Table 2. Kinetic parameters for WT and D219A GMPR.

Reactions were performed in 75 mM Tris, pH 7.8, 100 mM KCl, 1 mM DTT and 0.5 mM EDTA at 25 °C. NADPH consumption/formation was monitored as described in Methods. Values are the average and standard error of at least two independent experiments. “K_m(S)” denotes the K_m of GMP, dGMP, IMP or dIMP, as appropriate. “k” denotes k_cat for the reaction of dGMP+NADPH, V_m/[E] for the reaction of dIMP+NADP⁺+NH₃ and k_obs for the partial reaction of dIMP+NADP⁺.

Enzyme	Reaction	Km (S) (μM)	Km (cofactor) (μM)	k (s−1)
WT	GMP + NADPH a	3.2 ± 0.5	10.1 ± 0.8	0.35 ± 0.01
	dGMP + NADPH b	15 ± 3	16 ± 4	0.023 ± 0.002
D219A	GMP + NADPH c	380 ± 130	120 ± 20	0.013 ± 0.003
WT	IMP + NADP+ + NH3 d	23 ± 6	50 ± 16	0.018 ± 0.009
	dIMP + NADP+ + NH3 e	140 ± 20	109 ± 28	0.002 ± 0.001
D219A	IMP + NADP+ + NH3 f	1100 ± 240	420 ± 220	(4.0 ± 0.3) x 10−4
WT	IMP + NADP+ g	n.a.	n.a.	0.49 ± 0.01 (16%)
	dIMP + NADP+ g	n.a.	n.a.	0.064 ± 0.001 (10%)
D219A	IMP + NADP+ g	n.a.	n.a.	0.055 ± 0.003 (2.8%)

^aValues from ¹³.

^bReactions performed at saturating concentrations of the fixed substrate dGMP (200 μM) and NADPH (150 μM).

^cReactions performed at saturating concentrations of GMP (2 mM) and NADPH (600 μM).

^dReactions performed at saturating concentrations of IMP (500 μM), NADP⁺ (500 μM) and NH₄⁺ (20 mM).

^eReactions performed at saturating concentrations of the fixed substrate, IMP (1 mM) and NADP⁺ (500 μM).

^fReactions performed at saturating concentrations of the fixed substrate, IMP (10 mM) and NADP⁺ (3 mM).

^gReactions performed at saturating conditions as described above. The percentage of NADPH produced per active site at equilibrium is shown in parentheses; n.a., not applicable