Substrate Binding Is the Rate-limiting Step in Thromboxane Synthase Catalysis

Wang, Lee Ho; Tsai, Ah‐Lim; Hsu, Pei Yung

doi:10.1074/jbc.m009177200

Cited by 40 publications

(25 citation statements)

References 31 publications

(24 reference statements)

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“…The TXAS with hydrophilic N-terminus converts PGH 2 to TXA 2 , malondialdehyde and 12-hydroxy-5, 8, 10-heptadecatrienoic acid at a molar ratio of 1:1:1, with a turnover number, k cat , of 5,000 ± 300 min −1 and K M (PGH 2 ) of 29 ± 5 μM, comparable to the values for TXAS with hydrophobic N-terminus (k cat of 4,000 ± 300 min −1 ; K M , 21 ± 2 μM [20]). These results indicate that alteration of the N-terminal segment had little effect on the TXAS catalytic activity.…”

Section: Expression and Purification Of Txasmentioning

confidence: 64%

Reaction mechanisms of 15-hydroperoxyeicosatetraenoic acid catalyzed by human prostacyclin and thromboxane synthases

Yeh¹,

Tsai²,

Wang³

2007

Archives of Biochemistry and Biophysics

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Prostacyclin synthase (PGIS) and thromboxane synthase (TXAS) are atypical cytochrome P450s. They do not require NADPH or dioxygen for isomerization of prostaglandin H 2 (PGH 2 ) to produce prostacyclin (PGI 2 ) and thromboxane A 2 (TXA 2 ). PGI 2 and TXA 2 have opposing actions on platelet aggregation and blood vessel tone. In this report, we use a lipid hydroperoxide, 15-hydroperoxyeicosatetraenoic acid (15-HPETE), to explore the active site characteristics of PGIS and TXAS. The two enzymes transformed 15-HPETE not only into 8,, like many microsomal P450s, but also to 15-ketoeicosatetraenoic acid (15-KETE) and 15-hydroxyeicosatetraenoic acid (15-HETE). 13-OH-14,15-EET and 15-KETE result from homolytic cleavage of the O-O bond, whereas 15-HETE results from heterolytic cleavage, a common peroxidase pathway. About 80% of 15-HPETE was homolytically cleaved by PGIS and 60% was homolytically cleaved by TXAS. The V max of homolytic cleavage is 3.5-fold faster than heterolytic cleavage for PGIS-catalyzed reactions (1100 min −1 vs. 320 min −1 ) and 1.4-fold faster for TXAS (170 min −1 vs. 120 min −1 ). Similar K M values for homolytic and heterolytic cleavages were found for PGIS (∼60 μM 15-HPETE) and TXAS (∼80 μM 15-HPETE), making PGIS a more efficient catalyst for the 15-HPETE reaction.Keywords prostacyclin synthase; thromboxane synthase; cytochrome P450; peroxide bond cleavage; homolytic; heterolytic; hydroperoxides; epoxyalcohol Prostacyclin synthase (PGIS) and thromboxane synthase (TXAS) are members of the cytochrome P450 superfamily, which uses heme as the prosthetic group and has a cysteine thiolate proximal ligand [1,2]. PGIS and TXAS convert prostaglandin H 2 (PGH 2 ) to prostacyclin (PGI 2 ) and thromboxane A 2 (TXA 2 ), respectively. PGI 2 inhibits platelet activation and aggregation and induces vasodilation, whereas TXA 2 stimulates platelet secretion, aggregation and vasoconstriction [3]. Both PGI 2 and TXA 2 are rapidly hydrolyzed To whom correspondence should be addressed: Lee-Ho Wang Division of Hematology Department of Internal Medicine 6431 Fannin Houston, TX 77030 Tel: 713-500-6794 Fax: 713-500-6810 e-mail: lee-ho.wang@uth.tmc.edu 1 The abbreviations used are: PGIS, prostacyclin synthase; PGI 2 , prostacyclin; PGHS, prostaglandin H synthase; TXAS, thromboxane synthase; TXA 2 , thromboxane A 2 ; 8,11-eicosatrienoic acid; ESI/MS, electron-spray ionization/ mass spectrometry; EPR, electron paramagnetic resonance; SVD, singular value decomposition.Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. Hecker and Ullrich proposed a caged radical mechanism for TXAS ...

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Section: Expression and Purification Of Txasmentioning

confidence: 64%

Reaction mechanisms of 15-hydroperoxyeicosatetraenoic acid catalyzed by human prostacyclin and thromboxane synthases

Yeh¹,

Tsai²,

Wang³

2007

Archives of Biochemistry and Biophysics

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“…The Soret band at 406 nm for coral AOS [47] was, like CYP74C3, attributed to a major presence of high-spin haem iron. The positions of the α and β bands at 545 and 508 nm respectively in CYP74C3 are very different from those observed for most classical and non-classical cytochrome P450 enzymes (for example, at 569 and 535 nm in CYP2B4 [49] and at 567 and 535 nm in thromboxane synthase [5]). They are, however, more similar to those at 540 and 512 nm in flax AOS [48], and at 534 and 500 nm in coral AOS [47], both of which are CYP74 enzymes that were soluble in the absence of detergent.…”

Section: Discussionmentioning

confidence: 83%

“…14-fold increase in K d for cyanide (pK a 9.1 in water [51]) at pH 8.0 compared with that at pH 9.0 strongly suggests that CN − and not HCN is the form that binds. The K d (362 mM) for imidazole binding to CYP74C3 is notably four orders of magnitude higher than thromboxane synthase (33 µM) [5], which has also been shown to exhibit biphasic kinetics with this ligand.…”

Section: Discussionmentioning

confidence: 99%

“…CYP74 enzymes are very different from other cytochrome P450 enzymes, for example CYP73 plant enzymes like cinnamate hydroxylases [1], or classical cytochrome P450 enzymes of microbial [2] or mammalian [3] origin, in that they have an atypical reaction mechanism that requires neither oxygen nor an NADPH-reductase [4], and as a consequence have extraordinarily high catalytic-centre activity. In this sense, they have more in common with non-classical mammalian cytochrome P450 enzymes like thromboxane synthase [5]. HPL (hydroperoxide lyase), or hemiacetal synthase [6], is a member of this CYP74 subfamily and has an important role in oxylipin metabolism, plant defence and the food industry [7,8].…”

Section: Introductionmentioning

confidence: 99%

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Characterization of Medicago truncatula (barrel medic) hydroperoxide lyase (CYP74C3), a water-soluble detergent-free cytochrome P450 monomer whose biological activity is defined by monomer–micelle association

Hughes

Belfield

Muthusamay

et al. 2006

Biochemical Journal

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We describe the detailed biochemical characterization of CYP74C3 (cytochrome P450 subfamily 74C3), a recombinant plant cytochrome P450 enzyme with HPL (hydroperoxide lyase) activity from Medicago truncatula (barrel medic). Steady-state kinetic parameters, substrate and product specificities, RZ (Reinheitszahl or purity index), molar absorption coefficient, haem content, and new ligands for an HPL are reported. We show on the basis of gel filtration, sedimentation velocity (sedimentation coefficient distribution) and sedimentation equilibrium (molecular mass) analyses that CYP74C3 has low enzyme activity as a detergent-free, water-soluble, monomer. The enzyme activity can be completely restored by re-activation with detergent micelles, but not detergent monomers. Corresponding changes in the spin state equilibrium, and probably co-ordination of the haem iron, are novel for cytochrome P450 enzymes and suggest that detergent micelles have a subtle effect on protein conformation, rather than substrate presentation, which is sufficient to improve substrate binding and catalytic-centre activity by an order of magnitude. The kcat/K(m) of up to 1.6x10(8) M(-1) x s(-1) is among the highest recorded, which is remarkable for an enzyme whose reaction mechanism involves the scission of a C-C bond. We carried out both kinetic and biophysical studies to demonstrate that this effect is a result of the formation of a complex between a protein monomer and a single detergent micelle. Association with a detergent micelle rather than oligomeric state represents a new mechanism of activation for membrane-associated cytochrome P450 enzymes. Highly concentrated and monodispersed samples of detergent-free CYP74C3 protein may be well suited for the purposes of crystallization and structural resolution of the first plant cytochrome P450 enzyme.

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“…Nickel nitrilotriacetate agarose and hydroxyapatite were obtained from Qiagen and Bio-Rad, respectively. PGH 2 was synthesized from arachidonic acid using detergent-solubilized ovine prostaglandin H synthase-1 and was purified by normal phase chromatography [21]. …”

Section: Methodsmentioning

confidence: 99%

Functional analysis of human thromboxane synthase polymorphic variants

Chen

Poole

Ulrich

et al. 2012

Pharmacogenetics and Genomics

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Background Thromboxane A synthase (TXAS) metabolizes the cyclooxygenase product prostaglandin (PG) H2 into thromboxane A2 (TXA2), a potent inducer of blood vessel constriction and platelet aggregation. Non-synonymous polymorphisms in the TBXAS gene have the potential to alter TXAS activity and affect TXA2 generation. Objectives Assess the functional effects of genetic variants in the TXAS protein, including K258E, L357V, Q417E, E450K and T451N. Methods Wild-type TXAS and the variant proteins were expressed in a bacterial system and purified by affinity and hydroxyapatite chromatography. The two characteristic catalytic activities of TXAS were assayed in each of the purified recombinant proteins: isomerization of PGH2 to TXA2 and fragmentation of PGH2 to 12-hydroxyheptadecatrienoic acid and malondialdehyde. Results All of the variants exhibited both isomerization and fragmentation activities. The KM values of the variants ranged from 27–52 µM PGH2 (wild-type value: 32 µM PGH2); Vmax values of the variants ranged from 18–40 units/mg (wild-type value: 41 units/mg). The kinetic differences were largest for the L357V variant, whose Vmax / KM ratio was just 27% of the wild-type value. Conclusion The increased KM and decreased Vmax values observed with L357V suggest this variant may generate less TXA2 at the low levels of PGH2 expected in vivo, raising the possibility of attenuated signaling via the thromboxane pathway.

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Substrate Binding Is the Rate-limiting Step in Thromboxane Synthase Catalysis

Cited by 40 publications

References 31 publications

Reaction mechanisms of 15-hydroperoxyeicosatetraenoic acid catalyzed by human prostacyclin and thromboxane synthases

Reaction mechanisms of 15-hydroperoxyeicosatetraenoic acid catalyzed by human prostacyclin and thromboxane synthases

Characterization of Medicago truncatula (barrel medic) hydroperoxide lyase (CYP74C3), a water-soluble detergent-free cytochrome P450 monomer whose biological activity is defined by monomer–micelle association

Functional analysis of human thromboxane synthase polymorphic variants

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