Table 1.

Thermodynamic, structural, and catalytic properties of in vitro purified DHFR mutants

Mutant	(°C)	ΔG(H2O) (kcal/mol) 25 °C	kcat (s-1)	kcat/KM (s-1 μM-1)	ASA*	Conservation† (%)
						Native	Substitution
WT	51.7	−4.4	11.65	3.6
V40A	43.2	−3.31	24.05	3.22	0	43.6	0.34
I61V	53.4	−4.52	8.99	3.7	0	19.9	79
V75H	40.6	−2.18	15.5	4.57	0.05	32	0
V75I	39.2	−2.98	18.25	5.51	0.05	32	10.3
I91V	49.1	−3.14	17.87	5.13	0.07	33.3	29
I91L	41.4	−2.69	ND‡	ND‡	0.07	33.3	20
L112V	47.4	−2.7	11.45	2.34	0	34	27.5
W133F	46.3	−4.4	13.45	6.79	0.05	53.6	20.6
W133V	ND‡	−1.53	ND‡	ND‡	0.05	53.6	0.34
I155T	43.4	−3.68	11.27	2.72	0.12	15.8	21
I155L	45.8	−2.84	12.9	5.12	0.12	15.8	1
I155A	38.5	−2.42	11.95	2.71	0.12	15.8	1
I115V	51.4	−6	10.26	2.08	0.02	55.3	34.4
I115A	46.1	−3.4	7.59	0.5	0.02	55.3	0
V88I	44.3	−4.22	13.8	3.12	0.34	12.4	1.4
A145T	51.3	−4.13	10	3.6	0.89	12.4	1.4

^*ASA (accessible surface area) was calculated by Vadar package (http://vadar.wishartlab.com); cutoff for buried residues is around 0.25.

^†Conservation (%) of a native E. coli’s DHFR residue (left column) or a substituted residue (right column) in a given position of 290 aligned mesophylic prokaryotic DHFR sequences retrieved from the Optimal Growth Temperature database (http://pgtdb.csie.ncu.edu.tw).

^‡Not determined.