Structure and critical residues at the active site of spermidine/spermine-N1-acetyltransferase.
Spermidine/spermine-N1-acetyltransferase (SSAT) is a key enzyme in the degradation of polyamines. Alanine-scanning mutagenesis of all eight arginine residues was used to investigate the arginine residues involved in acetyl-CoA binding. The results indicate that Arg101, Arg142 and Arg143 are important for such binding. The apparent Km values for acetyl-CoA were significantly increased when any one of these residues was replaced by an alanine residue. These mutations also abolished the ability of acetyl-CoA to protect the protein from digestion by trypsin. Co-expression of the inactive R101A (Arg101 --> Ala) mutant and an E152K (Glu152 --> Lys) mutant, previously known to inactivate SSAT, led to restoration of activity, showing that the active enzyme is a dimer with residues contributed by both subunits. The double mutant R101A/E152K acted as a dominant negative when co-expressed with the wild-type SSAT. Transfection of COS-7 cells with a plasmid producing this mutant greatly attenuated the increase in SSAT activity brought about by N1, N12-bis(ethyl)spermine. These results indicate that the double mutant R101A/E152K-SSAT protein can be used to evaluate the importance of SSAT activity in response to exogenous polyamines or polyamine analogues.
Coleman
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Department of Cellular and Molecular Physiology, Milton S. Hershey Medical Center, Pennsylvania State University College of Medicine, Hershey 17033, USA.
Huang
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Pegg
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AE
D000105:Acetyl Coenzyme A / Q000378:metabolism; D000123:Acetyltransferases / Q000096:biosynthesis / Q000737:chemistry* / Q000378:metabolism*; D000409:Alanine; D000595:Amino Acid Sequence; D000818:Animals; D001120:Arginine; D001665:Binding Sites; D002522:Chlorocebus aethiops; D018698:Glutamic Acid; D007700:Kinetics; D008239:Lysine; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010957:Plasmids; D017354:Point Mutation; D014176:Protein Biosynthesis; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D014162:Transfection; D000099228:Diamine N-Acetyltransferase
D000105:Acetyl Coenzyme A; D000123:Acetyltransferases; D000409:Alanine; D001120:Arginine; D018698:Glutamic Acid; D008239:Lysine; D011994:Recombinant Proteins; D000099228:Diamine N-Acetyltransferase