TABLE 1

P450 content and enzyme kinetic analysis of CPA 4-hydroxylation in yeast microsomes expressing wild type CYP2B6 and site-directed mutants

P450 content was spectrally determined (Schoene et al., 1972). Data were calculated using GraphPad Prism software. Fifteen substrate concentrations from 0 to 25 mM were used for enzymatic analysis. Vmax values for CPA hydroxylase were expressed as moles of product formed per minute per mole of P450 included in the reaction. Km values were obtained after kinetic analysis derived from nonlinear regression.

Mutation
P450 Content
Vmax
Km
Vmax/Km
pmol/mg mol/min/mol P450 % of wt mM % of wt % of wt
CYP2B6wt 112 62.5 100 4.9 100 12.8 100
Mutations in active site
I114V 93 65.2 104 2.3 47 28.4 221
V367L 50 90.3 144 8.8 181 10.2 80
V367F 29 26.8 43 12.9 264 2.1 16
V367S 54 5.2 8 N.Q.
V367T 30 24.2 39 6.4 131 3.8 30
V367H 17 1.5 2 N.Q.
V477S 82 N.D N.Q.
V477T 76 37.2 59 8.9 182 4.2 33
V477Y 70 28.6 46 5.9 121 4.9 38
V477N 126 N.D N.Q.
V477D 53 2.5 4 N.Q.
V477E 72 1.8 3 N.Q.
V477I 53 77.4 124 3.2 66 24.2 189
V477F 104 81.1 130 3.3 68 24.5 191
V477W 203 99.9 160 2.8 57 35.7 278
G478A 75 28 45 6.3 129 4.4 35
G478V 81 36.3 58 2.6 53 14.2 110
G478S 139 7.5 12 N.Q.
G478E 48 5.4 9 N.Q.
Canine mutations
F107V 180 95.9 153 6.5 133 14.8 115
L199M 70 125.2 200 4.8 98 26.3 205
S207A 90 69.9 112 5.5 113 12.7 99
K236N 40 47 75 56.2 1153 0.8 7
M365I 340 12.9 21 3.8 77 3.4 27
C475I 190 1.7 3 4.7 96 0.4 3
Double mutant
I114V + V477W
120
58.5
94
1.1
23
52.7
411

  • N.Q., not quantifiable.