Table 1.

Steady-state kinetic parameters for Cj1424, Cj1425 and Cj1423^a

Enzyme	Substrate	kcat (s−1)	Km (μM)	kcat/Km (M−1 s−1)
Cj1424b	d-altro-heptulose-7-P (1)	0.14 ± 0.01	320 ± 25	(4.4 ± 0.4) × 102
Cj1425b	d-glycero-d-manno-heptose-7-P (2)c	0.48 ± 0.01	103 ± 9	(4.7 ± 0.4) × 103
Cj1152d	d-glycero-α-d-manno-heptose-1,7-bisphosphate (3)	0.12 ± 0.01	186 ± 25	(6.5 ± 1.0) × 102
GmhBe	d-glycero-α-d-manno-heptose-1,7-bisphosphate (3)	4.6 ± 0.1	67 ± 1	(7.0 ± 0.1) × 104
GmhBe	d-glycero-β-d-manno-heptose-1,7-bisphosphate	36.0 ± 0.2	5.0 ± 0.1	(7.2 ± 0.2) × 106
Cj1423f	d-glycero-α-d-manno-heptose-1-P (4)	1.35 ± 0.04	495 ± 65	(2.7 ± 0.4) × 103

^aErrors were calculated from the standard deviation of the fitting results.

^bReactions were monitored by UV coupled assay at pH 7.4 and 30 °C in the presence of NADH (300 μM), MgCl₂ (5 mM), ATP (2 mM), PEP (1.0 mM), 1 U of lactate hydrogenase and pyruvate kinase.

^cThe substrate for Cj1425, d-glycero-d-manno-heptose-7-P (2), was generated in situ using a 10-fold excess of Cj1424 and d-altro-heptulose-7-P (1). Kinetic constants were determined from the calculated concentrations of (2) using the equilibrium constant of 0.22 for the Cj1424 catalyzed reaction.

^dReaction was followed by discontinuous UV assay using malachite green free phosphate detection at pH 7.5 at 25°C.

^eReactions were monitored at pH 7.5 and 25 °C as previously reported (17).

^fReaction was monitored by HPLC at pH 7.4 and 25 °C with GTP (1.0 mM), MgCl₂ (2.0 mM) and pyrophosphatase (1 U).