The structural basis of aspirin activity inferred from the crystal structure of inactivated prostaglandin H2 synthase

Aspirin exerts its anti-inflammatory effects through selective acetylation of serine 530 on prostaglandin H2 synthase (PGHS). Here we present the 3.4 Å resolution X-ray crystal structure of PGHS isoform-1 inactivated by the potent aspirin analogue 2-bromoacetoxy-benzoic acid. Acetylation by this analogue abolishes cyclooxygenase activity by steric blockage of the active-site channel and not through a large conformational change. We observe two rotameric states of the acetyl-serine side chain which block the channel to different extents, a result which may explain the dissimilar effects of aspirin on the two PGHS isoforms. We also observe the product salicylic acid binding at a site consistent with its antagonistic effect on aspirin activity.

[1]  P. Kraulis.  A program to produce both detailed and schematic plots of protein structures , 1991 .

[2]  D. Dewitt,et al.  Differential inhibition of prostaglandin endoperoxide synthase (cyclooxygenase) isozymes by aspirin and other non-steroidal anti-inflammatory drugs. , 1993, The Journal of biological chemistry.

[3]  L. Marnett,et al.  Tyrosyl radicals and their role in hydroperoxide-dependent activation and inactivation of prostaglandin endoperoxide synthase. , 1992, Biochemistry.

[4]  J. Mancini,et al.  Mutation of serine‐516 in human prostaglandin G/H synthase‐2 to methionine or aspirin acetylation of this residue stimulates 15‐R‐HETE synthesis , 1994, FEBS letters.

[5]  P. Loll,et al.  Prostaglandin H synthase , 1994 .

[6]  F. Kuehl,et al.  Multiple sites on prostaglandin cyclooxygenase are determinants in the action of nonsteroidal antiinflammatory agents. , 1981, Proceedings of the National Academy of Sciences of the United States of America.

[7]  B. Vargaftig.  The inhibition of cyclo-oxygenase of rabbit platelets by aspirin is prevented by salicylic acid and by phenanthrolines. , 1978, European journal of pharmacology.

[8]  G. Gaetano,et al.  Non-steroidal anti-inflammatory drugs react with two sites on platelet cyclo-oxygenase. Evidence from "in vivo" drug interaction studies in rats. , 1982, Biochimica et biophysica acta.

[9]  P. Loll,et al.  The X-ray crystal structure of the membrane protein prostaglandin H2 synthase-1 , 1994, Nature.

[10]  C. Ji,et al.  Acetylation of human prostaglandin endoperoxide synthase-2 (cyclooxygenase-2) by aspirin. , 1994, The Journal of biological chemistry.

[11]  E. Dejana,et al.  Pharmacology of platelet inhibition in humans: implications of the salicylate-aspirin interaction. , 1985, Circulation.

[12]  J. Pflugrath,et al.  Crystal orientation and X-ray pattern prediction routines for area-detector diffractometer systems in macromolecular crystallography , 1987 .

[13]  B. Vargaftig,et al.  Structural requirements for preventing the aspirin- and the arachidonate-induced inactivation of platelet cyclo-oxygenase: additional evidence for distinct enzymatic sites. , 1984, European journal of pharmacology.

[14]  Collaborative Computational,et al.  The CCP4 suite: programs for protein crystallography. , 1994, Acta crystallographica. Section D, Biological crystallography.

[15]  J. Falgueyret,et al.  Overexpression of human prostaglandin G/H synthase-1 and -2 by recombinant vaccinia virus: inhibition by nonsteroidal anti-inflammatory drugs and biosynthesis of 15-hydroxyeicosatetraenoic acid. , 1994, Molecular pharmacology.

[16]  P. J. Wheatley.  1163. The crystal and molecular structure of aspirin , 1964 .

[17]  C. Walsh,et al.  The behavior and significance of slow-binding enzyme inhibitors. , 2006, Advances in enzymology and related areas of molecular biology.

[18]  C. Patrono,et al.  Selective cumulative inhibition of platelet thromboxane production by low-dose aspirin in healthy subjects. , 1982, The Journal of clinical investigation.

[19]  T. Shimokawa,et al.  Prostaglandin endoperoxide synthase. The aspirin acetylation region. , 1992, The Journal of biological chemistry.

[20]  G. de Gaetano,et al.  Salicylate reverses in vitro aspirin inhibition of rat platelet and vascular prostaglandin generation. , 1980, Biochemical pharmacology.

[21]  G. Roth,et al.  Isolation and covalent structure of the aspirin-modified, active-site region of prostaglandin synthetase. , 1983, Biochemistry.

[22]  Eveline,et al.  The aspirin and heme-binding sites of ovine and murine prostaglandin endoperoxide synthases. , 1990, The Journal of biological chemistry.

[23]  Wolfgang Kabsch,et al.  Evaluation of Single-Crystal X-ray Diffraction Data from a Position-Sensitive Detector , 1988 .

[24]  H. Ruf,et al.  A model for the catalytic mechanism of prostaglandin endoperoxide synthase. , 1993, Biochemical Society transactions.

[25]  A. Ristimäki,et al.  Cyclooxygenase Gene Expression in Inflammation and Angiogenesis a , 1993, Annals of the New York Academy of Sciences.

[26]  G J Roth,et al.  Acetylation of prostaglandin synthase by aspirin. , 1975, Proceedings of the National Academy of Sciences of the United States of America.

[27]  C. Benedetto.  Prostaglandins and related substances : a practical approach , 1987 .

[28]  J. Vane,et al.  Inhibition of prostaglandin synthesis as a mechanism of action for aspirin-like drugs. , 1971, Nature: New biology.

[29]  T. Shimokawa,et al.  Tyrosine 385 of prostaglandin endoperoxide synthase is required for cyclooxygenase catalysis. , 1990, The Journal of biological chemistry.

[30]  M. Holtzman,et al.  Identification of a pharmacologically distinct prostaglandin H synthase in cultured epithelial cells. , 1992, The Journal of biological chemistry.

[31]  S V Evans,et al.  SETOR: hardware-lighted three-dimensional solid model representations of macromolecules. , 1993, Journal of molecular graphics.

[32]  L. Rome,et al.  Structural requirements for time-dependent inhibition of prostaglandin biosynthesis by anti-inflammatory drugs. , 1975, Proceedings of the National Academy of Sciences of the United States of America.