Table 1

Kinetic constants determined from the concentration dependence of MDZ oxidation by CYP3A4 wild-type and mutants.

	1′-OH		4-OH		Ratio
	V_max	K_M	V_max/K_M	V_max	K_M	V_max/K_M	V_max/K_M(1′OH)/V_max/K_M(4-OH)
	min⁻¹	μM	μM/min	min⁻¹	μM	μM/min
WT	8.9 (0.3)	3.7 (0.9)	2.4	10.9 (0.3)	64.0 (4.0)	0.17	14
S119A^a	12.7 (0.6)	27.3 (4.5)	0.46	40.0 (2.2)	114.1 (12.8)	0.35	1.3
I120W^b ^1-c	23.5 (0.7)	1.9 (0.6)	12.1	8.5 (0.3)	93.7 (8.0)	0.09	134
L210A^a	20.5 (0.9)	22.3 (3.4)	0.92	28.6 (0.7)	42.0 (3.1)	0.68	1.4
F304A^a	12.6 (0.6)	12.9 (2.6)	0.98	23.9 (1.3)	65.3 (8.8)	0.37	2.7
F304W^b ^1-c	17.5 (0.4)	2.4 (0.5)	7.2	6.9 (1.0)	87.8 (28.3)	0.08	92
T309F	2.7 (0.1)	3.3 (0.9)	0.83				–
I369W				4.1 (0.4)	89.0 (21.3)	0.05	–
L373F^1-d	5.8 (0.3)	36.4 (6.6)	0.16	17.3 (0.7)	154.6 (12.1)	0.11	1.4
L479F^a	16.2 (0.7)	9.3 (2.1)	1.75	7.0 (0.9)	83.9 (23.6)	0.08	21

The values in parentheses show the average deviation obtained from the fit of the Michaelis-Menton equation to a single kinetic data set. The experiments were done multiple times (n = 2–4) using multiple protein preparations without significant change in the ratio ofV _max/K _M(1′-OH)/V _max/K _M(4-OH).

^a,b Due to very high rate of metabolite formation with some of the mutants, the incubations were done using 5 (a) and 3 (b) pmol of the enzyme in these cases.
↵1-c The reaction kinetics of 1′-OH MDZ formation by I120W and F304W possibly showed a small extent of inhibition at higher substrate concentrations (see Fig. 4) as reported for wild-type CYP3A4 (Gorski et al., 1994; Ghosal et al., 1996; Perloff et al., 2000). A reanalysis of the reaction kinetics by a model incorporating Michaelis-Menton kinetics with uncompetitive substrate inhibition showed no significant change in the K _M and V _maxvalues (shown by dotted lines in Fig. 4) compared with those determined using the Michaelis-Menton equation. The K _inbdetermined from these fits showed very large standard deviations and is therefore not reported.
↵1-d Eiselt et al. (2001).