QUINOENZYMES: BIOGENESIS, STRUCTURE AND FUNCTION

藜蛋白是一类新的催化剂，其辅因子来源于预先存在的氨基酸侧链。目前已确定的辅助因子有：原核生物中普遍存在的色氨酸、2,4,5-三羟基苯基丙氨酸醌（TPQ）和哺乳动物系统中普遍存在的赖氨酸酪氨酸醌。后两个辅因子是在P.I.的实验室中发现的。TPQ酶家族已被证明以自催化的方式催化辅因子的形成，这就提出了一个单一蛋白质结构如何支持辅因子生物发生和催化转换的双重功能的问题。我们选择了多态汉氏菌（Hansenula polymorpha， HPAO）中的酵母胺氧化酶作为实验系统，因为这种蛋白在酿酒酵母（用于催化转化的研究）和大肠杆菌（用于生物发生的研究）中都有表达。野生型酶的晶体结构在之前的授权期间得到了解决，许多突变形式的研究将在预计期间进行。我们计划利用WT和突变酶进行一系列的动力学和结构研究，目的是阐明生物发生中化学中间体的性质和限速步骤。在催化转换方面，提出了一种新的氧化半反应机制，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残基之间的相互作用；这些基序在生物发生和周转中的作用将被检查。赖氨酸氧化酶的生理功能是明确的，而在高等真核生物中含有tpq的分子的生理功能尚不清楚。其他的研究将包括：(i)研究与膜相关的TPQ酶在高等真核生物中的生理作用，（ii）赖氨酸氧化酶的结构功能研究，与HPAO进行比较，以及（iii）研究已知和潜在的新醌辅因子的模型化合物。

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.