Mechanism of Peptide Amidation: Structural and Kinetic Studies

智力优势：酰胺化肽是从海兔到人类的各种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都是通过双功能酶-氨基甘氨酸α-酰胺化单加氧酶(PAM)氧化裂解甘氨酸N-C(α)键而转化为活性酰胺化激素。PAM基因编码两个结构域，催化产生酰胺化肽的两个连续反应：甘氨酸的α-羟基化(PHM)和C(α)-N键的切除，得到α-酰胺化肽加乙醛(PAL)。PHM含有两个氧化还原活性铜原子，在抗坏血酸还原后，催化甘氨酸延伸底物羟基化的分子氧还原。PAL是一种含锌裂解酶，羟化后裂解C(α)-N键。作为先前工作的一部分，确定了还原和氧化的PHM的结构，包括单独的结构和带有底物的结构。还对PHM的小分子配体以及筛选出的突变体进行了结构和动力学表征。在一个非常重要的发展中，PAL催化核心结构的确定完成了。这导致了与非肽底物(羟基马尿酸)形成的络合物中PAL结构的确定，提出了一种机制，并设计和验证了PAL抑制剂。此外，还对筛选出的突变体进行了动力学研究，以证实这一机制。该项目包括使用单晶和溶液XAFS进一步表征PHM的铜中心，以及识别PHM中提取底物氢的物种。对PAL的研究集中在通过研究突变体、还原剂的影响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体。这两个领域之间的相互作用将使用求解方法来表征。该项目还将探索两步反应是随着PHM产物的释放而发生，还是通过从PHM到PAL的内部转移而连续发生。这些研究将促进对多肽酰胺化的理解，这是信号转导的基本步骤。此外，它们还将为理解长距离电子转移以及两步酶的过程提供一个杰出的范例。此外，PHM在序列和机制上与多巴胺β-单加氧酶同源，后者是一种将多巴胺转化为去甲肾上腺素的酶，去甲肾上腺素是关键的神经递质。更广泛的影响PI积极参与将科学的兴奋带给高中生、本科生和未被充分代表的少数群体。他在两个本科课程中授课，并与本科教学机构的两个实验室合作。他参加了一系列针对任职人数不足群体成员的外联活动。国际和平研究所的实验室每年都参加与巴尔的摩市学校系统一起为来自市中心的学生举办的全部门科学日活动。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.