Table 1:

Kinetic constants with YcjS and YcjQ and their respective substrates.

Enzyme	Substrate	pH	Cofactor	kcat (s−1)	Km (mM)	kcat/Km (M−1 s−1)
YcjS	1	8.0a	NAD+	0.65 ± 0.02	7.2 ± 0.8	90 ± 10
		9.0b		1.14 ± 0.06	8.9 ± 0.1	128 ± 7
	2	8.0a	NAD+	0.58 ± 0.01	5.6 ± 0.6	103 ± 11
		9.0b		1.2 ± 0.05	5.9 ± 0.7	200 ± 25
	3	8.0a	NAD+	0.4 ± 0.01	2.0 ± 0.1	191 ± 9
		9.0b		0.3 ± 0.01	1.6 ± 0.2	210 ± 20
	4	8.0a	NAD+	0.17 ± 0.01	2.1 ± 0.1	81 ± 4
		9.0b		0.4 ± 0.01	3.9 ± 0.4	100 ± 10
	5	6.5c	NADH	4.7 ± 0.3	0.5 ± 0.1	8500 ± 200
	6	7.0d	NADH	22 ± 0.8	0.95 ± 0.1	23100 ± 2800
		8.0e		18 ± 1.0	1.2 ± 0.3	15000 ± 3800
	7	7.0d	NADH	19 ± 1	0.80 ± 0.06	23400 ± 2200
		8.0e		12.5 ± 0.7	0.92 ± 0.08	13600 ± 1400
YcjQ	8	8.0f	NAD+	0.18 ± 0.01	5.1 ± 0.40	31 ± 2
		9.0b		0.23 ± 0.02	5.6 ± 0.8	41 ± 6
	9	8.0f	NAD+	0.35 ± 0.03	12.5 ± 1.5	28 ± 3
		9.0b		1.2 ± 0.05	12.0 ± 0.8	97 ± 6
	12g	7.0h	NADH	18.5 ± 1.6	9.4 ± 1.1	2000 ± 290
		8.0i		3.8 ± 0.1	1.5 ± 0.1	2600 ± 160
	11g	7.0h	NADH	7.0 ± 0.2	2.3 ± 0.1	3100 ± 200
		8.0i		9.7 ± 0.6	7.0 ± 0.6	1400 ± 160
YcjR	6	7.0j		-	-	240 ± 8k
		8.0l		19.2 ± 1.7	37 ± 4.4	520 ± 77
	7	7.0j		2.2 ± 0.3	34.5 ± 6.7	58 ± 14
		8.0l		4.0 ± 0.6	31 ± 6.5	130 ± 30

^aReactions were carried out at 30 °C in 50 mM NH₄⁺HCO₃⁻ buffer, pH 8.0 with 2.0 mM NAD⁺.

^bReactions were carried out at 30 °C in 50 mM Ches/K⁺ buffer, pH 9.0 with 2.0 mM NAD⁺.

^cReaction was carried out in 50 mM cacodylate/K⁺, pH 6.5 at 30 °C with 250 µM NADH.

^dReactions were carried out at 30 °C in 50 mM cacodylate/K⁺ , pH 7.0 with 0.3 mM NAD⁺.

^eReactions were carried out at 30 °C in 50 mM NH₄⁺HCO₃⁻ buffer, pH 8.0 with 0.3 mM NADH.

^fReactions were carried out at 30 °C in 50 mM HEPES/K⁺ buffer, pH 8.0 with 2.0 mM NAD⁺.

^g11 or 12 were generated in situ by the addition of excess YcjR (20 µM), 0.5 mM MnCl₂, and 7 or 6, respectively. Concentrations of 11 and 12 were corrected based on the measured equilibrium constant (K_eq = 4) between 7 and 11.

^hReactions were carried out at 30 °C in 50 mM HEPES/K⁺ buffer, pH 7.0 with 0.3 mM NADH.

ⁱReactions were carried out at 30 °C in 50 mM HEPES/K⁺ buffer, pH 8.0 with 0.3 mM NADH.

^jReactions were carried out at 30 °C in 50 mM HEPES/K⁺ buffer, pH 7.0 with 0.3 mM NADH, 0.5 mM MnCl₂, and excess YcjQ (10 µM).

^kValues based on fit of kinetic data to a linear equation with the slope giving the value of k_cat/K_m.

^lReactions were carried out at 30 °C in 50 mM HEPES/K⁺ buffer, pH 8.0 with 0.3 mM NADH, 0.5 mM MnCl₂, and excess YcjQ (10 µM).