Kinetic parameters for the glucuronidation of estradiol and fulvestrant by UGT1A isoenzymes

Kinetic studies were performed for 1 h using microsomal concentrations of proteins from HEK293 cells stably expressing human UGT1A1, -1A3, -1A4, and -1A8 proteins in the presence of UDPGA (2 mM) and concentrations of fulvestrant ranging from 0.5 to 200 μM. For estradiol, kinetic parameters were obtained from Lépine et al. (2004). Values represent the mean of two independent experiments ± S.D. Glucuronide conjugates were quantified using LC/MS-MS. Vmax values were normalized for the variable level of UGT1A protein expression in stably expressing HEK293 cells. Each normalized Vmax value was used to determine the glucuronidation efficiency (ratio Vmax/Km). Km is expressed as micromolar, Vmax as picomoles per minute per milligram of protein, and catalytic efficiency as microliters per minute per milligram of protein.

KmVmax Catalytic Efficiency Kinetic Profile
UGT1A1 23 93 2.0 S
UGT1A3 47 39 0.4 S
UGT1A4 N.A. N.A. N.A.
UGT1A8 38 195 5.1 H
UGT1A1 N.A. N.A. N.A.
UGT1A3 35 13 0.2 S
UGT1A4 N.A. N.A. N.A.
UGT1A8 N.A. N.A. N.A.
UGT1A1 1.9 9.30 4.89 I
UGT1A3 5.6 217.2 39.06 I
UGT1A4 0.5 19.50 36.79 I
UGT1A8 19 16.94 0.88 I
UGT1A1 N.A. N.A. N.A.
UGT1A3 N.A. N.A. N.A.
UGT1A4 N.A. N.A. N.A.
UGT1A8 25 0.74 0.03 I
  • N.A., no activity of glucuronidation; H, hyperbolic; S, sigmoid; I, substrate inhibition.