Thromboxane synthase: structure and function of protein and gene
Introduction
Thromboxane A synthase (TXAS) catalyzes roughly equal amounts of two distinct reactions with PGH2 as substrate [1] (, ).The first reaction is an isomerization of PGH2 to form thromboxane A2, the second a fragmentation of PGH2 to form 12-HHT and malondialdehyde. TXA2 has been implicated as an autocrine or paracrine lipid mediator in a wide variety of pathophysiological processes [2]. TXA2 generally acts via the TXA2 receptor, a protein which contains seven transmembrane domains and which is coupled to G-protein signaling pathways [3], [4]. TXAS activity was first described in platelets [5] and the enzyme was later purified to homogeneity as a 60 kDa hemoprotein with spectroscopic characteristics of a cytochrome P450 [6]. The assignment of TXAS to the P450 superfamily was confirmed when the cDNA was cloned and the functional importance of the cysteine ligand to the heme was demonstrated [7], [8], [9]. The focus of this review is on more recent studies of TXAS mechanism, kinetics, structure–function relationships, gene structure, and control of gene expression. The reader is encouraged to consult several excellent reviews in the past several years for more comprehensive coverage of other aspects of TXAS [10], [11], [12], [13], [14].
Section snippets
Heme environment
Initial work with TXAS purified from platelets established that the heme in the resting enzyme is in the ferric low spin state and that ligand-dependent shifts in the heme absorbance spectrum resemble those observed in other P450s [6]. The recent development of a bacterial system for large-scale expression of TXAS has permitted more detailed and extensive spectroscopic characterization of the heme environment [15]. The EPR spectrum of recombinant TXAS revealed signals for low spin ferric heme
Reaction mechanism and kinetics
Analysis of spectral shifts observed upon binding of PGH2 analogs that have a carbon substituted for one of the oxygens in the endoperoxide function (U44069 and U46619) led to the conclusion that the TXAS heme iron interacts with the oxygen attached to C-9 of PGH2 [16]. This interaction of ferric TXAS heme with the C-9 endoperoxide oxygen is the first step in the reaction mechanism proposed by Hecker and Ullrich ([16], Fig. 1). Stopped-flow kinetic studies with U44069 and recombinant TXAS
Structure–function studies
A structural model for TXAS has been constructed based on crystallographic data for P450BM3 [21]. This structural model has guided mutagenic studies targeted at residues predicted to be involved with heme or substrate binding [22], [23]. The relative positions of the mutated residues in the structural model are illustrated in Fig. 2. In agreement with an earlier report [9], the proximal heme ligand C480 was found to be critical for activity, with all substitutions at that position resulting in
Human TXAS variants
Eight missense mutations have been found in the TXAS gene in a sample of 200 normal volunteers [24]. This natural mutagenic study revealed R61H, D161E, N246S, L357V, Q417E, E450K, T451N, and R466Q substitutions. The activities of the mutant enzymes were not examined directly, but no clinical abnormalities were evident. Each of these TXAS variants involve residues which are at, or close to, the protein surface and well away from the active site in the TXAS structural model (Fig. 3). Of
Integration of TXAS into eicosanoid pathway
TXA2 synthesis in vivo obviously involves coordinated action of a series of enzymes, including phospholipases which release arachidonate from esterified precursors, PGH synthase-1 or -2 which convert arachidonate into PGH2, and TXAS itself. PGH2 can be converted into a number of other prostanoid signaling mediators besides TXA2, and the cellular controls which determine the actual profile of prostanoids produced in a given cell under a particular set of circumstances are beginning to be
Organization of the human TXAS gene
The human TXAS is a single copy gene, located at chromosome 7q34-35; isolation of the complete TXAS genomic DNA was hampered by its large size [28], [29]. A 193-kb contiguous DNA containing the entire human TXAS gene was mapped from a combination of yeast artificial chromosome and bacteriophage P1 libraries [30]. As shown in Fig. 4, the TXAS gene contains 13 exons with five large introns; two of 45-kb and one each of 35-, 25- and 17-kb, making it the largest cytochrome P450 gene characterized
TXAS gene expression
The TXAS gene is transcribed as a 2.1-kb mRNA (0.14-kb of 5′-untranslated region, 1.6-kb of coding region and 0.4-kb of 3′-untranslated region) in hematopoietic cells, such as platelets, macrophages, monocytes and leukocytes, as well as in various tissues, particularly in lung, kidney, liver, spleen, prostate, placenta and thymus [28], [33]. Single nucleotide polymorphisms were found in the promoter, protein coding region and 3′-untranslated region. In the promoter, a G to T mutation at −386 in
Transcriptional regulation of the TXAS gene
The transcriptional start site of human TXAS gene was identified in the same exon as the translational start site, and mapped 137–140 nucleotides upstream from the translational start site [28], [30]. The core promoter contains two putative elements, a TATA box and an initiator, located at −29/−26 and −3/+3, respectively (Fig. 5). The sequence of the pyrimidine-rich TXAS initiator, CACA+1TT, resembles a weak initiator consensus sequence PyPyA+1NT/APyPy (A is the transcriptional start site and
Acknowledgements
Work in the authors’ laboratories has been supported by United States National Institutes of Health grants GM52170, HL60625 and NS23327. We thank Drs. Ah-Lim Tsai and Masahiro Yaekashiwa for critical reading of the manuscript and Drs. Ke-He Ruan and Jiaxin Wu for help with the TXAS structural model.
References (60)
- et al.
Conversion of prostaglandin endoperoxides to C17-hydroxy acids catalyzed by human platelet thromboxane synthase
FEBS Lett.
(1977) - et al.
Isolation and characterization of thromboxane synthase from human platelets as a cytochrome P-450 enzyme
J. Biol. Chem.
(1985) - et al.
Molecular cloning of human platelet thromboxane A synthase
Biochem. Biophys. Res. Commun.
(1991) - et al.
Primary structure of human thromboxane synthase determined from the cDNA sequence
J. Biol. Chem.
(1992) - et al.
Prostacyclin and thromboxane synthases
J. Lipid Mediat. Cell Signal.
(1995) - et al.
New physiological and pathophysiological aspects on the thromboxane A2-prostacyclin regulatory system
Biochim. Biophys. Acta
(2001) - et al.
Expression, purification, and spectroscopic characterization of human thromboxane synthase
J. Biol. Chem.
(1999) - et al.
On the mechanism of prostacyclin and thromboxane A2 biosynthesis
J. Biol. Chem.
(1989) - et al.
Substrate binding is the rate-limiting step in thromboxane synthase catalysis
J. Biol. Chem.
(2001) - et al.
Identification of the substrate interaction site in the N-terminal membrane anchor segment of thromboxane A2 synthase by determination of its substrate analog conformational changes using high resolution NMR technique
J. Biol. Chem.
(2000)