The effect of reciprocal active site mutations in human cytochromes P450 1A1 and 1A2 on alkoxyresorufin metabolism.
Five reciprocal active site mutants of P450 1A1 and 1A2 and an additional mutant, Val/Leu-382 --> Ala, were constructed, expressed in Escherichia coli, and purified by Ni-NTA affinity chromatography. In nearly every case, the residue replacement led to loss of 7-methoxy- and 7-ethoxyresorufin O-dealkylase activity compared to the wild-type enzymes, except for the P450 1A1 S122T mutation which increased both activities. Mutations at position 382 in both P450 1A1 and 1A2 shifted substrate specificity from one enzyme to another, confirming the importance of this residue. Changes in activity of P450 1A enzymes upon amino acid replacement were, in general, consistent with molecular dynamics analyses of substrate motion in the active site of homology models.
Liu
Jianguo
J
Department of Basic Pharmaceutical Sciences, School of Pharmacy, West Virginia University, Morgantown, WV 26506-9530, USA.
Ericksen
Spencer S
SS
Sivaneri
Meena
M
Besspiata
Dan
D
Fisher
Charles W
CW
Szklarz
Grazyna D
GD
D019943:Amino Acid Substitution; D001665:Binding Sites; D019363:Cytochrome P-450 CYP1A1 / Q000235:genetics* / Q000378:metabolism*; D019388:Cytochrome P-450 CYP1A2 / Q000235:genetics* / Q000378:metabolism*; D003640:Dealkylation; D004926:Escherichia coli / Q000378:metabolism; D006801:Humans; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D010078:Oxazines / Q000737:chemistry / Q000378:metabolism*; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity
D010078:Oxazines; D011994:Recombinant Proteins; C014180:resorufin; D019363:Cytochrome P-450 CYP1A1; D019388:Cytochrome P-450 CYP1A2