Mechanistic and Structural Analysis of Human Spermidine/Spermine N¹-Acetyltransferase^,

Abstract

The N1-acetylation of spermidine and spermine by spermidine/spermine acetyltransferase (SSAT) is a crucial step in the regulation of the cellular polyamine levels in eukaryotic cells. Altered polyamine levels are associated with a variety of cancers as well as other diseases, and key enzymes in the polyamine pathway, including SSAT, are being explored as potential therapeutic drug targets. We have expressed and purified human SSAT in Escherichia coli and characterized its kinetic and chemical mechanism. Initial velocity and inhibition studies support a random sequential mechanism for the enzyme. The bisubstrate analogue, N1-spermine-acetyl-coenzyme A, exhibited linear, competitive inhibition against both substrates with a true Ki of 6 nM. The pH-activity profile was bell-shaped, depending on the ionization state of two groups exhibiting apparent pKa values of 7.27 and 8.87. The three-dimensional crystal structure of SSAT with bound bisubstrate inhibitor was determined at 2.3 A resolution. The structure of the SSAT-spermine-acetyl-coenzyme A complex suggested that Tyr140 acts as general acid and Glu92, through one or more water molecules, acts as the general base during catalysis. On the basis of kinetic properties, pH dependence, and structural information, we propose an acid/base-assisted reaction catalyzed by SSAT, involving a ternary complex.

Complex N-Acetylation of Triethylenetetramine

“…The acetylation rate of N 1 AcTETA was approximately 10% at 10 mM compared with TETA at 10 mM using the spectrophotometric method (Coleman et al, 2004) (data not shown). Furthermore, we determined the kinetic values for acetylation of TETA using hSSAT1 to compare the properties of the human and mouse recombinant enzymes that show high structural homology (Hegde et al, 2007;Montemayor and Hoffman, 2008 (Weisell et al, 2010).…”

Paramecium bursaria Chlorella Virus 1 Encodes a Polyamine Acetyltransferase

“…Therefore, we propose the reaction mechanism (Fig. 2e), which resembles the described catalytic mechanism for lysine acetyltransferases (19).…”

“…In mammalian cells, polyamine acetylation is the first ratelimiting step in polyamine catabolism, and the human acetyltransferase, spermidine/spermine N 1 -acetyltransferase, is highly regulated due to the association of polyamine abundance with cellular proliferation (19,20,28). Through our newly determined crystal structure of vPAT and characterization of its catalytic activity as a polyamine acetyltransferase, we describe for the first time a virally encoded polyamine acetyltransferase with a catalytic efficiency (K m ϭ 21 M and k cat ϭ 131 min…”

“…acetyltransferase spermidine/spermine N 1 -acetyltransferase (K m ϭ 5.7 M and k cat ϭ 155 min Ϫ1 ) (10) and more efficient than that of a bacterial spermine acetyltransferase PaiA (K m ϭ 76 M and k cat ϭ 19 min Ϫ1 ) (19,32). Therefore, our study suggests a new basic molecular mechanism by which a eukaryotic virus depletes polyamines in the host, presumably for its own advantage for replication.…”

Catalytic synthesis of 1,3-propylenediamines

“…[1][2][3]. Recent research showed that the polyamine derivatives based on spermidine and spermine exhibit high activity against Alzheimer's disease, cystic fi brosis, and also of various tumors and cancer cells [4][5][6][7]. 1,3-Propylenediamine fragment are included into the structure of many drugs used in the treatment of the central nervous system [e.g., antidepressant imipramine (tofranil)] [3].…”