Mechanism of Peptide Amidation: Structural and Kinetic Studies

智力优势：酰胺化肽是信号系统的一部分，从人类到动物都有。在所有情况下，甘氨酸延伸的中间体通过双功能酶肽基甘氨酸α-酰胺化单加氧酶（PAM）氧化裂解甘氨酸N-C（α）键转化为活性酰胺化激素。PAM基因编码两个结构域，其催化产生酰胺化肽的两个连续反应;甘氨酸（PHM）的α-羟基化和C（α）-N键的切除以产生α-酰胺化肽加乙醛酸（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后，催化分子氧的还原以用于甘氨酸扩展的底物的羟基化。PAL是一种含锌裂解酶，在羟基化后裂解C（alpha）-N键。作为以前工作的一部分，结构的还原和氧化PHM，单独和基板，进行了测定。PHM的小分子配体以及选定的突变体的结构和动力学表征也完成了。在一个非常重要的进展中，完成了PAL催化核心结构的测定。这导致了PAL与非肽底物（羟马尿酸）复合的结构的确定，提出了一种机制，以及PAL抑制剂的设计和验证。此外，选择的突变体的动力学研究进行了确认的机制。该项目涉及进一步表征的PHM铜中心使用单晶和解决方案XAFS，和识别的物种，抽象的基板氢PHM。对PAL的研究集中在通过研究突变体、还原剂的影响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结构域的完整构建体以及两个结构域的复合物。两个域之间的相互作用将使用解决方案的方法。该项目还将探讨两步反应是否发生与释放的PHM产品，或者如果它发生的内部转移从PHM到PAL。这些研究将促进对肽酰胺化的理解，这是信号传导的基本步骤。此外，他们将提供一个优秀的范例，了解远程电子转移以及在两步酶的持续合成能力。此外，PHM在序列和机制上与多巴胺β-单加氧酶同源，多巴胺β-单加氧酶是将多巴胺转化为去甲肾上腺素的酶，去甲肾上腺素是一种关键的神经递质。PI积极参与将科学的兴奋带给高中生，本科生和代表性不足的少数民族。他教授两门本科课程，并与本科教学机构的两个实验室合作。他参加了针对任职人数不足群体成员的广泛外联活动。PI的实验室每年参加与巴尔的摩市学校系统一起为来自内城的学生组织的全系科学日。PI在西班牙裔科学界和拉丁美洲科学界有很强的影响力。他是拉丁美洲生物药理学家协会（SOBLA）的创始成员和前任主席。PI目前在他的实验室培训四名少数民族学生。

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.