Mechanism of Peptide Amidation: Structural and Kinetic Studies

智力优势：从澳大利亚到人类，酰胺肽是物种信号系统的一部分。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酰胺化单加氧酶（PAM）氧化裂解甘氨酸N-C（α）键，转化为活性修饰激素。PAM基因编码两个结构域，催化产生酰胺肽的两个连续反应；甘氨酸的-羟基化（PHM）和C(α)-N键的切除得到α酰胺肽和乙醛酸盐（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，在羟基化后裂解C(α)-N键。作为先前工作的一部分，还原和氧化PHM的结构，单独和与底物，被确定。还完成了PHM小分子配体和选择突变体的结构和动力学表征。在一个非常重要的进展中，完成了PAL催化核结构的测定。这导致了与非肽底物（羟基马尿酸）配合物中PAL结构的测定，提出了一种机制，以及PAL抑制剂的设计和验证。此外，还对所选突变体进行了动力学研究，以证实其机理。该项目包括使用单晶和溶液XAFS进一步表征PHM铜中心，并确定在PHM中提取底物氢的物种。对PAL的研究主要集中在通过研究突变体、还原剂的作用和一种新的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结构域的完整结构，以及这两个结构域的复合物。这两个领域之间的相互作用将使用求解方法进行表征。该项目还将探索两步反应是随着PHM产物的释放而发生，还是通过PHM到PAL的内部转移而逐步发生。这些研究将促进对肽酰胺化的理解，这是信号转导的基本步骤。此外，它们将为理解长距离电子转移以及两步酶的处理性提供一个杰出的范例。此外，PHM在序列和机制上与多巴胺-单加氧酶（一种将多巴胺转化为去甲肾上腺素的酶）是同源的，去甲肾上腺素是一种关键的神经递质。更广泛的影响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，并与两个本科教学机构的实验室合作。他参加了针对代表性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联合组织的全系科学日，为来自内城的学生举办。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目前在他的实验室里培养四名少数民族学生。

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.