Mechanism of Peptide Amidation: Structural and Kinetic Studies

AmzelMCB 9982945许多生物活性多肽必须在其羧基末端进行酰胺化才能显示全部活性。令人惊讶的是，这些酰胺不是由转氨基反应产生的。相反，这些激素是从甘氨酸延伸的中间体合成的，这些中间体通过甘氨酸N-钙键的氧化裂解转化为活性酰胺化激素。在高等生物中，双功能酶氨基甘氨酸a-酰胺化单加氧酶(PAM)催化这一反应。PAM基因编码两个结构域(PHM和PAL)，当通过切割或独立表达分离时，它们保持各自的酶活性。它们共同催化产生酰胺化肽的两个连续反应：甘氨酸的A-羟基化(PHM)和切割Ca-N键以得到a-酰胺化肽产品和乙醛(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是一种含锌的裂解酶，在钙羟化后裂解钙-氮键。本项目将利用X射线衍射和动力学技术来解决PHM和PAL广泛底物专一性的起源问题，鉴定对PHM和PAL活性至关重要的残基，并提出这两种酶的酶机制。多肽酰胺化是一种基本的生物学过程。在从海兔到人类的各种物种的信号系统中都发现了酰胺化的多肽。用于该反应的生物合成途径允许有机体严格调节这些多肽的生产。了解这一反应的化学过程，特别是第一步反应的化学过程，不仅将有助于多肽酰胺化领域的研究，而且将为理解远程电子转移和防止有害氧物种的产生提供一个杰出的范例。

AmzelMCB 9982945Many bioactive peptides must be amidated at their carboxy terminus to exhibit full activity. Surprisingly, the amides are not generated by a transamination reaction. Instead, the hormones are synthesized from glycine-extended intermediates that are transformed into active amidated hormones by oxidative cleavage of the glycine N-Ca bond. In higher organisms, the bifunctional enzyme peptidylglycine a-amidating monooxygenase (PAM) catalyzes this reaction. The PAM gene encodes two domains (PHM and PAL) that when separated, either through cleavage or through independent expression, retain their individual enzymatic activities. Together they catalyze the two sequential reactions that produce amidated peptide: a-hydroxylation of the glycine (PHM) and excision of the Ca-N bond to give a-amidated peptide product and glyoxylate (PAL). PHM contains two redox active copper atoms that, after reduction by ascorbate, catalyze the reduction of molecular oxygen for the hydroxylation of glycine-extended substrates. PAL is zinc containing lyase that cleaves the Ca-N bond after hydroxylation of the Ca. This project will use of x-ray diffraction and kinetic techniques to address questions about the origin of the broad substrate specificity of PHM and PAL, identify residues critical for PHM and PAL activity, and propose enzymatic mechanisms for both enzymes. Peptide amidation is a fundamental biological process. Amidated peptides have been found in the signaling systems of species ranging from Aplysia to humans. The biosynthetic path used for this reaction allows the organism to strictly regulate the production of these peptides. Understanding the chemistry of this reaction, especially that of the first step, will not only contribute to the field of peptide amidation, but will also provide an outstanding paradigm for understanding long range electron transfers and the prevention of production of deleterious oxygen species.