Arg126 and Asp49 Are Essential for the Catalytic Function of Microsomal Prostaglandin E2 Synthase 1 and Ser127 Is Not.
BACKGROUND
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.


OBJECTIVE
The objective of this study was to investigate which amino acids play a key role in the catalytic mechanism of MPGES1.


METHODS
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.


CONCLUSIONS
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.
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.
Joan
http://orcid.org/0000-0001-6808-7024
J
Raouf
Institute of Environmental Medicine, Division of Biochemical Toxicology, Karolinska Institutet, Stockholm, Sweden.
Nazmi
N
Rafique
Department of Chemistry, Vanderbilt University School of Medicine, Nashville, United States of America.
Michael Christopher
MC
Goodman
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.
Helena
H
Idborg
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.
Filip
F
Bergqvist
Department of Chemistry, Vanderbilt University School of Medicine, Nashville, United States of America.
Richard N
RN
Armstrong
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.
Per-Johan
PJ
Jakobsson
Institute of Environmental Medicine, Division of Biochemical Toxicology, Karolinska Institutet, Stockholm, Sweden.
Ralf
R
Morgenstern
Institute of Environmental Medicine, Division of Biochemical Toxicology, Karolinska Institutet, Stockholm, Sweden.
Linda
L
Spahiu
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