Determination of kinetic constants for variants A7P and I58V using isoAsp-DSIP as methyl acceptor.

All assays were carried out in triplicate as described in Materials and methods. For WT (both panels) and A7P (top panel), isoAsp-DSIP was varied over the range of 3–30 μM. For I58V (bottom panel), isoAsp-DSIP was varied over the range of 5–100 μM, but data points for 100 μM are not shown. Initial rates measured at each concentration of isoAsp-DSIP are superimposed on a curve defined by the Michaelis-Menten equation using values of Vmax and Km calculated via the Direct Linear Plot method of Eisenthal and Cornish-Bowden [17, 18]. The colored vertical dashed lines intersect the X-axis at the corresponding value of the calculated Km for isoAsp-DSIP.