Microsomal prostaglandin E 2 synthase type 1 (mPGES-1) is responsible for the formation of the potent lipid mediator prostaglandin E 2 under proinflammatory conditions, and this enzyme has received considerable attention as a drug target. Recently, a high-resolution crystal structure of human mPGES-1 was presented, with Ser-127 being proposed as the hydrogen-bond donor stabilizing thiolate anion formation within the cofactor, glutathione (GSH). We have combined site-directed mutagenesis and activity assays with a structural dynamics analysis to probe the functional roles of such putative catalytic residues. We found that Ser-127 is not required for activity, whereas an interaction between Arg-126 and Asp-49 is essential for catalysis. We postulate that both residues, in addition to a crystallographic water, serve critical roles within the enzymatic mechanism. After characterizing the size or charge conservative mutations Arg-126-Gln, Asp-49-Asn, and Arg-126-Lys, we inferred that a crystallographic water acts as a general base during GSH thiolate formation, stabilized by interaction with Arg-126, which is itself modulated by its respective interaction with Asp-49. We subsequently found hidden conformational ensembles within the crystal structure that correlate well with our biochemical data. The resulting contact signaling network connects Asp-49 to distal residues involved in GSH binding and is ligand dependent. Our work has broad implications for development of efficient mPGES-1 inhibitors, potential anti-inflammatory and anticancer agents.is an abundant lipid mediator that signals via four receptors (EP1-4) to induce an array of important biological actions in physiology as well as pathophysiology (1). Under proinflammatory conditions, biosynthesis of PGE 2 proceeds from arachidonic acid, which is converted to the unstable endoperoxide PGH 2 by cyclooxygenase type 2 (COX-2). PGH 2 is further isomerized into PGE 2 by microsomal PGE synthase type 1 (mPGES-1) (2, 3). mPGES-1 is encoded by PTGES and is up-regulated by mitogens and cytokines in a pathway that is functionally coupled to COX-2 (2, 4). Because of its key role in PGE 2 synthesis, mPGES-1 has attracted attention as a potential drug target in the areas of inflammation, pain, fever, and cancer (5).mPGES-1 is a member of the MAPEG (Membrane-Associated Proteins in Eicosanoid and Glutathione metabolism) superfamily of enzymes (6), which also includes two key proteins in the leukotriene (LT) cascade, viz. 5-lipoxygenase activating protein and LT C 4 synthase (LTC4S). All MAPEG members are integral, homotrimeric membrane proteins, and structural information on this family has been scarce. However, significant progress has recently been made in this area with several high-resolution structures being solved by X-ray crystallography (7-9). In particular, the crystal structures of human LTC4S provided detailed structural information, including an arginine residue that was later shown to activate the glutathione (GSH) thiolate (10, 11). We have previously proposed ...
