Table 2.

Kinetic constants for PEP³⁻, MgADP complex and Mg²⁺_free of WT-RMPK, K114Q-RMPK, E117K-RMPK, T113L/E117K-RMPK and T113L/K114Q/E117K-RMPK. The experiments for PEP³⁻ were carried out in the presence of saturating MgADP complex and saturating Mg²⁺_free, whereas those for MgADP and Mg²⁺_free were performed in the presence of saturating concentrations of the other substrates. The experimental conditions were as in Figure 2 except for the absence of monovalent cations for pyruvate kinases mutants with K117 (K⁺-independent enzymes) and the inclusion of (CH₃)₄NCl to maintain ionic strength at 250 mM. Kinetic data (not shown) were fitted to the Hill equation (v = V_max Sⁿ/K_0.5ⁿ + Sⁿ) and to the Michaelis–Menten equation (v = V_max S/K_m + S) (Origin version 7.0). The mean and standard deviation from three experiments are shown. k_cat/^aK values are expressed in log form. Data of ^aT113L and ^bT113L were taken from Table S1 and Table S2 of Supplementary material, respectively, [21].

RMPK & RMPK Mutants	With E117				RMPK Mutants	With K117
	kcat (s−1)	K (mM)	n	Log kcat/K (s−1 M−1)		kcat (s−1)	K (mM)	n	Log kcat/K (s−1 M−1)
	PEP					PEP
WT	703 ± 28	0.063 ± 0.008	__	7.05	E117K	249 ± 16	0.063 ± 0.00	__	6.60
aT113L	725 ± 49	0.108 ± 0.018	__	6.83	T113L/E117K	280 ± 8	0.048 ± 0.002	__	6.77
K114Q	805 ± 26	0.15 ± 0.014	__	6.74	T113L/K114Q/E117K	363 ± 4	0.067 ± 0.00	1.82 ± 0.17	6.73
MgADP						MgADP
WT	691 ± 16	0.23 ± 0.010	__	6.48	E117K	261 ± 12	0.360 ± 0.035	1.23 ± 0.09	5.86
bT113L	831 ± 120	1.07 ± 0.26	__	5.89	T113L/E117K	363 ± 28	0.100 ± 0.030	__	6.56
K14Q	690 ± 19	0.54 ± 0.034	__	6.10	T113L/K114Q/E117K	419 ± 8	0.640 ± 0.030	1.82 ± 0.17	5.82
Mg2+free						Mg2+free
WT	865 ± 12	0.17 ± 0.010	1.46 ± 0.1	6.71	E117K	284 ± 4	4.9 ± 0.3	__	4.76
K114Q	N.D.	N.D.	N.D.	N.D.	T113L/E117K	458 ± 75	1.6 ± 0.5	1.39 ± 0.3	5.46
T113L	N.D.	N.D.	N.D.	N.D.	T113L/K114Q/E117K	371 ± 16	8.7 ± 0.7	1.44 ± 0.1	4.63

K represents the K_0.5 and K_m for the data fitted to the Hill and Michaelis–Menten equations, respectively.