Properties of the spermidine/spermine N¹-acetyltransferase mutant L156F that decreases cellular sensitivity to the polyamine analogue N¹, N¹¹-Bis(ethyl)norspermine

Properties of a mutant form of spermidine/spermine N¹-acetyltransferase, L156F (L156F-SSAT), that is present in Chinese hamster ovary cells selected for resistance to the polyamine analogue N^1, N¹¹-bis(ethyl) norspermine (BE 3-3-3) were investigated. Increased K_m values, decreased V_max values, and decreased k_cat values with both polyamine substrates, spermidine and spermine, indicated that L156F-SSAT is an inferior and less efficient acetyltransferase than wild-type SSAT. Transfection of L156F-SSAT into C55.7Res cells indicated that cellular SSAT activity per nanogram of SSAT protein correlated well with the in vitro data and was also ∼20-fold less for the mutant protein than for wild-type SSAT. Increased expression of L156F-SSAT was unable to restore cellular sensitivity to BE 3-3-3 despite providing measurable basal SSAT activity. Only a 4-fold induction of L156F-SSAT activity resulted from the exposure of cells to the polyamine analogue, whereas wild-type SSAT was induced-300-fold. Degradation studies indicated that BE 3-3-3 cannot prevent ubiquitination of L156F-SSAT and is therefore unable to protect the mutant protein from degradation. These studies indicate that the decreased cellular sensitivity to BE 3-3-3 is caused by the lack of SSAT activity induction in the presence of the analogue due to its inability to prevent the rapid degradation of the L156F-SSAT protein.