TABLE 3

Enzyme kinetic properties of recombinant WT and mutant CYP2C9 proteins for tolbutamide hydroxylation and in silico functional prediction of CYP2C9 variants

These data represent the mean ± S.D. of three independently performed catalytic assays. Concordance is defined as agreement of the kinetic assay result with at least two of three in silico software results.

cDNAVmax ± S.D. × 10−3Km ± S.D.CLINT × 10−4 [Relative CLINT (%WT)]PolyphenSIFTPROVEANEnzyme Activity in Relation to WTConcordance
(Yes/No)
Vivid*MS*
nmol/mg per minuteµMµl/mgprotein per minute%%
WT13.5 ± 0.6451 ± 10.512.71 ± 0.41
218C>T4.11 ± 0.2267.8 ± 16.10.62 ± 0.11 (23)**Probably damagingDamagingDeleterious3123Yes
229C>A17.7 ± 0.6176.2 ± 7.492.33 ± 0.16 (86)Probably damagingDamagingNeutral8886No
343A>C7.12 ± 0.79283 ± 49.20.26 ± 0.04 (9.6)**Probably damagingDamagingDeleterious1110Yes
707_delAa0.42 ± 0.04141 ± 183Possibly damagingN/AN/A00N/A
709G>C13.9 ± 3.36219 ± 1270.73 ± 0.24 (29)**BenignToleratedNeutral2627No
707_709delinsCCa0.06N/AN/AN/A
791T>C5.83 ± 0.4150.1 ± 5.621.17 ± 0.09 (43.6)*Possibly damagingToleratedDeleterious7243Yes
801C>T14.7 ± 0.4656.5 ± 10.62.66 ± 0.42 (98.1)ToleratedNeutral11198Yes
*32.80 ± 0.81149 ± 1570.32 ± 0.24 (10.3)**110
  • CLINT, intrinsic clearance; Km, Michaelis constant; MS, mass spectrometry; N/A, not applicable.

  • a The kinetic parameters for tolbutamide hydroxylation of CYP2C9 variants 707_delA and 707_709delinsCC could not be determined because the amount of produced metabolite was at or below the detection limit at the lower substrate concentrations.

  • * P < 0.05; **P < 0.01 compared with CYP2C9 WT.