Structure and critical residues at the active site of spermidine/spermine-N-1-acetyltransferase

Spermidine/spermine-N-1-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 Arg(101), Arg(142) and Arg(143) 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 R1O1A (Arg(101) --> Ala) mutant and an E152K (Glu(152) --> 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 N-1,N-12-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.