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: 0920288
• 负责人: L. Mario Amzel
• 金额: $ 115.64万
• 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: 2009
• 资助国家: 美国
• 起止时间: 2009-08-01 至 2015-07-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0920288&HistoricalAwards=false
• 关键词: Mechanism; Peptide; Amidation; Structural; Kinetic

Intellectual Merit: Amidated peptides are part of signaling systems in species ranging from Aplysia to humans. In all cases, glycine-extended intermediates are transformed into active amidated hormones by oxidative cleavage of the glycine N-C(alpha) bond by a bifunctional enzyme, peptidylglycine alpha-amidating monooxygenase (PAM). The PAM gene encodes two domains that catalyze the two sequential reactions producing amidated peptide; alpha-hydroxylation of the glycine (PHM) and excision of the C(alpha)-N bond to give the alpha-amidated peptide plus glyoxylate (PAL). PHM contains two redox active copper atoms that, after reduction by ascorbate, catalyze the reduction of molecular oxygen for hydroxylation of glycine-extended substrates. PAL is a zinc-containing lyase that cleaves the C(alpha)-N bond after hydroxylation. As part of previous work, the structures of reduced and oxidized PHM, alone and with substrates, were determined. Structural and kinetic characterization of small molecule ligands of PHM as well as selected mutants were also accomplished. In a very important development, the determination of the structure of the catalytic core of PAL was accomplished. This led to the determination of the structure of PAL in complex with a non-peptidic substrate (hyrdoxyhippuric acid), proposing a mechanism, and the design and validation of a PAL inhibitor. In addition, kinetic studies of selected mutants were carried out to confirm the mechanism. This project involves further characterization of the PHM copper centers using single crystal and solution XAFS, and identification of the species that abstracts the substrate hydrogen in PHM. Studies on PAL are centered on addressing additional features of its mechanism by studying mutants, the effects of reductants, and the kinetics of a new inhibitor. The structure of PAM will be determined as a complete construct containing the PHM and PAL domains and as a complex of the two domains. The interaction between the two domains will be characterized using solution methods. The project will also explore whether the two-step reaction takes place with release of the PHM product or if it occurs processively by an internal transfer from PHM to PAL. The studies will advance understanding of peptide amidation which is a fundamental step in signal transduction. They will, in addition, provide an outstanding paradigm for understanding long range electron transfers as well as processivity in two step enzymes. Moreover, PHM is homologous in sequence and mechanism to dopamine beta-monooxygenase -the enzyme that converts dopamine to norepinephrine, a key neurotransmitter. Broader Impact The PI is actively involved in bringing the excitement of science to high school students, undergraduates and underrepresented minorities. He teaches classes in two undergraduate courses, and has collaborations with two laboratories at undergraduate teaching institutions. He participated in a broad range of outreach activities that have been directed at members of underrepresented groups. The laboratory of the PI participates annually in the Department-wide Science Day organized in conjunction with the Baltimore City school system for students from the inner city. The PI has a strong presence in the Hispanic scientific community and in Latin American science. He is a founding member and past president of the Society for Latin American Biophysicists (SOBLA). The PI currently trains four minority students in his laboratory.

智力优点：胺化肽是从高脂到人类的物种中信号系统的一部分。在所有情况下，通过双功能酶，肽基甘氨酸α-抗甘露甘氨酸α-增强单氧酶（PAM）的氧化裂解，将甘氨酸延伸的中间体转化为活性胺激素。 PAM基因编码了两个结构域，这些结构域催化了两个产生胺化肽的顺序反应。甘氨酸（PHM）的α-羟基化和C（alpha）-n键的切除，得出α添加的肽加甘酰基（PAL）。 PHM含有两个氧化还原活性铜原子，在抗坏血酸还原后，它们催化了分子氧的还原甘氨酸扩张底物的羟基化。 PAL是一种含锌的石酶，在羟基化后切割C（alpha）-n键。作为先前工作的一部分，确定了单独的和氧化的PHM的结构，并确定了底物。还完成了PHM小分子配体以及选定突变体的结构和动力学表征。在非常重要的发展中，确定了PAL催化核心的结构。这导致了与非肽底物（氢化乳酸）复合物中PAL结构的确定，提出了一种机制，以及对PAL抑制剂的设计和验证。此外，对选定的突变体进行了动力学研究以确认该机制。该项目涉及使用单晶和溶液XAFS对PHM铜中心的进一步表征，以及鉴定在PHM中抽象底物氢的物种。关于PAL的研究集中在通过研究突变体，还原剂的作用和新抑制剂的动力学来解决其机制的其他特征。 PAM的结构将被确定为包含PHM和PAL结构域以及两个域的复合物的完整构造。使用解决方案方法将表征两个域之间的相互作用。该项目还将探索两步反应是通过释放PHM产物发生的，还是通过从PHM到PAL的内部转移进行过程。研究将提高对肽症的了解，这是信号转导的基本步骤。另外，他们将提供出色的范式，以了解两个步骤酶中的远程电子传输以及加工性。此外，PHM在序列和机制上是多巴胺β-单氧化酶的机理 - 将多巴胺转化为脱甲肾上腺素的酶，它是一种关键的神经递质。 PI更广泛的影响力积极参与将科学兴奋带给高中生，本科生和代表性不足的少数民族。他在两门本科课程中教课程，并与本科教学机构的两个实验室合作。他参加了针对代表性不足的团体成员的广泛推广活动。 PI的实验室每年参加部门范围内的科学日，与巴尔的摩市学校的学校系统一起组织，适合来自内城的学生。 PI在西班牙裔科学界和拉丁美洲科学方面具有很强的作用。他是拉丁美洲生物物理学家协会（SOBLA）的创始成员和前任主席。 PI目前在他的实验室中训练四名少数族裔学生。