Identification of Thromboxane A2 Synthase Active Site Residues by Molecular Modeling-guided Site-directed Mutagenesis

Abstract

Human thromboxane A 2 synthase (TXAS) exhibits spectral characteristics of cytochrome P450 but lacks monooxygenase activity. Its distinctive amino acid sequence makes TXAS the sole member of family 5 in the P450 superfamily. To better understand the structurefunction relationship of this unusual P450, we have recently constructed a three-dimensional model for TXAS using P450 BM-3 as the template (Ruan, K.-H., Milfeld, K., Kulmacz, R. J., and Wu, K. K. (1994) Protein Eng. 7, 1345-1551) and have identified a potential active site region. The catalytic roles of several putative active site residues were evaluated using selectively mutated recombinant TXAS expressed in COS-1 cells. Mutation of Ala-408 to Glu or Arg-413 to Gly led to a complete loss of enzyme activity despite expression of mutant protein levels equivalent to that of the wild-type TXAS. Mutation of Ala-408 to Gly or Leu retained the enzyme activity at levels of 30 or 40%, respectively. This suggests that Ala-408 provides a hydrophobic environment for substrate binding. Mutation of Arg-413 to Lys or Gln completely abolished the enzyme activity, indicating that this residue is essential to catalytic activity and supports its identification as an active site residue. Mutation of Arg-410 to Gly or Glu-433 to Ala resulted in >50% reduction in the enzyme activity without appreciably altering mutant protein expression, consistent with a more subtle effect of these residues on TXAS catalytic efficiency. Mutation of residues predicted to be involved in binding the heme prosthetic group, including the heme thiolate ligand Cys-480, Arg-478, Phe-127, and Asn-110, each markedly reduced the expressed protein level and abolished enzyme activity. This suggests that proper heme binding is important to synthesis or stability of recombinant TXAS. Mutation of Ile-346, which corresponds to P450 cam -Thr-252, an essential amino acid involved in dioxygen bond scission, to Thr increased the enzymatic activity by 40%, suggesting that oxygen bond cleavage is not a rate-limiting step in thromboxane A 2 biosynthesis. The present results from site-directed mutagenesis support the overall structure of the TXAS active site predicted by homology modeling and have allowed refinement of the position of bound substrate.Thromboxane A 2 (TXA 2 ) 1 is a potent mediator of platelet aggregation, vasoconstriction, and bronchoconstriction and plays an important role in major human diseases, including myocardial infarction, stroke, septic shock, and asthma (1, 2). TXA 2 biosynthesis is catalyzed in succession by phospholipase A 2 , which liberates arachidonic acid from membrane phospholipids, prostaglandin H synthase, which converts arachidonate to prostaglandin H 2 (PGH 2 ), and TXA 2 synthase (TXAS, EC 5.3.99.5), which converts PGH 2 to TXA 2 as well as to 12L-hydroxy-5,8,10-heptadecatrienoic acid (3). Because PGH 2 can also be converted to several other prostanoids, TXAS plays a pivotal role in determining the amount of TXA 2 synthesized. In mammalian tissues, TXAS is a membrane-bound hemop...

Metabolism of the Endocannabinoids, 2-Arachidonylglycerol and Anandamide, into Prostaglandin, Thromboxane, and Prostacyclin Glycerol Esters and Ethanolamides

“…8). This finding is consistent with site-directed mutagenesis studies and computer modeling that have shown that TXAS interactions with the carboxylate moiety of PGH 2 -like compounds are critical in thromboxane production (40). In addition, it is noteworthy that platelets, expressing only the COX-1 isoform, are the predominant source of thromboxanes in vivo (41,42).…”

Substrate access channel topology in membrane-bound prostacyclin synthase

“…On the basis of the crystallographic structure of the P450 BM-$ , we had previously constructed a detailed 3-D structural working model of TXAS using the QUANTA-CHARMm protein-modelling package (Molecular Simulations Inc., San Diego, CA, U.S.A.) on a Silicon Graphics (Mountain View, CA, U.S.A.) workstation [14]. The accuracy of the 3-D model has partially been confirmed by site-directed mutagenesis for the several important residues predicted from the 3-D working model, which are involved in the making up of the substrate-or haembinding sites of TXAS [16]. The observation that the crystallographic structure of P450 BM-$ serves as a suitable template for construction of the 3-D model of TXAS has supported the creation of a 3-D working model for human PGIS (Figure 1) using a protein-modelling process similar to that employed for TXAS.…”

“…This orientation places the substrate access opening facing the membrane. Our working models developed for thromboxane A # synthase (TXAS) and PGIS by homology modelling strategies using P450 BM-$ , a bacterial P450, as a template, which has a similar substrate-binding cavity [14][15][16], have indicated that the substrate openings are near the Nterminal membrane-anchor domains. These imply that the substrate access channel openings of PGIS and TXAS face the ER membrane.…”

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

“…TXAS is more efficient in HHT/MDA synthesis not only because its active site is hydrophobic but it also has a greater affinity for PGH 2 . Furthermore, several TXAS active site amino acid residues, as we have previously shown (29), may be involved in the reaction. It is intriguing to note that prostacyclin synthase, although it has a high affinity for PGH 2 (K d is ϳ10 M), does not catalyze HHT/MDA formation (11).…”