Mechanism of Peptide Amidation: Structural and Kinetic Studies

AmzelMCB 9982945许多生物活性肽必须在其羧基末端酰胺化才能表现出完整的活性。 令人惊讶的是，酰胺不是通过转氨基反应产生的。 相反，激素是从甘氨酸延伸的中间体合成的，甘氨酸延伸的中间体通过甘氨酸N-Ca键的氧化裂解转化为活性酰胺化激素。 在高等生物中，双功能酶肽酰甘氨酸α-酰胺化单加氧酶（PAM）催化该反应。 PAM基因编码两个结构域（PHM和PAL），当通过切割或通过独立表达分离时，保留其各自的酶活性。 它们一起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Ca-N键的切除以产生α-酰胺化肽产物和乙醛酸酯（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是含锌的裂解酶，其在Ca的羟基化后裂解Ca-N键。该项目将使用X射线衍射和动力学技术来解决PHM和PAL的广泛底物特异性的起源问题，确定PHM和PAL活性的关键残基，并提出两种酶的酶促机制。肽酰胺化是一个基本的生物过程。 酰胺化肽已经在从Aesthesia到人类的物种的信号系统中被发现。 用于该反应的生物合成途径允许生物体严格调节这些肽的产生。 了解这个反应的化学，特别是第一步，不仅有助于肽酰胺化领域，而且还将提供一个优秀的范例，了解远程电子转移和防止有害氧的产生。

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.