TABLE 1. Steady-state kinetics parameters of recombinant BcUGlcAE and BcUGlcDH.

Kinetics of UGlcAE were measured with varied concentrations of UDP-GlcA (0.1 – 1.0 mM) or TDP-GlcA (0.08 – 0.5 mM) and 1 mM NAD⁺ after 8 min at standard conditions. The reciprocal initial velocity was plotted against the reciprocal UDP-GlcA or TDP-GlcA concentration according to Lineweaver and Burk to calculate the corresponding K_m values. The data presented are the average K_m values from three experiments. Kinetics of BcUGlcDH were measured with 2 mM NAD⁺ and varied concentrations of UDP-Glc (0.1 – 1.0 mM) after 15 min or with varied concentrations of TDP-Glc (0.1–1.0 mM) after 25 min under standard conditions. Kinetics of BcUGlcDH were also measured with varied concentrations of NAD⁺ (0.1–2 mM) with 1 mM UDP-Glc after 15 min or 1 mM TDP-Glc after 25 min under standard conditions. The data presented are the average K_m values from two experiments.

	Km (mM)	kcat (s−1)	kcat/Km (mM−1s−1)
UDP-GlcAE activity	0.12 ± 0.01	3.2 ± 0.1	26 ± 1.4
TDP-GlcAE activity	0.18 ± 0.01	2.1 ± 0.1	12 ± 0.4
UDP-GlcDH Activity – (UDP-Glc)	0.137 ± 0.019	0.974 ± 0.031	7.1 ± 1.0
UDP-GlcDH Activity – (NAD+)	0.042 ± 0.006	0.898 ± 0.014	21.4 ± 3.1
TDP-GlcDH Activity* – (TDP-Glc)	2.07 ± 0.39	0.646 ± 0.091	0.31 ± 0.073
TDP-GlcDH Activity- (NAD+)	0.664 ± 0.050	0.307 ± 0.012	0.46 ± 0.039

^*The kinetic value for TDP-Glc and NAD are an estimate and likely qualitative rather than quantitative, since the reaction with TDP-Glc could not be done at saturation. This in turn may affect the Kcat value for NAD. Nonetheless, the data provide sufficient information showing that the dehydrogenase is more efficient with UDP-Glc as the substrate.