Table 1.

k_catand k_cat/K_mvalues determined for the various reactions catalyzed by wild-type RePC ^a

	Variable Substrateb	kcat (min−1)	Km (mM)	kcat/Km (min−1 mM−1)	% kcat of pyruvate carboxylation
Pyruvate carboxylation	MgATPc	396 ± 10d	0.145 ± 0.009	2670 ± 120	(100)
	HCO3−	700 ± 20	10.8 ± 0.4	64.8 ± 0.9	---
	pyruvate	440 ± 8d	0.15 ± 0.01	2900 ± 150	---
Full reverse reaction	MgADP	0.835 ± 0.005	0.12 ± 0.02	6.95 ± 0.002	0.2
Oxamate-induced decarboxylation of oxaloacetate	NDe	9.03 ± 0.04f	---	----	2.0
HCO3−- dependent ATPase	MgATP	2.58 ± 0.09	0.022 ± 0.003	115 ± 2	0.6
ADP phosphorylation	Carbamoyl phosphate	2.41 ± 0.01	2.6 ± 0.5	0.93 ± 0.02	0.6
	Acetyl phosphate	0.56 ± 0.02	0.34 ± 0.02	1.70 ± 0.05	0.1

^aKinetic parameters were determined from data fits to eqn (1).

^bVaried substrate indicated, all other substrates were held constant at saturating concentrations. Reaction conditions: 50 mM Bis-tris, 25 mM Tricine, 25 mM Glycine (pH 7.5), 25° C, 0.25 mM acetyl-CoA. For more detailed reaction conditions see Methods section.

^ck_cat (min⁻¹) and k_cat/K_m (min⁻¹ mM⁻¹) were determined from fits of the initial rates of pyruvate carboxylation at varying concentrations of MgATP and fixed Mg²⁺ to the equation for an equilibrium ordered pattern (eqn (2)) and the K_{ia Mg2+} = 1.2 ± 0.2 mM . All other k_cat and k_cat/K_m values were determined from fits to the eqn (1).

^dApparent k_cat.

^eND = not determined.

^fSpecific activity, determined in triplicate with saturating concentrations of all substrates and activators.