Studies of the acetyl-CoA-binding site of rat liver spermidine/spermine N1-acetyltransferase.
Rat liver spermidine/spermine N1-acetyltransferase was found to be strongly inhibited by the dyes Cibacron F3GA, Coomassie Brilliant Blue and Congo Red. Inhibition was competitive with respect to acetyl-CoA and Ki values of 0.7 microM and 52 microM were determined for Cibacron F3GA and Coomassie Brilliant Blue respectively. The enzyme was strongly retained by columns of Affi-Gel Blue, which contains Cibacron F3GA linked to agarose. It was not eluted from this adsorbent in the presence of 10 mM-spermidine/0.5 M-NaCl/50 mM-Tris/HCl, pH 7.5, but was released by 1 mM-CoA in 10 mM-spermidine/50 mM-Tris/HCl, pH 7.5. These results are consistent with the presence in the enzyme of a dinucleotide fold that binds acetyl CoA and has a high affinity for Cibacron F3GA. The spermidine/spermine N1-acetyltransferase was irreversibly inactivated by exposure to butane-2,3-dione in sodium borate, pH 7.8, or by exposure to phenylglyoxal or camphorquinone-10-sulphonic acid. All of these reagents are known to interact with arginine residues in proteins under the conditions in which they inactivated the acetyltransferase. Inactivation was prevented by the presence of acetyl-CoA or CoA, but to a lesser extent by 3'-dephospho-CoA and not at all by NAD or adenosine. This protection suggests that an arginine residue at the active site is involved in the binding of the acetyl-CoA substrate. Treatment of the assay mixture but not the spermidine N1-acetyltransferase with alkaline phosphatase prevented the reaction taking place. This suggests that the apparent loss of enzyme activity in response to alkaline phosphatase reported by Matsui, Otani, Kamei & Morisawa [(1982) FEBS Lett. 150, 211-213] is due to dephosphorylation of the acetyl-CoA substrate and that the 3'-phosphate group is essential for activity.
D000105:Acetyl Coenzyme A / Q000378:metabolism*; D000107:Acetylation; D000123:Acetyltransferases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D000469:Alkaline Phosphatase / Q000494:pharmacology; D000818:Animals; D001665:Binding Sites; D004396:Coloring Agents / Q000494:pharmacology; D008099:Liver / Q000201:enzymology*; D009705:Nucleosides / Q000494:pharmacology; D010658:Phenylglyoxal / Q000494:pharmacology; D051381:Rats; D014227:Triazines / Q000494:pharmacology; D000099228:Diamine N-Acetyltransferase
D000105:Acetyl Coenzyme A; D000123:Acetyltransferases; D000469:Alkaline Phosphatase; D004396:Coloring Agents; D009705:Nucleosides; D010658:Phenylglyoxal; D014227:Triazines; D000099228:Diamine N-Acetyltransferase; C012842:Cibacron Blue F 3GA