. 2014 May 12;136(23):8333–8341. doi: 10.1021/ja501936d

Table 4. Association and Dissociation Rate Constants (k_on and k_off, Respectively) That Describe the Interactions between Different Ligands and ecDHFR Species (l-, Light DHFR; h-, Heavy DHFR), As Measured by Rapid Mixing of the Ligand with ecDHFR or a Binary Complex at 25 °C, pH 7.

	ligand	enzyme species		k_on (μM^–1 s^–1)	k_off (s^–1)	K_d = k_off/k_on (μM)
(a)	NADPH	DHFR	l-	17.3 ± 0.4	3 ± 2^a	0.18 ± 0.09
(a)	NADPH	DHFR	h-	16.8 ± 0.4	5 ± 2^a	0.28 ± 0.09
(b)	DHF	DHFR	l-	41.0 ± 0.5	54 ± 4	1.3 ± 0.1
(b)	DHF	DHFR	h-	30.1 ± 0.6	113 ± 6	3.8 ± 0.2
(c)		DHFR·NADP⁺	l-	42.1 ± 0.5	10 ± 3	0.24 ± 0.07
(c)		DHFR·NADP⁺	h-	38.5 ± 0.6	11 ± 4	0.3 ± 0.1
(d)	FA	DHFR	l-	34.3 ± 0.4	110 ± 3	3.22 ± 0.09
(d)	FA	DHFR	h-	35.9 ± 0.5	125 ± 4	3.5 ± 0.1
(e)		DHFR·NADPH	l-	5.9 ± 0.1	90.0 ± 0.8	15.2 ± 0.3
(e)		DHFR·NADPH	h-	6.0 ± 0.1	91.7 ± 0.8	15.4 ± 0.3
(f)	methotrexate	DHFR	l-	28.4 ± 0.3	2.0 ± 0.6	0.07 ± 0.02
(f)	methotrexate	DHFR	h-	28.1 ± 0.4	1.9 ± 0.6	0.07 ± 0.02
(g)		DHFR·NADPH	l-	52.2 ± 0.4	–0.7 ± 0.7^b	–0.01 ± 0.01
(g)		DHFR·NADPH	h-	47.2 ± 0.4	0.9 ± 0.8^b	0.02 ± 0.02

Small differences within the limits of experimental error may exist for k_off of NADPH between l- and h-DHFR.

The k_off for methotrexate dissociating from the DHFR·NADPH·methotrexate ternary complex cannot be accurately determined by this method.

Table 4. Association and Dissociation Rate Constants (kon and koff, Respectively) That Describe the Interactions between Different Ligands and ecDHFR Species (l-, Light DHFR; h-, Heavy DHFR), As Measured by Rapid Mixing of the Ligand with ecDHFR or a Binary Complex at 25 °C, pH 7.