Identification of thromboxane A(2), synthase active site residues by molecular modeling-guided site-directed mutagenesis

被引:36
作者
Wang, LH [1 ]
MatijevicAleksic, N [1 ]
Hsu, PW [1 ]
Ruan, KH [1 ]
Wu, KK [1 ]
Kulmacz, RJ [1 ]
机构
[1] UNIV TEXAS,SCH MED,DEPT INTERNAL MED,VASC BIOL RES CTR,HOUSTON,TX 77030
关键词
D O I
10.1074/jbc.271.33.19970
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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 structure-function 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.
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页码:19970 / 19975
页数:6
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