QUINOENZYMES: BIOGENESIS, STRUCTURE AND FUNCTION

藜麦蛋白是一类新型催化剂，其辅因子来源于已有的氨基酸侧链。到目前为止，已确定的辅因子包括仅限于原核生物的色氨酸色氨酸基醌、普遍存在的2，4，5-三羟基苯丙氨酸醌(TPQ)和仅限于哺乳动物系统的赖氨酸酪氨酸醌。后两种辅因子是在P.I.的实验室中发现的。TPQ酶家族已被证明以自动催化的方式催化辅因子的形成，这提出了一个问题，即单一的蛋白质结构如何支持辅因子生物生成和催化周转的双重功能。多态汉逊酵母的酵母胺氧化酶(HPAO)是我们选择的实验系统，因为该蛋白在酿酒酵母(用于催化周转研究)和大肠杆菌(用于生物发生研究)中都有表达。野生型酶的晶体结构在之前的授权期内已经解决，许多突变形式的研究将在计划的时期内继续进行。计划使用WT和突变酶进行一系列动力学和结构研究，目的是阐明生物发生中化学中间体的性质和限速步骤。关于催化周转，提出的氧化半反应的新机理将通过诱变实验进行测试。已经确定了几个结构基序，涉及催化二聚体相对亚基上的残基之间的相互作用；我们将研究这些基序在生物发生和周转中的作用。赖氨酰氧化酶的生理功能已经明确，而高等真核生物中含有TPQ的分子的生理功能尚不清楚。其他研究将涉及(I)膜相关TPQ酶在高等真核生物中的生理作用的研究，(Ii)赖氨酰氧化酶的结构功能研究，以与HPAO进行比较，以及(Iii)已知的和潜在的新的奎诺辅酶因子的模型化合物的研究。

Quinoproteins constitute a new class of catalysts in which their cofactor is derived from a pre-existing amino acid side chain. Cofactors identified thus far are tryptophan tryptophanylquinone, restricted to prokaryotes, 2,4,5-trihydroxyphenylalanine quinone (TPQ), found to be ubiquitous and lysine tyrosylquinone, restricted to mammalian systems. The latter two cofactors were discovered in the laboratory of the P.I. The family of TPQ enzymes has been demonstrated to catalyze cofactor formation in an auto-catalytic manner, raising the question of a how a single protein structure supports the dual functionalities of cofactor biogenesis and catalytic turnover. The yeast amine oxidase from Hansenula polymorpha (HPAO) is our experimental system of choice, since this protein has been expressed in both S. cerevisiae (for studies of catalytic turnover) and in E. coli (for studies of biogenesis). A crystal structure of the wild type enzyme was solved during the previous granting period and studies of numerous mutant forms will be pursued during the projected period. A series of kinetic and structural studies are planned using both WT and mutant enzymes, with the goal of elucidating the nature of chemical intermediates and rate limiting steps in biogenesis. With regard to catalytic turnover, a new mechanism proposed for the oxidative half reaction will be tested via mutagenesis experiments. Several structural motifs have been identified that involve interaction between residues on opposite subunits of the catalytic dimer; the role of these motifs in biogenesis and turnover will be examined. The physiological function of lysyl oxidase is well defined, whereas that of TPQ-containing molecules in higher eukaryotes remains unknown. Additional studies will involve (i) the investigation of the physiological role of the membrane associated TPQ enzymes in higher eukaryotes, (ii) structure function studies of lysyl oxidase, for comparison to HPAO, and (iii) studies of model compounds for both known and potentially new quinocofactors.