Dbf2-Mob1 peptide substrate requires arginine at position -3. (A) Synthetic peptides based on the predicted optimal substrate of Dbf2-Mob1. The underlined residues represent the predicted preferred amino acids for Dbf2-Mob1 substrate specificity; the asterik denotes the single amino acid substitution. (B) The various peptides denoted in (A) at a concentration of 250 μM were treated with ~13 ng of FHHDbf2 bound to H6Mob1TM9. Aliquots of the kinase reaction were quenched at the indicated timepoints to determine the amount of phosphorylation by liquid scintillation. The Optimal peptide was also treated with the kinase-inactive FHHDbf2(N305A)-H6Mob1TM9 complex, as denoted by D2M. Similar results were obtained in 4 independent experiments. (C) Using the conditions in (B), Km and Vmax was determined for each peptide with the exception of R-3K, R-3A, negative control, and the Optimal peptide treated with kinase-inactive FHHDbf2(N305A)-H6Mob1TM9, due to low phosphorylation.