Quinoproteins: Characterization and Mechanism

本提案的目的是利用生化和生物物理方法进一步表征一类含有辅酶的酶，称为藜蛋白。重点研究哺乳动物奎诺蛋白、血浆单胺氧化酶、肾二胺氧化酶和赖氨酸氧化酶。所有这些酶都被认为含有细菌的辅助因子，吡咯喹啉醌，PQQ，但这一结论现在受到质疑。本提案的目的是：1)表征二胺氧化酶和赖氨酸氧化酶的活性位点辅助因子肽。这些酶可能与血浆单胺氧化酶（即6-羟多巴）具有相同的辅助因子；2)克隆基因，建立血浆单胺氧化酶和肾二胺氧化酶表达系统；3)利用各种生物物理方法表征这三种酶的Cu2+和辅因子位点的接近性；4)开展6-羟多巴生物合成来源的研究。这些研究将为这些金属藜蛋白的机制和结构特征提供一个清晰的认识，并导致对哺乳动物来源的藜蛋白中羰基辅因子的性质的共识。

The aims of this proposal are to employ biochemical and biophysical methods to further characterize a class of cofactor-containing enzymes called quinoproteins. The research will focus on the mammalian quinoproteins, plasma monoamine oxidase, kidney diamine oxidase and lysyl oxidase. All of these enzymes had been thought to contain the bacterial cofactor, pyrroloquinoline quinone, PQQ, but this conclusion is now in doubt. The objectives of this proposal are 1) to characterize the active site cofactor-containing peptides of diamine oxidase and lysyl oxidase. It is possible that these enzymes have the same cofactor as plasma monoamine oxidase, i.e., 6-hydroxydopa; 2) to clone the genes and develop an expression system for plasma monoamine oxidase and kidney diamine oxidase; 3) to characterize the proximity of the Cu2+ and cofactor sites of all three enzymes using a variety of biophysical methods; and 4) to initiate an investigation of the biosynthetic origin of 6-hydroxydopa. These studies will provide a clear understanding of the mechanistic and structural features of these metalloquinoproteins and lead to a consensus concerning the nature of the carbonyl cofactor in quinoproteins of mammalian origin.