Raouf Joan, Rafique Nazmi, Goodman Michael Christopher, Idborg Helena, Bergqvist Filip, Armstrong Richard N, Jakobsson Per-Johan, Morgenstern Ralf, Spahiu Linda
Unit of Rheumatology, Department of Medicine Solna, Karolinska Institutet, SE-171 76, Stockholm, Sweden.
Unit of Rheumatology, Karolinska University Hospital, SE-171 76, Stockholm, Sweden.
PLoS One. 2016 Sep 29;11(9):e0163600. doi: 10.1371/journal.pone.0163600. eCollection 2016.
Prostaglandins are signaling molecules that regulate different physiological processes, involving allergic and inflammatory responses and cardiovascular control. They are involved in several pathophysiological processes, including inflammation and cancer. The inducible terminal enzyme, microsomal prostaglandin E synthase 1 (MPGES1), catalyses prostaglandin E2 production during inflammation. MPGES1 has therefore been intensively studied as a pharmaceutical target and many competitive inhibitors targeting its active site have been developed. However, little is known about its catalytic mechanism.
The objective of this study was to investigate which amino acids play a key role in the catalytic mechanism of MPGES1.
Based on results and predictions from previous structural studies, the amino acid residues Asp49, Arg73, Arg126, and Ser127 were chosen and altered by site-directed mutagenesis. The mutated enzyme variants were cloned and expressed in both the E. coli and the Baculovirus expression systems. Their catalytic significance was evaluated by activity measurements with prostanoid profiling.
Our study shows that Arg126 and Asp49 are absolutely required for the catalytic activity of MPGES1, as when exchanged, the enzyme variants loose activity. Ser127 and Arg73 on the other hand, don't seem to be central to the catalytic mechanism because when exchanged, their variants retain considerable activity. Our finding that the Ser127Ala variant retains activity was surprising since high-resolution structural data supported a role in glutathione activation. The close proximity of Ser127 to the active site is, however, supported since the Ser127Cys variant displays 80% lowered activity.
前列腺素是调节不同生理过程的信号分子，涉及过敏和炎症反应以及心血管控制。它们参与多种病理生理过程，包括炎症和癌症。诱导性末端酶微粒体前列腺素E合酶1（MPGES1）在炎症过程中催化前列腺素E2的产生。因此，MPGES1作为药物靶点受到了深入研究，并且已经开发出许多靶向其活性位点的竞争性抑制剂。然而，对其催化机制知之甚少。
本研究的目的是探究哪些氨基酸在MPGES1的催化机制中起关键作用。
基于先前结构研究的结果和预测，选择天冬氨酸49、精氨酸73、精氨酸126和丝氨酸127这几个氨基酸残基，并通过定点诱变进行改变。将突变的酶变体在大肠杆菌和杆状病毒表达系统中进行克隆和表达。通过前列腺素谱分析的活性测量来评估它们的催化意义。
我们的研究表明，精氨酸126和天冬氨酸49是MPGES1催化活性绝对必需的，因为交换后，酶变体失去活性。另一方面，丝氨酸127和精氨酸73似乎对催化机制不是核心，因为交换后，它们的变体保留了相当的活性。我们发现丝氨酸127丙氨酸变体保留活性这一结果令人惊讶，因为高分辨率结构数据支持其在谷胱甘肽激活中的作用。然而，丝氨酸127与活性位点的紧密接近得到了支持，因为丝氨酸127半胱氨酸变体的活性降低了80%。
