Mechanism of Peptide Amidation: Structural and Kinetic Studies

酰胺化肽是在从拟南芥到人类的物种中发现的基本信号分子。 令人惊讶的是，肽酰胺化不是通过转氨基反应进行的，而是通过氧化裂解、甘氨酸延伸的前体进行的。 一种双功能酶，肽基甘氨酸α-酰胺化单加氧酶（PAM）催化该反应。 PAM基因编码两个结构域（PHM和PAL），其催化两个连续反应：甘氨酸的α-羟基化（PHM）和C α-N键的切除以产生α-酰胺化肽（PAL）。 PHM含有两个氧化还原活性铜原子，其在被抗坏血酸盐还原时催化分子氧的还原以用于甘氨酸延伸的底物的羟基化。 PAL是一种含锌裂解酶，在C α羟基化后裂解C α-N键。 本计画将利用X光绕射、动力学实验、胜肽设计及定点突变来阐明PHM与PAL催化反应之机制。了解PAM的化学，特别是反应的第一步，不仅有助于肽酰胺化领域，而且还将提供一个杰出的范例，了解远程电子转移和防止有害氧的产生。 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。少数民族学生的辅导将通过参加马里兰州巴尔的摩校区的迈耶霍夫学者方案来实现。该项目还将包括参加与巴尔的摩市学校系统联合举办的科学日活动。

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.