Structure and critical residues at the active site of spermidine/spermine-N1-acetyltransferase

Catherine Coleman, Huatao Huang, Anthony Pegg

Research output: Contribution to journalArticle

48 Citations (Scopus)

Abstract

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 K(m) 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.

Original languageEnglish (US)
Pages (from-to)697-701
Number of pages5
JournalBiochemical Journal
Volume316
Issue number3
DOIs
StatePublished - Jun 15 1996

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Acetyltransferases
Spermidine
Spermine
Catalytic Domain
Acetyl Coenzyme A
Polyamines
Alanine
Arginine
Mutagenesis
COS Cells
Enzymes
diamine N-acetyltransferase
Dimers
Trypsin
Proteolysis
Restoration
Transfection
Proteins
Plasmids
Scanning

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

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title = "Structure and critical residues at the active site of spermidine/spermine-N1-acetyltransferase",
abstract = "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 K(m) 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.",
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Structure and critical residues at the active site of spermidine/spermine-N1-acetyltransferase. / Coleman, Catherine; Huang, Huatao; Pegg, Anthony.

In: Biochemical Journal, Vol. 316, No. 3, 15.06.1996, p. 697-701.

Research output: Contribution to journalArticle

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