Role of conserved Asp293 of cytochrome P450 2C9 in substrate recognition and catalytic activity.
Human cytochrome P450 2C9 (CYP2C9) is important in the metabolism of non-steroidal anti-inflammatory compounds such as diclofenac, the antidiabetic agent tolbutamide and other clinically important drugs, many of which are weakly acidic. Multiple sequence alignment of CYPs identified CYP2C9 Asp(293) as corresponding to Asp(301) of CYP2D6, which has been suggested to play a role in the binding of basic substrates to the latter enzyme. Replacement of Asp(293) with Ala (D293A) decreased activity by more than 90%, and led to an approx. 3- to 10-fold increase in K (m) values for the three test substrates tolbutamide, dextromethorphan and diclofenac. Conservative replacement of the carboxyl side chain in a Glu (D293E) mutant produced no significant changes in K (m) values and slight increases in k (cat) values. Changes in regiospecificity were observed for both the Ala and Glu substitutions; low levels of both dextromethorphan O- and N-demethylation were observed in the D293A mutant, whereas increased preference for O-demethylation was observed for the D293E mutant. Expression of constructs coding for Asn (D293N) and Gln (D293Q) substitutions failed to form a P450 correctly. Our analysis suggests a structural role for the carboxyl side chain of Asp(293) in CYP2C9 substrate binding and catalysis. The conservation of an Asp residue in other CYP families in a position equivalent to Asp(293) indicates a common mechanism for maintaining the active-site architecture.
Flanagan
Jack U
JU
Biomedical Research Centre, Ninewells Hospital & Medical School, University of Dundee, Dundee DD1 9SY, UK.
McLaughlin
Lesley A
LA
Paine
Mark J I
MJ
Sutcliffe
Michael J
MJ
Roberts
Gordon C K
GC
Wolf
C Roland
CR
D000595:Amino Acid Sequence; D000818:Animals; D000894:Anti-Inflammatory Agents, Non-Steroidal / Q000378:metabolism; D000996:Antitussive Agents / Q000378:metabolism; D001189:Aryl Hydrocarbon Hydroxylases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D001224:Aspartic Acid / Q000235:genetics / Q000378:metabolism*; D065729:Cytochrome P-450 CYP2C9; D003915:Dextromethorphan / Q000378:metabolism; D004008:Diclofenac / Q000378:metabolism; D006801:Humans; D007004:Hypoglycemic Agents / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D015394:Molecular Structure; D016297:Mutagenesis, Site-Directed; D009251:NADPH-Ferrihemoprotein Reductase / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D017433:Protein Structure, Secondary; D011817:Rabbits; D016415:Sequence Alignment; D013379:Substrate Specificity; D014044:Tolbutamide / Q000378:metabolism
D000894:Anti-Inflammatory Agents, Non-Steroidal; D000996:Antitussive Agents; D007004:Hypoglycemic Agents; D004008:Diclofenac; D001224:Aspartic Acid; D003915:Dextromethorphan; D014044:Tolbutamide; C450260:CYP2C9 protein, human; D065729:Cytochrome P-450 CYP2C9; D001189:Aryl Hydrocarbon Hydroxylases; D009251:NADPH-Ferrihemoprotein Reductase