TABLE 3

Kinetic constants for daunorubicin 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		427 ± 16	897 ± 51	0.27 ± 0.01	329 ± 14
	I15F	405 ± 12	939 ± 50	0.25 ± 0.01	277 ± 15
	H42L	414 ± 56	781 ± 91	0.27 ± 0.04	343 ± 60
	L73V	382 ± 38	775 ± 85	0.25 ± 0.02	320 ± 48
	K90E	389 ± 61	725 ± 65	0.25 ± 0.04	348 ± 66
	G204S	456 ± 51	790 ± 78	0.29 ± 0.03	375 ± 68
	T288I	440 ± 37	839 ± 88	0.29 ± 0.02	339 ± 47
AKR1B10		1829 ± 77	447 ± 37	1.23 ± 0.06	2752 ± 382
	P87S	1700 ± 91	404 ± 30	1.14 ± 0.07	2822 ± 360
	M286T	1654 ± 98	452 ± 28	1.11 ± 0.07	2453 ± 287
	D313N	1790 ± 84	410 ± 32	1.20 ± 0.06	2927 ± 391
AKR1C1		87 ± 9	551 ± 41	0.057 ± 0.006	103 ± 19
	R170H	77 ± 7	585 ± 57	0.051 ± 0.006	87 ± 16
	Q172L	72 ± 10	534 ± 39	0.047 ± 0.006	88 ± 17
AKR1C2		114 ± 17	1236 ± 168	0.076 ± 0.01	62 ± 7
	F46Y	120 ± 19	1322 ± 152	0.080 ± 0.02	61 ± 10
AKR1C3		1570 ± 37	122 ± 16	1.09 ± 0.03	8937 ± 701
	H5Q	1640 ± 76	114 ± 9	1.15 ± 0.04	10,066 ± 770
	R66Q	1534 ± 28	122 ± 14	1.07 ± 0.02	8854 ± 844
	A106T	1082 ± 103^*	118 ± 19	0.76 ± 0.07^*	6440 ± 740^*
	R170C	837 ± 53^*	108 ± 21	0.58 ± 0.05^*	5348 ± 676^*
	P180S	1211 ± 46^*	134 ± 21	0.85 ± 0.03^*	6432 ± 584^*
AKR1C4		377 ± 28	1096 ± 78	0.23 ± 0.02	228 ± 21
	G135E	340 ± 25	1156 ± 64	0.22 ± 0.02	194 ± 25
	S145C	356 ± 22	1122 ± 89	0.23 ± 0.02	210 ± 19
	L311V	199 ± 24^*	1060 ± 91	0.13 ± 0.02^*	125 ± 18^*
AKR7A2		3659 ± 294	329 ± 33	2.39 ± 0.19	7369 ± 858
	V135M	3883 ± 240	411 ± 62	2.54 ± 0.16	6322 ± 661
	A142T	1418 ± 83^*	842 ± 77^*	0.93 ± 0.05^*	1109 ± 117^*
	Q157H	3363 ± 163	379 ± 26	2.24 ± 0.15	5836 ± 510
	E180K	3204 ± 159	366 ± 20	2.10 ± 0.10	5750 ± 412
	G198S	3557 ± 174	353 ± 37	2.33 ± 0.11	6652 ± 638
	S255N	3274 ± 294	339 ± 21	2.14 ± 0.19	6354 ± 718
CBR4		9 ± 1	673 ± 94	0.0044 ± 0.0007	7 ± 2
	L70M	10 ± 1	664 ± 86	0.0049 ± 0.0007	7 ± 2

↵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).