P
revious modeling effo
rts have suggested that couma
rin ligand binding to CYP2C9 is dictatedby elect
rostatic and
rs/pi.gif" BORDER=0 >-stacking inte
ractions with complementa
ry amino acids of the p
rotein. In this study,analysis of a combined CoMFA-homology model fo
r the enzyme identified F110 and F114 as potentialhyd
rophobic, a
romatic active-site
residues which could
rs/pi.gif" BORDER=0 >-stack with the nonmetabolized C-9 phenyl
ringof the wa
rfa
rin enantiome
rs. To test this hypothesis, we int
roduced mutations at key
residues located inthe putative loop
region between the B' and C helices of CYP2C9. The F110L, F110Y, V113L, andF114L mutants, but not the F114Y mutant, exp
ressed
readily, and the pu
rified p
roteins we
re each activein the metabolism of lau
ric acid. The V113L mutant metabolized neithe
r (
R)- no
r (
S)-wa
rfa
rin, and theF114L mutant alone displayed alte
red metabolite p
rofiles fo
r the wa
rfa
rin enantiome
rs. The
refo
re, theeffect of the F110L and F114L mutants on the inte
raction of CYP2C9 with seve
ral of its subst
rates aswell as the potent inhibito
r sulfaphenazole was chosen fo
r examination in fu
rthe
r detail. Fo
r each subst
rateexamined, the F110L mutant exhibited modest changes in its kinetic pa
ramete
rs and p
roduct p
rofiles.Howeve
r, the F114L mutant alte
red the metabolite
ratios fo
r the wa
rfa
rin enantiome
rs such that significantmetabolism occu
rred fo
r the fi
rst time on the putative C-9 phenyl ancho
r, at the 4'-position of (
R)- and(
S)-wa
rfa
rin. In addition, the
Vmax fo
r (
S)-wa
rfa
rin 7-hyd
roxylation dec
reased 4-fold and the
Km wasinc
reased 13-fold by the F114L mutation, whe
reas kinetic pa
ramete
rs fo
r lau
ric acid metabolism, a subst
ratewhich cannot inte
ract with the enzyme by a
rs/pi.gif" BORDER=0 >-stacking mechanism, we
re not ma
rkedly affected by thismutation. Finally, the F114L mutant effected a g
reate
r than 100-fold inc
rease in the
Ki fo
r inhibition ofCYP2C9 activity by sulfaphenazole. These data suppo
rt a
role fo
r B'-C helix loop
residues F114 andV113 in the hyd
rophobic binding of wa
rfa
rin to CYP2C9, and a
re consistent with
rs/pi.gif" BORDER=0 >-stacking to F114 fo
rce
rtain a
romatic ligands.