IDENTIFICATION OF THROMBOXANE A(2), SYNTHASE ACTIVE-SITE RESIDUES BY MOLECULAR MODELING-GUIDED SITE-DIRECTED MUTAGENESIS

Human thromboxane A(2) synthase (TXAS) exhibits spectral characteristi cs of cytochrome P450 but lacks monooxygenase activity, Its distinctiv e amino acid sequence makes TXAS the sole member of family 5 in the P4 50 superfamily. To better understand the structure-function relationsh ip of this unusual P450, we have recently constructed a three-dimensio nal model for TXAS using P450(BM-3) as the template (Ruan, K.-H., Milf eld, K., Kulmacz, R. J., and Wu, K. K. (1994) Protein Eng. 7, 1345-155 1) and have identified a potential active site region. The catalytic r oles of several putative active site residues were evaluated using sel ectively mutated recombinant TXAS expressed in COS-1 cells. Mutation o f Ala-408 to Glu or Arg-413 to Gly led to a complete loss of enzyme ac tivity 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 t hat Ala-408 provides a hydrophobic environment for substrate binding. Mutation of Arg-413 to Lys or Gln completely abolished the enzyme. act ivity, indicating that this residue is essential to catalytic activity and supports its identification as an active site residue. Mutation o f 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 catal ytic efficiency, Mutation of residues predicted to be involved in bind ing the heme prosthetic group, including the heme thiolate ligand Cys- 480, Arg-478, Phe-127, and Asn-110, each markedly reduced the expresse d protein level and abolished enzyme activity, This suggests that prop er 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 increa sed the enzymatic activity by 40%, suggesting that oxygen bond cleavag e is not a rate-limiting step in thromboxane A(2) biosynthesis. The pr esent results from site-directed mutagenesis support the overall struc ture of the TXAS active site predicted by homology modeling and have a llowed refinement of the position of bound substrate.