Mounier C E, Shi J, Sirimanne S R, Chen B H, Moore A B, Gill-Woznichak M M, Ping D, May S W
School of Chemistry and Biochemistry, Georgia Institute of Technology, Atlanta, Georgia 30332, USA.
J Biol Chem. 1997 Feb 21;272(8):5016-23. doi: 10.1074/jbc.272.8.5016.
Carboxyl-terminal amidation, a required post-translational modification for the bioactivation of many neuropeptides, entails sequential enzymatic action by peptidylglycine monooxygenase (PAM, EC 1.14.17.3) and peptidylamidoglycolate lyase (PGL, EC 4.3.2.5). The monooxygenase, PAM, first catalyzes conversion of a glycine-extended pro-peptide to the corresponding alpha-hydroxyglycine derivative, and the lyase, PGL, then catalyzes breakdown of this alpha-hydroxyglycine derivative to the amidated peptide plus glyoxylate. We now introduce the first potent inhibitors for peptidylamidoglycolate lyase. These inhibitors, which can be viewed as pyruvate-extended N-acetyl amino acids, constitute a novel class of compounds. They were designed to resemble likely transient species along the reaction pathway of PGL catalysis. A general synthetic procedure for preparation of pyruvate-extended N-acetyl amino acids or peptides is described. Since these compounds possess the 2,4-dioxo-carboxylate moiety, their solution tautomerization was investigated using both NMR and high performance liquid chromatography analyses. The results establish that freshly prepared solutions of N-Ac-Phe-pyruvate consist predominantly of the enol tautomer, which then slowly tautomerizes to the diketo form when left standing for several days in an aqueous medium; upon acidification, formation of the hydrate tautomer occurs. Kinetic experiments established that these novel compounds are highly potent, pure competitive inhibitors of PGL. Kinetic experiments with the ascorbate-dependent copper monooxygenases, PAM and dopamine-beta-monooxygenase, established that these compounds also bind competitively with respect to ascorbate; however, pyruvate-extended N-acyl-amino acid derivatives possessing hydrophobic side chains are much more potent inhibitors of PGL than of PAM. Selective targeting of N-Ac-Phe-pyruvate so as to inhibit the lyase, but not the monooxygenase, domain was demonstrated with the bifunctional amidating enzyme of Xenopus laevis. The availability of potent inhibitors of PGL should facilitate studies regarding the possible biological role of alpha-hydroxyglycine-extended peptides.
羧基末端酰胺化是许多神经肽生物激活所需的翻译后修饰，它需要肽基甘氨酸单加氧酶（PAM，EC 1.14.17.3）和肽基酰胺基乙醇酸裂解酶（PGL，EC 4.3.2.5）的顺序酶促作用。单加氧酶PAM首先催化甘氨酸延伸的前体肽转化为相应的α-羟基甘氨酸衍生物，然后裂解酶PGL催化该α-羟基甘氨酸衍生物分解为酰胺化肽和乙醛酸。我们现在介绍了肽基酰胺基乙醇酸裂解酶的首批强效抑制剂。这些抑制剂可被视为丙酮酸延伸的N-乙酰氨基酸，构成了一类新型化合物。它们的设计类似于PGL催化反应途径中可能的瞬态物种。描述了制备丙酮酸延伸的N-乙酰氨基酸或肽的一般合成方法。由于这些化合物具有2,4-二氧代羧酸盐部分，因此使用核磁共振和高效液相色谱分析对其溶液互变异构进行了研究。结果表明，新制备的N-乙酰苯丙氨酸丙酮酸溶液主要由烯醇互变异构体组成，当在水性介质中放置数天后，它会缓慢互变异构为二酮形式；酸化后，会形成水合物互变异构体。动力学实验表明，这些新型化合物是PGL的高效、纯竞争性抑制剂。用抗坏血酸依赖性铜单加氧酶PAM和多巴胺-β-单加氧酶进行的动力学实验表明，这些化合物也与抗坏血酸竞争性结合；然而，具有疏水侧链的丙酮酸延伸的N-酰基氨基酸衍生物对PGL的抑制作用比对PAM的抑制作用要强得多。非洲爪蟾的双功能酰胺化酶证明了N-乙酰苯丙氨酸丙酮酸对裂解酶结构域而非单加氧酶结构域的选择性靶向抑制作用。PGL强效抑制剂的可用性应有助于研究α-羟基甘氨酸延伸肽可能的生物学作用。
