些生物免受有毒和诱变的 氧化应激的影响。这些建议的长期目标 研究旨在阐明这两种物质各自的催化机制。 AhpR系统的蛋白AhpC和AhpF，以获得更好的 了解这些蛋白质在保护环境中的生理作用 细胞大分子对抗活性氧物种。一个更广泛的 目的是定义这种酶活性的分布范围有多广 在好氧和厌氧生物中。这个系统已经无处不在， 通过鉴定来自广泛的AhpR同源物的一些建议 来源多种多样。 要检验的主要假设是氧化还原活性 半胱氨酸残基参与血红素非依赖性催化 过氧化氢还原，在考虑到另外两种机制的情况下，这是可以预期的 已知的非血红素过氧化氢还原酶、NADH过氧化物酶和谷胱甘肽 过氧化物酶。具体目标包括L)示范的具体内容 每种AhpR蛋白的催化作用及催化活性鉴定 每种蛋白质中的重要氨基酸，包括半胱氨酸残基， 2)N-末端的207个氨基酸是否 AhpF，在其他同源硫氧还蛋白中没有对应的 还原酶蛋白质，是催化或服务于任何其他 可辨别的功能，3)每个蛋白质的结构特征， 包括X射线结晶学研究是否可以获得高质量的晶体 生成，以及4)分析其他AhpC同源物的能力 支持过氧化氢还原。解决这些具体目标的努力将 包括酶活性的热力学和动力学研究。 每种蛋白质结合化学修饰和定点 和随机诱变研究。 AhpR系统在其化学方面具有相当大的兴趣，因为 另一个可能的例子是一种不寻常的氧化形式的参与 半胱氨酸，半胱氨酸磺酸(R-SOH)，在催化。从一个方面来说 AhpR的生理重要性可能是在帮助致病 沙门氏菌，可能还有其他人类病原体(包括 溶组织内阿米巴、幽门螺杆菌和禽分枝杆菌)，以 逃脱宿主吞噬细胞产生的有毒氧气物种的杀戮。 在各种各样的生物体中鉴定这种酶系统， 正如AHP同系物的广泛分布所表明的那样，可能导致 对AhpR作为一种抗氧化酶具有更广泛的意义 系统。抗氧化系统在高等生物体中是至关重要的 对抗与氧化相关的过程，如致癌、炎症 以及与年龄相关的疾病。

The alkyl hydroperoxide reductase (AhpR) enzyme system of Salmonella typhimurium serves to protect these organisms from the toxic and mutagenic effects of oxidative stress. The long-term objective of these proposed studies is to elucidate the catalytic mechanisms for each of the two proteins of the AhpR system, AhpC and AhpF, in order to gain a better understanding of the physiological role these proteins play in protecting cellular macromolecules against reactive oxygen species. A broader objective is to define how widely this enzymatic activity is distributed among aerobic and anaerobic organisms. Ubiquity of this system has been suggested by the identification of a number of AhpR homologues from a wide variety of sources. The primary hypothesis to be tested is whether or not redox-active cysteine residues are involved in the heme-independent catalysis of peroxide reduction, as might be expected given mechanisms of two other known non-heme peroxide reductases, NADH peroxidase and glutathione peroxidase. Specific aims include l) demonstration of the specific catalytic roles of each AhpR protein and identification of catalytically important amino acids, including cysteine residues, within each protein, 2) determination of whether or not the 207 amino acids at the N-terminus of AhpF, which have no counterpart in the otherwise homologous thioredoxin reductase protein, are required for catalysis or serve any other discernible function, 3) structural characterization of each protein, including X-ray crystallographic studies if high-quality crystals can be generated, and 4) analysis of the ability of other AhpC homologues to support peroxide reduction. Efforts to address these specific aims will include thermodynamic and kinetic studies of the enzymatic activities of each protein in conjunction with chemical modification and site-directed and random mutagenesis studies. The AhpR system is of considerable interest in terms of its chemistry as another possible example of the participation of an unusual oxidized form of cysteine, cysteine sulfenic acid (R-SOH), in catalysis. One aspect of the physiological importance of AhpR may be in helping pathogenic Salmonella, and possibly other human pathogens as well (including Entamoeba histolytica, Helicobacter pylori and Mycobacterium avium), to escape killing by toxic oxygen species produced by the host's phagocytes. The identification of this enzyme system in a wide variety of organisms, as has been suggested by the wide distribution of Ahp homologues, may lead to a more generalized significance for AhpR as an antioxidant enzyme system. Antioxidant systems are critical in higher organisms in countering such oxidation-linked processes as carcinogenesis, inflammation and age-related diseases.