Identification of thromboxane synthase amino acid residues involved in heme-propionate binding.
Thromboxane A2 synthase (TXAS) is a member of the cytochrome P450 superfamily and catalyzes an isomerization reaction that converts prostaglandin H2 to thromboxane A2. As a step toward understanding the structure/function relationships of TXAS, we mutated amino acid residues predicted to bind the propionate groups of A- and D-pyrrole rings of the heme. These mutations at each of these residues (Asn-110, Trp-133, Arg-137, Arg-413, and Arg-478) resulted in altered heme binding, as evidenced by perturbation of the absorption spectra and EPR. The mutations, although causing no significant changes in the secondary structure of the proteins, induced tertiary structural changes that led to increased susceptibility to trypsin digestion and alteration of the intrinsic protein fluorescence. Moreover, these mutant proteins lost their binding affinity to the substrate analog, had a lower heme content and retained less than 5% of the wild-type catalytic activity. However, mutations at the neighboring amino acid of the aforementioned residues yielded mutant proteins retaining the biochemical and biophysical properties of the wild type TXAS. Aligning the TXAS sequence with the structurally known P450s, we proposed that in TXAS the A-ring propionate of the heme is hydrogen bonded to Asn-110, Arg-413, and Arg-478, whereas D-ring propionate is hydrogen bonded to Trp-133 and Arg-137. Furthermore, both A- and D-ring propionates bulge away from the heme plane and both lie on the proximal face of heme plane, a structure similar to P450terp.
Hsu
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Department of Internal Medicine, University of Texas Medical School, Houston 77030, USA.
Tsai
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Wang
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D000595:Amino Acid Sequence; D000596:Amino Acids / Q000378:metabolism; D001665:Binding Sites; D006418:Heme / Q000378:metabolism*; D008969:Molecular Sequence Data; D009154:Mutation; D011485:Protein Binding; D016415:Sequence Alignment; D013379:Substrate Specificity; D013930:Thromboxane-A Synthase / Q000235:genetics / Q000378:metabolism*
D000596:Amino Acids; D006418:Heme; D013930:Thromboxane-A Synthase