TABLE 4

Kinetic constants for doxorubicin metabolism by recombinant 6×His-tagged AKR and CBR4 wild-type and variant enzymes

Values correspond to mean ± S.D. obtained from three experiments performed with three independent enzyme preparations (n = 9) for each isoform.

Enzyme	Variant	V_max	K_m	k_cat^{^a}	k_cat/K_m
		nmol/min · mg protein	μM	s⁻¹	s⁻¹ · M⁻¹
AKR1B1		193 ± 12	813 ± 17	0.12 ± 0.01	153 ± 10
	I15F	191 ± 23	851 ± 16	0.12 ± 0.01	144 ± 18
	H42L	167 ± 8	840 ± 11	0.11 ± 0.01	127 ± 10
	L73V	167 ± 19	829 ± 19	0.11 ± 0.01	129 ± 14
	K90E	168 ± 19	854 ± 23	0.11 ± 0.01	126 ± 12
	G204S	160 ± 15	844 ± 17	0.10 ± 0.01	123 ± 21
	T288I	160 ± 17	805 ± 26	0.10 ± 0.01	128 ± 16
AKR1B10		11 ± 1	508 ± 64	0.007 ± 0.001	14 ± 3
	P87S	9 ± 1	530 ± 48	0.006 ± 0.001	11 ± 3
	M286T	15 ± 2	521 ± 34	0.010 ± 0.002	19 ± 4
	D313N	12 ± 2	489 ± 40	0.008 ± 0.001	16 ± 3
AKR1C1		17 ± 2	643 ± 52	0.011 ± 0.002	17 ± 4
	R170H	14 ± 1	670 ± 48	0.009 ± 0.001	13 ± 3
	Q172L	19 ± 1	651 ± 34	0.012 ± 0.001	18 ± 3
AKR1C2		56 ± 11	780 ± 67	0.038 ± 0.007	49 ± 12
	F46Y	63 ± 16	708 ± 104	0.043 ± 0.011	62 ± 21
AKR1C3		546 ± 27	142 ± 29	0.38 ± 0.02	2514 ± 268
	H5Q	570 ± 18	161 ± 12	0.40 ± 0.01	2485 ± 241
	R66Q	509 ± 14	158 ± 14	0.36 ± 0.01	2266 ± 194
	A106T	527 ± 16	138 ± 13	0.37 ± 0.01	2692 ± 297
	R170C	323 ± 23^*	188 ± 17	0.23 ± 0.02^*	1208 ± 115^*
	P180S	306 ± 27^*	273 ± 20^*	0.21 ± 0.02^*	786 ± 95^*
AKR1C4		73 ± 13	962 ± 64	0.049 ± 0.008	51 ± 9
	G135E	70 ± 12	974 ± 37	0.047 ± 0.008	48 ± 8
	S145C	71 ± 11	992 ± 63	0.047 ± 0.007	48 ± 9
	L311V	69 ± 12	935 ± 41	0.046 ± 0.008	49 ± 8
AKR7A2		237 ± 15	340 ± 25	0.16 ± 0.01	457 ± 17
	V135M	215 ± 24	361 ± 20	0.14 ± 0.02	393 ± 56
	A142T	141 ± 19^*	501 ± 23^*	0.09 ± 0.01^*	184 ± 24^*
	Q157H	214 ± 16	362 ± 20	0.14 ± 0.01	385 ± 26
	E180K	226 ± 28	367 ± 19	0.15 ± 0.02	404 ± 52
	G198S	222 ± 19	374 ± 17	0.15 ± 0.01	389 ± 33
	S255N	220 ± 13	377 ± 18	0.14 ± 0.01	383 ± 38
CBR4		63 ± 8	139 ± 16	0.031 ± 0.005	223 ± 55
	L70M	71 ± 9	125 ± 19	0.035 ± 0.004	280 ± 61

↵a k_cat calculated from M_r 38500 (AKR1B1), 40300 (AKR1B10), 39400 (AKR1C1), 40200 (AKR1C2), 41900 (AKR1C3), 39700 (AKR1C4), 39300 (AKR7A2), and 29600 (CBR4).
↵* Variants significantly different from their respective wild-type enzymes (P < 0.05).