Table 1. In vitro properties of DHFR mutant proteins ^a.

	Wild type	D27F	D27N	D27G	D27C
kcat (s−1)	13 ± 2	(5 ± 1)×10−4	(2.6 ± 0.9) × 10−2	(1.8 ± 0.4)×10−2	3 ± 0.8
KM (μM)	0.8 ± 0.2	(1.2 ± 0.6)×102	(4 ± 1)×101	23 ± 7	(7 ± 2)×101
kcat/KM (s−1 μM−1)	16 ± 1	(4 ± 1)×10−6	(7 ± 0.8)×10−4	(8 ± 2)×10−4	(4.2 ± 0.8)×10−2
kcat/KM relative to wild type	-	2 × 10−7	4 × 10−5	3 × 10−5	2 × 10−3
Ki (nM)	1.0 ± 0.3	ND	ND	ND	(1.7 ± 0.7)×104
ΔTm (0C)	-	+7.6 ± 0.1 b	+1.1 ± 0.3	+1.2 ± 0.2	+0.6 ± 0.6
bis-ANS	1	1.1 ± 0.1	2.9 ± 0.3	1.7 ± 0.3	2.2 ± 0.5

^a Kinetic properties for dihydrofolate reductase catalytic activity (k_cat = enzymatic turnover number, K_M = Michaelis Menten constant, k_cat/K_M = catalytic efficiency, K_i = inhibition constant for trimethoprim) and protein stability properties (ΔT_m = difference in melting temperature of folding with respect to wild type, bis-ANS = relative fluorescence from binding of bis-ANS to molten-globule intermediates with respect to wild type protein).