Figure 1.

P472L mutation increases p97 ATPase activity and desensitizes the enzyme to ATP-competitive and allosteric inhibitors. A, p97 mutations found in NMS-873- or CB-5083–resistant HCT116 cells are localized to the D1–D2 linker and D2 and are distinct from disease-causing MSP-1 mutations. N615V and N616F are mutations shown to disrupt NMS-873 analog binding from cross-linking studies (37). Residues implicated in NMS-873 (pink) and CB-5083 (cyan) resistance are shown (PDB code 5FTL) with inhibitor-binding sites indicated. Additional mutations found in HCT116 cells resistant to CB-5083 and NMS-873 during this study are in bold. B, ATP titration experiments were performed with p97 and the P472L mutant to obtain steady-state kinetic parameters. Resulting Michaelis-Menten constants (K_M), turnover numbers (k_cat), and catalytic efficiencies (k_cat/K_M) are shown in Table 1. C, sensitivities of the ATPase activities of p97 and the P472L mutant to CB-5083 or NMS-873 were evaluated in inhibitor titration experiments. IC₅₀ values obtained from dose-response curves are shown in Table 1. Data points represent the mean (n = 4) with standard deviation (S.D.) error.