Mutational and X-ray Crystallographic Analysis of the Interaction of Dihomo-γ-linolenic Acid with Prostaglandin Endoperoxide H Synthases

Thuresson, Elizabeth D.; Malkowski, Michael G.; Lakkides, Karen M.; Rieke, Caroline Jill; Mulichak, A. M.; Ginell, Stephan L.; Garavito, R. Michael; Smith, William L.

doi:10.1074/jbc.m009378200

Cited by 44 publications

(88 citation statements)

References 55 publications

(84 reference statements)

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“…Consistent with previous, related observations (11)(12)(13)(14)(15), the specific COX activity of the G533A huPGHS-2 homodimer with AA as the substrate was Ϸ5% of the specific activity of the native huPGHS-2 homodimer (Fig. 2).…”

supporting

confidence: 78%

“…However, observation of the crystal structures of PGHS-1 and PGHS-2 dimers suggests that both subunits are identical and both bind substrates and inhibitors (14,15,17,24,25). We performed differential scanning calorimetr y (DSC) on the native huPGHS-2 homodimer, the R120Q huPGHS-2 homodimer, and the native͞R120Q huPGHS-2 heterodimer in the presence and absence of FBP, a time-dependent COX inhibitor, and Sibuprofen (S-IBP), a simple competitive inhibitor, to determine whether we could obtain physical evidence for a nonuniformity of subunits (Fig.…”

Section: Physical Properties Of the Native͞r120q Hupghs-2 Heterodimermentioning

confidence: 99%

“…Gly-533 is at the distal end of the COX binding channel and plays a key role in aligning the 8 proS hydrogen of fatty acid substrates with the Tyr-385 radical involved in COX catalysis (11). G533A mutants of PGHSs oxygenate fatty acids with low efficiencies (11)(12)(13)(14)(15). Arg-120 is found near the mouth of the COX active site of PGHSs and is involved in binding the carboxyl group of fatty acid substrates (11,16,17).…”

mentioning

confidence: 99%

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Partnering between monomers of cyclooxygenase-2 homodimers

Yuan

Rieke

Rimon

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

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Prostaglandin endoperoxide H synthases (PGHSs) 1 and 2 convert arachidonic acid to prostaglandin H2 in the committed step of prostanoid biosynthesis. These enzymes are pharmacological targets of nonsteroidal antiinflammatory drugs and cyclooxygenase (COX) 2 inhibitors. Although PGHSs function as homodimers and each monomer has its own COX and peroxidase active sites, the question of whether there is cross-talk between monomers has remained unresolved. Here we describe two heterodimers in which a native subunit of human PGHS-2 has been coupled to a subunit having a defect within the COX active site at some distance from the dimer interface. Native͞G533A PGHS-2, a heterodimer with a COX-inactive subunit, had the same specific COX activity as the native homodimer. Native͞R120Q PGHS-2, a heterodimer in which both subunits can oxygenate arachidonic acid but in which the R120Q subunit cannot bind the COX inhibitor flurbiprofen, was inhibited by flurbiprofen to about the same extent as native PGHS-2. These results imply that native PGHS-2 exhibits half-ofsites reactivity. Isothermal titration calorimetry established that only one monomer of the native PGHS-2 homodimer binds flurbiprofen tightly. In short, binding of ligand to the COX site of one monomer alters its companion monomer so that it is unable to bind substrate or inhibitor. We conclude that PGHS monomers comprising a dimer, although identical in the resting enzyme, differ from one another during catalysis. The nonfunctioning subunit may provide structural support enabling its partner monomer to catalyze the COX reaction. This subunit complementarity may prove to be characteristic of other dimeric enzymes having tightly associated monomers.aspirin ͉ flurbiprofen ͉ heme ͉ ibuprofen ͉ prostaglandin P rostaglandin endoperoxide H synthases (PGHSs) 1 and 2 catalyze the committed step in prostaglandin biosynthesis (1-3). They are both targets of nonsteroidal antiinflammatory drugs, and PGHS-2 is the target of cyclooxygenase (COX) 2 inhibitors (4, 5). The enzymes have two catalytic activities: (i) a COX that converts arachidonic acid (AA) to a prostaglandin endoperoxide, prostaglandin G 2 (PGG 2 ), and (ii) a peroxidase (POX) that reduces PGG 2 to PGH 2 . Oxidation of the heme group at the POX active site by a peroxide is required to activate the COX by generating a tyrosyl radical at Tyr-385 at the COX active site (1-3). The Tyr-385 radical is involved in abstracting the hydrogen atom from the fatty acid substrate in the initial and rate-determining step in COX catalysis.PGHS-1 and PGHS-2 function only as homodimers although each subunit has both a POX and a COX active site (1-3, 6). It is not clear whether each monomer functions independently or whether cross-talk occurs between monomers comprising a dimer. Titrations of PGHS-1 with time-dependent COX inhibitors such as flurbiprofen (FBP) and indomethacin indicate that complete inhibition requires only one inhibitor molecule to be bound per dimer (7). Other studies with PGHS-1 using low concentrations of PGHS-2-spe...

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supporting

confidence: 78%

Section: Physical Properties Of the Native͞r120q Hupghs-2 Heterodimermentioning

confidence: 99%

mentioning

confidence: 99%

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Partnering between monomers of cyclooxygenase-2 homodimers

Yuan

Rieke

Rimon

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

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113

142

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“…The cyclooxygenase reaction occurs within a hydrophobic channel that extends from the membrane binding domain of the enzyme into the core of the globular domain. The fatty acid substrate is positioned within this site in an extended L-shaped conformation (15,24). Cyclooxygenase catalysis begins with abstraction of the 13-pro-S hydrogen from AA by a tyrosyl radical centered on Tyr-385 in the rate-determining step to generate an arachidonyl radical (25)(26)(27).…”

Section: Prostaglandin Endoperoxide H Synthase (Pghs)mentioning

confidence: 99%

“…Oligonucleotides used in the preparation of various mutants were reported previously (24,35,49). Plasmids used for transfections were purified by CsCl gradient ultracentrifugation, and mutations were reconfirmed by double-stranded sequencing of the pSVT7 constructs using Sequenase version 2.0 (U. S. Biochemical Corp.) and the protocol described by the manufacturer.…”

Section: Materials-fattymentioning

confidence: 99%

Structure of Eicosapentaenoic and Linoleic Acids in the Cyclooxygenase Site of Prostaglandin Endoperoxide H Synthase-1

Malkowski

Thuresson

Lakkides

et al. 2001

Journal of Biological Chemistry

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101

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Prostaglandin endoperoxide H synthases-1 and -2 (PGHSs) can oxygenate 18 -22 carbon polyunsaturated fatty acids, albeit with varying efficiencies. Here we report the crystal structures of eicosapentaenoic acid (EPA, 20:5 n-3) and linoleic acid (LA, 18:2 n-6) bound in the cyclooxygenase active site of Co 3؉ protoporphyrin IX-reconstituted ovine PGHS-1 (Co 3؉ -oPGHS-1) and compare the effects of active site substitutions on the rates of oxygenation of EPA, LA, and arachidonic acid (AA). Both EPA and LA bind in the active site with orientations similar to those seen previously with AA and dihomo-␥-linolenic acid (DHLA). For EPA, the presence of an additional double bond (C-17/C-18) causes this substrate to bind in a "strained" conformation in which C-13 is misaligned with respect to Tyr-385, the residue that abstracts hydrogen from substrate fatty acids. Presumably, this misalignment is responsible for the low rate of EPA oxygenation. Prostaglandin endoperoxide H synthase (PGHS)1 converts arachidonic acid (AA) to prostaglandin H 2 in the committed step of prostaglandin and thromboxane biosynthesis (1, 2). Two isoforms of PGHS exist in mammalian tissues. PGHS-1 is constitutively expressed and generates prostaglandins in response to hormone stimulation, whereas PGHS-2 is an inducible enzyme that is expressed in response to growth factors, tumor promotors, or cytokines (1-11). Both isoforms are quite similar structurally (12-15) and mechanistically (1, 2, 16), with only subtle kinetic differences in substrate (17, 18) and inhibitor (19, 20) specificities, and hydroperoxide activator requirements (21-23).Both PGHS-1 and -2 catalyze two different reactions: a cyclooxygenase reaction and a peroxidase reaction. Structural studies indicate that the active sites are spatially distinct from each other and separated by a heme group (12-15). The cyclooxygenase reaction occurs within a hydrophobic channel that extends from the membrane binding domain of the enzyme into the core of the globular domain. The fatty acid substrate is positioned within this site in an extended L-shaped conformation (15,24). Cyclooxygenase catalysis begins with abstraction of the 13-pro-S hydrogen from AA by a tyrosyl radical centered on Tyr-385 in the rate-determining step to generate an arachidonyl radical (25-27). Two molecules of O 2 are then sequentially added to the arachidonyl radical to form the bicyclic hydroperoxide prostaglandin G 2 (PGG 2 ). This intermediate diffuses to the peroxidase active site where the 15-hydroperoxyl group undergoes a two-electron reduction to the alcohol prostaglandin H 2 . Although the peroxidase activity can function independently of the cyclooxygenase activity (28), activation of the cyclooxygenase requires a functional peroxidase (1,2,23).AA is the preferred substrate for PGHS-1 and PGHS-2 (17). Both isoforms will, however, oxygenate a variety of n-3 and n-6 18 -22 carbon fatty acids albeit with reduced catalytic efficiencies (17, 29 -36). Most of these alternative fatty acid substrates have somewhat higher K m...

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X‐Ray Crystallography in Drug Discovery

Livingston¹,

Buchanan²,

D’Amico³

et al. 2003

Burger's Medicinal Chemistry and Drug Discovery

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The focus of this chapter is the methods, recent advances, and the applications of X‐ray protein crystallography in the discovery of new drug targets and candidate drugs. It begins with a basic description of the various steps involved in solving a structure, including recent advances in technique and technology, and the major challenges that are faced in executing the process. The underlying theory, various crystallization methods, data collection equipment and techniques, and computational reduction of the data to give the final structure are all covered in this section. The accessible databases that contain large collections of structures are also described. The application to drug discovery is then briefly addressed with a discussion of co‐crystallization methods and issues, which is followed by a comprehensive table enumerating the published structures of the known drug targets and close homologs. Applications to discovering and validating protein targets of therapeutic relevance are then covered in a section on the very new field of structural genomics. Genome annotation by structural homology, elucidating pathways and the potential for the rational design of multitarget drugs, and advances in the field of protein structure modeling that are enabled by crystallographic databases are described and exemplified with very recent results from the field.

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Mutational and X-ray Crystallographic Analysis of the Interaction of Dihomo-γ-linolenic Acid with Prostaglandin Endoperoxide H Synthases

Cited by 44 publications

References 55 publications

Partnering between monomers of cyclooxygenase-2 homodimers

Partnering between monomers of cyclooxygenase-2 homodimers

Structure of Eicosapentaenoic and Linoleic Acids in the Cyclooxygenase Site of Prostaglandin Endoperoxide H Synthase-1

X‐Ray Crystallography in Drug Discovery

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