Terminal enzymes of heme synthesis

描述（由申请人提供）：拟议工作的总体目标是获得数据，以便在分子水平上描述末端两种血红素合成途径酶原卟啉原氧化酶（PRO）和亚铁螯合酶的功能。目前的建议的目标是确定催化和结构特征的人PRO和亚铁螯合酶和细菌氧非依赖性PRO。涉及这些酶以及细胞色素c和共济失调蛋白的潜在蛋白质-蛋白质相互作用将被检查。所获得的数据将描述人类自然发生的突变的催化后果，这些突变导致称为卟啉症的遗传性疾病，这应该对患者治疗和咨询有价值。这些数据还将识别和表征人类和微生物酶之间存在的任何关键差异。这些结果可能为开发新的抗微生物剂类别奠定基础。本提案的具体目标是：1）通过使用动力学、定点诱变和基于结构的研究来定义人PRO的结构-功能特征，确定细胞色素c是否是PPO的真正电子受体并表征这种相互作用，以及分离和表征细菌氧非依赖性原卟啉原氧化酶。2.）通过使用动力学、定点诱变和基于结构的研究来定义亚铁螯合酶的结构-功能特征，表征亚铁螯合酶：共济失调蛋白相互作用并确定其与具有环状铁粒幼细胞的ERR患者的相关性，以及通过定点诱变和物理检查来表征动物和细菌亚铁螯合酶中的[2Fe-2S]簇。3.）第三章鉴定和表征亚铁螯合酶和PPO之间蛋白质-蛋白质相互作用。

DESCRIPTION (provided by applicant): The overall goals of the proposed work are to obtain data that will allow descriptions at the molecular level how the terminal two heme synthetic pathway enzymes, protoporphyrinogen oxidase (PRO) and ferrochelatase, function. The goals of the current proposal are to define catalytic and structural features of human PRO and ferrochelatase and bacterial oxygen-independent PRO. Potential protein-protein interactions involving these enzymes as well as cytochrome c and frataxin will be examined. The data obtained will describe the catalytic consequences of naturally occurring mutations in humans that lead to the inherited diseases known as porphyrias, and this should be of value in patient treatment and counseling. The data will also identify and characterize any key differences that exist between the human and microbial enzymes. These results may set the stage for development of new classes of antimicrobial agents. Specific aims of this proposal are to: 1.) define structure-function characteristics of human PRO through the use of kinetics, site-directed mutagenesis, and structure-based studies, determine if cytochrome c is a bona fide electron acceptor for PPO and characterize this interaction, and isolate and characterize bacterial oxygen-independent protoporphyrinogen oxidase. 2.) define structure-function characteristics of ferrochelatase through the use of kinetics, site-directed mutagenesis, and structure-based studies, characterize the ferrochelatase:frataxin interaction and determine its relevance to ERR patients with ringed sideroblasts, and characterize the [2Fe-2S] cluster in animal and bacterial ferrochelatases through site-directed mutagenesis and physical examination. 3.) identify and characterize protein-protein interactions between ferrochelatase and PPO.