Mechanism of Peptide Amidation: Structural and Kinetic Studies

肽酰胺化机制：结构和动力学研究

• 批准号: 0450465
• 负责人: L. Mario Amzel
• 金额: $ 71.95万
• 依托单位: Johns Hopkins University
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-02-01 至 2010-01-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0450465&HistoricalAwards=false
• 关键词: Mechanism; Peptide; Amidation; Structural; Kinetic

Amidated peptides are fundamental signaling molecules found in species ranging from Aplysia to humans. Surprisingly, peptide amidation is not carried out by a transamination reaction, but by oxidative cleavage, glycine-extended precursors. A bifunctional enzyme, peptidylglycine alpha-amidating monooxygenase (PAM) catalyzes this reaction. The PAM gene encodes two domains (PHM and PAL) that catalyze two sequential reactions: alpha-hydroxylation of the glycine (PHM) and excision of the C alpha-N bond to give alpha-amidated peptide (PAL). PHM contains two redox active copper atoms that, upon reduction by ascorbate, catalyze the reduction of molecular oxygen for the hydroxylation of glycine-extended substrates. PAL is a zinc containing lyase that cleaves the C alpha-N bond after hydroxylation of the C alpha. This project will employ X-ray diffraction, kinetic experiments, peptide design, and site directed mutagenesis to elucidate important aspects of the mechanism of reactions catalyzed by PHM and PAL. Understanding the chemistry of PAM, especially that of the first step of the reaction, will not only contribute to the field of peptide amidation, but will also provide an outstanding paradigm for understanding long range electron transfer and the prevention of production of deleterious oxygen species. Broader Impacts: The PI of this project is actively involved in teaching as well as in bringing the excitement of science to underrepresented minorities. Mentoring of minority students will be achieved through participation in the Meyerhoff Scholars Programs of the University of Maryland Baltimore Campus. The project will also involve participation in the Science Day organized in conjunction with the Baltimore City school system.

酰胺肽是一种基本的信号分子，存在于从澳大利亚到人类的物种中。令人惊讶的是，肽酰胺化不是通过转氨化反应进行的，而是通过氧化裂解，甘氨酸延伸前体进行的。一种双功能酶，肽酰甘氨酸酰胺化单加氧酶（PAM）催化这一反应。PAM基因编码两个结构域（PHM和PAL），催化两个连续反应：甘氨酸的α -羟基化（PHM）和C - α - n键的切除得到α -酰胺肽（PAL）。PHM含有两个氧化还原活性铜原子，在被抗坏血酸还原后，催化分子氧的还原，用于甘氨酸延伸底物的羟基化。PAL是一种含锌裂解酶，在C α羟基化后裂解C α - n键。本项目将采用x射线衍射、动力学实验、多肽设计和定点诱变来阐明PHM和PAL催化反应机理的重要方面。了解PAM的化学性质，特别是反应第一步的化学性质，不仅有助于肽酰胺化领域的研究，但也将为理解远距离电子转移和防止有害氧的产生提供一个杰出的范例。更广泛的影响：该项目的PI积极参与教学，并将科学的兴奋带给代表性不足的少数民族。少数民族学生的指导将通过参与马里兰大学巴尔的摩校区的迈耶霍夫学者项目来实现。该项目还包括参与与巴尔的摩市学校系统联合组织的科学日。