An Examination by Site-Directed Mutagenesis of Putative Key Residues in the Determination of Coenzyme Specificity in Clostridial NAD+-Dependent Glutamate Dehydrogenase

Table 2

Comparison of kinetic parameters between wildtype, and F238S, P262S and F238S/P262S mutant enzymes. To determine kinetic parameters for NAD(P)H, NH₄Cl, and oxoglutarate concentrations were kept constant at 100 mM and 20 mM, respectively, over a range of NAD(P)H concentrations (0.001–0.3 mM) under standard assay conditions. All experiments were repeated in triplicate, and the kinetic parameters and their standard errors (±SE) were calculated by the Wilkinson nonlinear regression method [10] with Enzpack version 3.0 (Biosoft Ltd., UK).

			NADH			NADPH
	pH	𝑘cat  (s^−1)	𝐾𝑚 (mM)	𝑘cat/𝐾𝑚 (s^−1 mM)	𝑘cat  (s^−1)	𝐾𝑚 (mM)	𝑘cat/𝐾𝑚 (s^−1 mM)
Wildtype	6.0	19.9±0.3	0.036±0.011	553	3.59±0.23	0.061±0.01	58.9
A242G	6.0	11.2±1.3	0.034±0.010	329	1.43±0.01	0.074±0.01	19.3
F238S	6.0	13.6±0.7	0.028±0.004	485	5.00±0.33	1.0±0.02	5.00
P262S	6.0	14.4±1.0	0.018±0.005	800	14.8±1.1	1.1±0.16	13.5
F238S/P262S	6.0	13.9±1.1	0.048±0.012	289	11.2±1.5	2.00±0.46	5.75
D263K	6.0	14.6±0.6	0.022±0.001	663	20.0±1.6	0.068±0.010	294
Wildtype	7.0	130.2±3.1	0.020±0.001	6500	2.87±0.11	0.090±0.011	31.9
A242G	7.0	65.1±1.6	0.025±0.001	2604	1.87±0.01	0.386±0.05	4.84
F238S	7.0	139±5	0.040±0.003	3475	11.1±0.21	0.23±0.05	48.3
P262S	7.0	160±8	0.047±0.103	3404	62.5±1.6	0.11±0.01	568
F238S/P262S	7.0	105±4	0.035±0.010	3000	83.3±3.5	0.33±0.02	252
D263K	7.0	80±2.5	0.023±0.002	3478	26.3±0.35	0.107±0.02	246
Wildtype	8.0	232±22	0.078±0.013	2974	0.82±0.11	0.53±0.07	1.54
A242G	8.0	177±9	0.085±0.010	2082	~0.15	ND	ND
F238S	8.0	402±15	0.230±0.018	1747	3.61±0.21	0.462±0.087	7.81
P262S	8.0	490±37	0.142±0.016	3450	88.5±1.15	0.330±0.108	268
F238S/P262S	8.0	133±3	0.204±0.064	665	142±2.2	0.56±0.12	221
D263K	8.0	150±11	0.091±0.010	1648	37.5±0.17	0.154±0.06	232