QUINOENZYMES: BIOGENESIS, STRUCTURE AND FUNCTION

醌酶：生物发生、结构和功能

• 批准号: 6041722
• 负责人: JUDITH P KLINMAN
• 金额: $ 40.97万
• 依托单位: UNIVERSITY OF CALIFORNIA BERKELEY
• 依托单位国家: 美国
• 财政年份: 1988
• 资助国家: 美国
• 起止时间: 1988-02-01 至 2004-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6041722
• 关键词: Escherichia coli; Saccharomyces; X ray crystallography; active sites; amine oxidase (copper); amine oxidoreductase; cofactor; enzyme activity; enzyme biosynthesis; enzyme mechanism; enzyme structure; enzyme substrate; gene expression; hydroquinones; ligands; metalloenzyme; phenylalanine analog; protein sequence

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.

醌蛋白是一类新的催化剂，其中它们的辅因子来自于预先存在的氨基酸侧链。到目前为止鉴定的辅因子是仅限于原核生物的色氨酸苯丙氨酸醌、发现普遍存在的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酰醌。 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方式催化辅因子形成，这提出了单个蛋白质结构如何支持辅因子生物发生和催化周转的双重功能的问题。 来自多形汉逊酵母的酵母胺氧化酶（HPAO）是我们选择的实验系统，因为这种蛋白质已经在两种酵母中表达。cerevisiae（用于催化周转的研究）和E.大肠杆菌（用于生物发生的研究）。 野生型酶的晶体结构在上一个资助期内得到了解决，在计划期间将继续研究许多突变形式。 一系列的动力学和结构研究计划使用WT和突变酶，目的是阐明化学中间体的性质和生物发生中的限速步骤。 关于催化营业额，一个新的机制提出的氧化半反应将通过诱变实验进行测试。 已经确定了几个结构图案，涉及催化二聚体的相对亚基上的残基之间的相互作用，这些图案在生物发生和营业额的作用将被检查。 赖氨酰氧化酶的生理功能已得到明确定义，而高等真核生物中含TPQ分子的生理功能仍不清楚。其他研究将涉及（i）膜相关的TPQ酶在高等真核生物中的生理作用的调查，（ii）赖氨酰氧化酶的结构功能研究，比较HPAO，和（iii）模型化合物的研究已知的和潜在的新quinocofactors。