Role of the carboxyl terminal MATEE sequence of spermidine/spermine N1-acetyltransferase in the activity and stabilization by the polyamine analog N1,N12-bis(ethyl)spermine.
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-bis(ethyl)spermine (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.(ABSTRACT TRUNCATED AT 250 WORDS)
Coleman
C S
CS
Department of Cellular and Molecular Physiology, Milton S. Hershey Medical Center, Pennsylvania State University College of Medicine, Hershey 17033, USA.
Huang
H
H
Pegg
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AE
D000123:Acetyltransferases / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D000970:Antineoplastic Agents / Q000494:pharmacology; D001665:Binding Sites; D003001:Cloning, Molecular; D004926:Escherichia coli; D006801:Humans; D007700:Kinetics; D008322:Mammals; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D013096:Spermine / Q000031:analogs & derivatives* / Q000494:pharmacology; D000099228:Diamine N-Acetyltransferase
D000123:Acetyltransferases; D000970:Antineoplastic Agents; D011994:Recombinant Proteins; D013096:Spermine; D000099228:Diamine N-Acetyltransferase; C052614:N(1), N(12)-diethylspermine