Table 1. Kinetic parameters of PPIP5K2^KD:PP-InsP synthesis.

Kinetic parameters were determined under pseudo first-order conditions [38] for phosphorylation of InsP₆ or 5-InsP₇ for both ATP and InsP substrates (see the Materials and methods section). When fixed, ATP was 10 mM and InsP 10 μM. Initial velocities were measured by conversion of an appropriate [³H]-radiolabelled inositol phosphate tracer (4000 DPM) as analysed by Partisphere 5 SAX HPLC (e.g., see Figures 3A and 3C) for each data point. The Michaelis–Menten equation was fit to plots of initial velocity against substrate concentration using non-linear regression (GraphPad Prism v5.03). No substrate inhibition was observed (results not shown). Results are presented as the means±S.E.M. (n≥2 independent experiments).

		Km (μM)				kcat/Km (M−1·s−1)
Substrates		Nucleotide	InsP	Vmax (nmol·min−1·mg−1)	kcat (s−1)	Nucleotide	InsP
ATP	InsP6	37±5	0.39±0.09	43±2	0.030±0.001	7.9×102	7.6×104
ATP	5-InsP7	22±7	0.060±0.009	190±10	0.13±0.01	5.9×103	2.2×106