Protein- and Peptide-Derived Cofactors

描述（由申请人提供）：已发现许多与医学相关的哺乳动物蛋白质含有源自氨基酸侧链的新型氧化还原辅助因子。这些辅助因子是铜胺氧化酶蛋白家族中的2,4,5三羟基苯丙烯基醌（TPQ）和赖氨酸酪氨酸醌（LTQ），存在于赖氨酸氧化酶的活性位点。另外两种酪氨酸衍生的辅助因子也在真菌和原核生物中被发现。它们是半乳糖氧化酶中蛋白质结合的半胱氨酸酪氨酸自由基（CTR）和多种细菌产生的肽衍生的吡罗喹啉醌（PQQ）。本提案的一个主要目标是阐明每一种酪氨酸衍生的辅因子的生物发生途径，并确定共同策略（或未）用于实现非常不同的最终辅因子结构的程度。在两个实例（TPQ和CTR）中，有证据表明生物发生仅通过向前体蛋白添加02和Cu 2+而发生。LTQ可能也是如此，而PQQ的产生需要许多基因产物。提出的研究生物发生的实验策略包括前体蛋白或多肽的表达（在PQQ的情况下连同附属蛋白的表达），然后应用动力学和光谱学来检测和表征生物发生的中间体。

DESCRIPTION (provided by applicant): A number of medically relevant mammalian proteins have been found to contain novel redox cofactors that are derived from amino acid side chains. These cofactors are 2,4,5 trihydroxyphenylalanyl quinone (TPQ), discovered in the copper amine oxidase family of proteins, and the lysine tyrosyl quinone (LTQ), present in the active site of lysyl oxidases. Two other tyrosine-derived cofactors have also been described in fungi and prokaryotes. These are the protein bound cysteine tyrosyl radical (CTR) in galactose oxidase and the peptide derived pyrroloquinoline quinone (PQQ) produced by a variety of bacteria. A major goal of this proposal is to elucidate the pathway for the biogenesis of each of these tyrosine-derived cofactors and to determine the degree to which common strategies have (or have not) been employed to achieve the very different final cofactor structures. In two instances (TPQ and CTR), evidence has been presented that biogenesis occurs solely by the addition of 02 and Cu 2+ to precursor protein. This is likely to also be true for LTQ, while PQQ production requires a number of gene products. The proposed experimental strategy for studying biogenesis involves the expression of precursor protein or polypeptide (together with the expression of accessory proteins in the case of PQQ), followed by the application of kinetics and spectroscopy to detect and characterize biogenesis intermediates. The impact of cofactor production on cellular physiology may be very significant. In the case of the lysyl oxidases, it is known that many disease states correlate with enzyme activity that will be affected by copper availability and the rate and extent of cofactor production. The cellular roles of the mammalian copper amine oxidases are only now becoming apparent. Experiments are described that address the role of the copper amine oxidase located on the outer surface of the endothelium and the adipocyte. Experimental protocols will focus on the characterization of the endothelial enzyme that we have recently expressed in high yield in insect cells and the use of an adipocyte cell line to follow the impact of copper amine oxidase activity on cellular function.