Table 3 Summary of steady-state kinetic and inhibition constants of EcDHFR, DfrA1, and DfrA5.

	EcDHFR	DfrA1	DfrA5	EcDHFR D27E	EcDHFR L28Q	EcDHFR D27E/L28Q
DHF steady-state kinetics
K_M (µM)	3.2 ± 0.2	9.7 ± 2.1	24.2 ± 6.6	15.4 ± 2.1	5.2 ± 1.0	56.6 ± 8.0
k_cat (s⁻¹)	6.7 ± 0.1	12.0 ± 0.6	31.4 ± 3.2	12.0 ± 0.6	5.1 ± 0.2	1.7 ± 0.09
k_cat/K_M (µM⁻¹*s⁻¹)	2.1 ± 0.14	1.2 ± 0.3	1.3 ± 0.4	0.80 ± 0.1	0.99 ± 0.2	0.03 ± 0.004
NADPH steady-state kinetics
K_M (µM)	5.8 ± 0.8	9.3 ± 1.7	19.9 ± 6.1	5.8 ± 0.9	7.6 ± 1.3	11.1 ± 1.6
k_cat (s⁻¹)	3.2 ± 0.1	11.9 ± 0.6	34.3 ± 3.3	5.9 ± 0.3	4.4 ± 0.2	3.8 ± 0.2
k_cat/K_M (µM⁻¹*s⁻¹)	0.6 ± 0.07	1.3 ± 0.2	1.7 ± 0.5	1.0 ± 0.2	0.6 ± 0.1	0.4 ± 0.05
Ki data (nM)^a
UCP1223	1.58 ± 0.26	20.33 ± 2.56	16.76 ± 0.29	12.75 ± 0.56	7.37 ± 0.72	187.14 ± 5.50
UCP1228	2.73 ± 0.09	30.06 ± 3.35	20.08 ± 0.39	11.80 ± 0.40	10.53 ± 0.69	189.28 ± 5.36
Trimethoprim	0.43 ± 0.02	1332 ± 149	393.76 ± 81.8	7.25 ± 0.25	6.26 ± 0.30	171.07 ± 33.5
Iclaprim	1.64 ± 0.21	1143.7 ± 69	99.02 ± 13.9	–	–	–
Methotrexate	0.34 ± 0.05	177.36 ± 21	578.75 ± 73.8	–	–	–

Kinetic and inhibition parameters are based on the rate of conversion of NADPH and DHF to NADP⁺ and THF, monitored by the changes in the absorbance reading at 340 nm. The kinetic constant, K_M (µM) was generated by fitting initial velocity data as a function of DHF or NADPH concentration to the Michaelis–Menten model. The same model was used to determine the catalytic constant k_cat (s⁻¹) where the enzyme concentration was constrained to a constant value. Average values from triplicates with standard error for each parameter are shown. The inhibition constants (Ki) were derived from Cheng-Prusoff equation (Eq. 1). The kinetic data demonstrate decreased affinity for DHF and NADPH in DfrA enzymes, concomitant with an increase in rate of catalysis resulting in similar catalytic efficiency. The inhibition data reveals a high level of resistance for clinical antifolates.
^aCalculated as K_i = IC₅₀/(1 + [S]/K_M) (Eq. 1).

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