Role of the Carboxyl Terminal MATEE Sequence of Spermidine/Spermine N¹-Acetyltransferase in the Activity and Stabilization by the Poly amine Analog N¹,N¹²-Bis(ethyl)sperminet

Purified recombinant spermidine/spermine N¹-acetyltransferase (SSAT) was found to be unstable in the absence of polyamines, but the loss of activity could be prevented or reversed by the addition of the polyamine analog and potential antitumor agent N¹.N¹²-bislethyllspermine (BE-3-4-3), which is known to be a potent inducer of SSAT in mammalian cells. Addition of BE-3-4-3 prevented the loss of SSAT activity and the digestion of the protein by the proteases trypsin, Lys-C, or Glu-C. In the absence of BE-3-4-3, this digestion occurred at the sequence Lys¹⁴¹Arg¹⁴²Arg¹⁴³ for trypsin or Lys-C and at the sequence Glu¹⁵¹Glu¹⁵² for Glu-C. When these sites were altered by mutation to residues which are not substrates for these proteases, cleavage in the absence of BE-3-4-3 occurred at residues Lys¹⁶¹, Lys¹⁶⁶, and Glu¹⁶². These results indicate that the structure of SSAT contains a region that binds to the polyamine analog, BE-3-4-3, and that binding alters the configuration of the protein to prevent protease access to the region from amino acid residue 141 to the carboxyl terminal end (residue 171) of the SSAT. In order to determine the nature of the regulatory sites, specific mutations were made in the SSAT amino acid sequence, and the activity of the resulting SSAT protein and the sensitivity to proteases in the presence and absence of BE-3-4-3 was determined. The results indicate that the carboxyl terminal domain, MATEE, is critical for activity and for protection by BE-3-4-3. Deletion of the five amino acids at the carboxyl terminal increased the apparent K_m for spermidine by 40-fold, reduced the maximal activity by 90%, and abolished protection by BE-3-4-3. Residue Glu¹⁵² was also important in the activity and protection by BE-3-4-3 since its conversion to Gin or Lys removed the response to BE-3-4-3 and increased the K_m by 4- or 9-fold, respectively. These results suggest that BE-3-4-3 binds to SSAT via an interaction with Glu¹⁵² and the MATEE sequence at residues 167-171 and that this binding leads to a conformational change that increases the affinity for the polyamine substrate and protects the protein from protease digestion. This effect may play a major role in the increased SSAT activity in cells treated with BE-3-4-3 and related compounds.