Characterization of substrate binding to cytochrome P450 1A1 using molecular modeling and kinetic analyses: case of residue 382.
Key residue Val-382 in P450 1A1 has been predicted to interact with the alkoxy chain of resorufin derivatives. Therefore, we undertook a detailed analysis of substrate mobility in the active site of the P450 1A1 homology model and assessed the effect of mutations at position 382. Dynamic trajectories of 7-methoxy-, 7-ethoxy-, and 7-pentoxyresorufin indicated that 7-ethoxyresorufin would be oxidized most efficiently by the wild-type enzyme. The Val-382-->Ala mutation would increase the O-dealkylation of 7-pentoxyresorufin but decrease the oxidation of other substrates. In the case of the V382L mutant, the large bulk of Leu would block alkoxyresorufins from productive binding orientations leading to lowered activities. Binding free energy calculations for three substrates with 1A1 WT and two mutants indicated that binding constants would be similar for all enzyme-substrate combinations. Modeling predictions were tested experimentally. The plasmid containing the cDNA for human P450 1A1 modified for bacterial expression was altered to include a C-terminal PCR-generated six histidine domain to facilitate enzyme purification. The V382A and V382L mutants were constructed by site-directed mutagenesis and Escherichia coli-expressed enzymes purified using Ni-NTA affinity chromatography. The activity of the WT 1A1 was highest toward 7-ethoxyresorufin and lowest toward 7-pentoxyresorufin. Both mutants displayed a decrease in V(max) with 7-methoxy- and 7-ethoxyresorufin, whereas for the V382A mutant, V(max) with 7-pentoxyresorufin was increased. No significant changes in K(m) were observed relative to the wild-type enzyme. The experimental results are thus in good agreement with modeling predictions.
Liu
Jianguo
J
Department of Basic Pharmaceutical Sciences, School of Pharmacy, West Virginia University, Morgantown, WV 26506-9530, USA.
Ericksen
Spencer S
SS
Besspiata
Dan
D
Fisher
Charles W
CW
Szklarz
Grazyna D
GD
D001665:Binding Sites; D003001:Cloning, Molecular; D019363:Cytochrome P-450 CYP1A1 / Q000235:genetics / Q000378:metabolism*; D017931:DNA Primers; D015870:Gene Expression; D006801:Humans; D006859:Hydrogen; D066298:In Vitro Techniques; D007700:Kinetics; D007930:Leucine / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D008968:Molecular Conformation; D016297:Mutagenesis, Site-Directed; D010078:Oxazines / Q000493:pharmacokinetics; D010084:Oxidation-Reduction; D013379:Substrate Specificity; D013997:Time Factors; D014633:Valine / Q000235:genetics / Q000378:metabolism
D017931:DNA Primers; D010078:Oxazines; D006859:Hydrogen; D019363:Cytochrome P-450 CYP1A1; D007930:Leucine; D014633:Valine