FIGURE 6.

Cdc14 selectivity for Ser(P) arises from the structure of Cdc14 active site. A, the active site of human Cdc14B (Protein Data Bank (PDB) ID: 1OHE) (17) with the bound peptide substrate modified in silico to contain a Thr(P) side chain. A surface representation of the protein and peptide is depicted with purple indicating the Thr(P) side chain methyl group and the mesh delineating the surface of Ala-316. The distance between the carbon atoms of the methyl groups on Ala 316 and the Thr(P) side chain is 2.3 Å, a value that is substantially less than the sum of the Van der Waals radii of the two atoms, indicating severe steric clash. The surface after a Gly (orange) substitution at 316 is also shown. The side chain of Lys-315 was hidden for optimal visualization of the active site pocket. MacPyMOL (30) was used to visualize the hCdc14B structure, mutate the bound phosphopeptide substrate by utilizing the site mutagenesis function, and measure distances between atoms. B, relative rates of Cdc6pT7 dephosphorylation by wild-type budding yeast Cdc14 (●) and the Cdc14 A285G mutant (■) were measured as a function of peptide concentration. Activities were normalized to activity on Cdc6pS7 to directly compare differences in selectivity. Data are averages of three trials and were fit with the Michaelis-Menten equation. C, catalytic efficiencies (k_cat/K_m) determined from B and similar experiments on Cdc6pS7. Selectivity (Ser(P)/Thr(P) (pS/pT)) is k_cat/K_m for Cdc6pS7 divided by k_cat/K_m for Cdc6pT7.