Identification of Thromboxane Synthase Amino Acid Residues Involved in Heme-Propionate Binding

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

doi:10.1006/abbi.2000.2041

Cited by 12 publications

(9 citation statements)

References 25 publications

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“…This proposed side-chain-disruption model is further supported by our experiments on imidazole, miconazole, and U46619 bound TXAS and by site-directed mutation experiments (42). As shown in Figure 4, line d, the propionate bending vibration pattern does not change upon binding of imidazole.…”

Section: Spin and Coordination States Of Resting And Ligand-supporting

confidence: 74%

“…Although large variations in the dissociation constants, K d , measured for these substrate analogues have been reported, it is commonly believed that the affinity of TXAS for U44069 is higher than for U46619, indicating that the heme iron prefers to bind the O(9) rather than O(11) of the substrate (6,8,41,42). On the basis of this observation, an isomerization mechanism of PGH 2 has been proposed, in which binding of O( 9) to the heme iron initiates a series of reactions, leading to the formation of TXA 2 , while coordination of O (11) to the heme iron was not considered (8,9).…”

Section: Spin and Coordination States Of Resting And Ligand-mentioning

confidence: 99%

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Resonance Raman Investigation of the Interaction of Thromboxane Synthase with Substrate Analogues

2003

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Thromboxane synthase is a hemethiolate enzyme that catalyzes the isomerization of prostaglandin H2 to thromboxane A2. We report the first resonance Raman (RR) spectra of recombinant human thromboxane synthase (TXAS) in both the presence and the absence of substrate analogues U44069 and U46619. The resting enzyme and its U44069 complex are found to have a 6-coordinate, low spin (6c/ls) heme, in agreement with earlier experiments. The U46619-bound enzyme is detected as a 6c/ls heme too, which is in contradiction with a previous conclusion based on absorption difference spectroscopy. Two new vibrations at 368 and 424 cm(-1) are observed upon binding of the substrate analogues in the heme pocket and are assigned to the second propionate and vinyl bending modes, respectively. We interpret the changes in these vibrational modes as the disruption of the protein environment and the hydrogen-bonding network of one of the propionate groups when the substrate analogues enter the heme pocket. We use carbocyclic thromboxane A2 (CTA2) to convert the TXAS heme cofactor to its 5-coordinate, high spin (5c/hs) form, as is confirmed by optical and RR spectroscopy. In this 5c/hs state of the enzyme, the Fe-S stretching frequency is determined at 350 cm(-1) with excitation at 356.4 nm. This assignment is supported by comparison to the spectrum of resting enzyme excited at 356.4 nm and by exciting at different wavelengths. Implications of our findings for substrate binding and the catalytic mechanism of TXAS will be discussed.

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Section: Spin and Coordination States Of Resting And Ligand-supporting

confidence: 74%

Section: Spin and Coordination States Of Resting And Ligand-mentioning

confidence: 99%

Resonance Raman Investigation of the Interaction of Thromboxane Synthase with Substrate Analogues

2003

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“…The initial 3D structural model for TBXAS1 was constructed using a homology-modeling approach using other P450s as templates (Ruan et al 1994). The TBXAS structural model was later modified based on results from site-directed mutagenesis (Hsu et al 2000).…”

Section: Phenotypic Impact Of Polymorphismsmentioning

confidence: 99%

“…PolyPhen creates a composite of these calculations and returns a prediction of probably damaging, possibly damaging, benign, or unknown. The potential functional impact of individual amino-acid substitutions was also examined by a molecular modeling approach (Hsu et al 2000). The initial 3D structural model for TBXAS1 was constructed using a homology-modeling approach using other P450s as templates (Ruan et al 1994).…”

Section: Phenotypic Impact Of Polymorphismsmentioning

confidence: 99%

Thromboxane synthase (TBXAS1) polymorphisms in African-American and Caucasian populations: evidence for selective pressure

Ulrich

Carlson²,

Sibert

et al. 2005

Hum. Mutat.

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Thromboxane synthase (TBXAS1), a cytochrome P450 enzyme, converts prostaglandin H2 into thromboxane A2, a potent vasoconstrictor and inducer of platelet aggregation. Thromboxane A2 has been implicated in modulating cell cytotoxicity and in tumor growth and metastasis. Twelve coding-region variants were identified in the human TBXAS1 gene in 48 African-American and 46 Caucasian individuals, of which eight were amino-acid substitutions. The latter were confirmed in an independent Caucasian population (n=94 unrelated individuals). We performed an evolutionary analysis of patterns of nucleotide diversity and identified patterns of amino acid replacement in human-mouse comparisons consistent with purifying selection on an inter-species time scale using the McDonald-Kreitman test. We also observed patterns of nucleotide diversity within humans consistent with purifying selection acting on existing polymorphism using Tajima's D within coding regions. These evolutionary tests suggest that some of the rare coding variations observed in the human population are deleterious. We used two sequence-homology-based software programs and molecular modeling to predict the potential impact of these polymorphisms on TBXAS1 function. The c.772C>T (p.Lys258Glu), c.1249C>G (p.Gln417Glu), and c.1348G>A (p.Glu450Lys) substitutions are predicted as most likely to alter protein function; another, c.1352C>A (p.Thr451Asn), may also affect function. Given the evolutionary evidence, these variants may be functional and therefore of relevance for disease endpoints related to inflammation and angiogenesis, as well as for the pharmacogenetics of non-steroidal anti-inflammatory drugs.

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“…Two of them, R413 and G482, were shown to localize adjacent to the catalytic site heme. The importance of residue R413 for catalysis is known from a previous study, which revealed that a R413E TXAS mutant retains about 1% of the catalytic activity (8). The G482W substitution introduces an aromatic residue that creates steric hindrance near the catalytic site, and the L83P and L488P mutations are thought to introduce coils in the protein secondary structure.…”

mentioning

confidence: 99%

Ghosal syndrome – genetics unveiled

Stein

2008

Clinical Genetics

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Identification of Thromboxane Synthase Amino Acid Residues Involved in Heme-Propionate Binding

Cited by 12 publications

References 25 publications

Resonance Raman Investigation of the Interaction of Thromboxane Synthase with Substrate Analogues

Resonance Raman Investigation of the Interaction of Thromboxane Synthase with Substrate Analogues

Thromboxane synthase (TBXAS1) polymorphisms in African-American and Caucasian populations: evidence for selective pressure

Ghosal syndrome – genetics unveiled

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