TABLE 2

Kinetic analysis of glucuronide formation for UGT1A9-overexpressing cell microsomes

Kinetic data are reported as mean ± S.D. for three independent experiments. K_m represents apparent K_m. V_max values are adjusted per microgram of the corresponding UGT1A9 protein as determined by Western blot.

UGT1A9	Substrate	K_m or K_S^a	V_max	V_max/K_m or K_S	Constant Values for Eq. 2^b or 3^a
		μM	pmol min⁻¹ mg⁻	μl min⁻ mg⁻¹
Wild-type	Benzidine	2234 ± 439	14 ± 3.4	0.0063 ± 0.0004
33Thr		1672 ± 336	9.0 ± 1.1	0.0055 ± 0.0005
167Ala		2295 ± 282	19 ± 2.1	0.0086 ± 0.0017
183Gly		1971 ± 479	21 ± 4.2	0.011 ± 0.0039
Wild-type	4-ABP	221 ± 13	48 ± 1.9	0.22 ± 0.02
33Thr		945 ± 114***	29 ± 1.7***	0.03 ± 0.004***
167Ala		140 ± 29*	43 ± 9.3	0.31 ± 0.12
183Gly		280 ± 4.5***	93 ± 19**	0.33 ± 0.06*
Wild-type	4-MU	5.3 ± 1.3	54 ± 7.3	10 ± 1.5
33Thr		985 ± 40***	415 ± 3.1***	0.42 ± 0.016***
167Ala		6.0 ± 1.7	108 ± 14**	19 ± 3.8*
183Gly		7.1 ± 0.91	87 ± 16*	12 ± 3.8
Wild-type	11-OH-DB[a,l]P	0.25 ± 0.076	13 ± 3.2	51 ± 9.7
33Thr		0.32 ± 0.12	19 ± 5.0	59 ± 8.2
167Ala		0.31 ± 0.15	12 ± 2.2	47 ± 21
183Gly		0.84 ± 0.09***	26 ± 2.2***	31 ± 2.0*	K_i = 6.6 ± 3.2^a
Wild-type^b	3-OH-B[a]P	0.12 ± 0.022	40 ± 0.56	337 ± 54	K_i = 0.42 ± 0.06^b
33Thr^a		0.02 ± 0.083	3.6 ± 10.7**	261 ± 223	α = 48.9 ± 171^a
					β = 5.87 ± 11.1^a
167Ala^b		0.22 ± 0.034*	52 ± 5.3*	246 ± 57	K_i = 0.42 ± 0.16^b
183Gly^a		0.02 ± 0.050*	7.8 ± 9.9**	525 ± 350	α = 78.2 ± 175^a
					β = 6.72 ± 5.90^a

↵a Data were fitted to eq. 3 (see Materials and Methods), where K_S is the substrate dissociation constant and α and β are constants.
↵b Data were fitted to eq. 2 (see Materials and Methods), where K_i is the substrate inhibition constant as a micromolar concentration.
↵* P < 0.05, relative to wild-type UGT1A9 (UGT1A9^{33Met/167Val/183Cys}).
↵** P < 0.01.
↵*** P < 0.001.