Purification and characterization of propylamine transferase from Sulfolobus solfataricus, an extreme thermophilic archaebacterium

The enzyme propylamine transferase, catalyzing the transfer of the propylamine moiety from S-adenosyl(5′)-3-methylthiopropylamine to several amine acceptors, has been purified 643-fold in 20% yield from Sulfolobus solfataricus, an extreme thermophilic archaebacterium optimally growing at 87°C. The purified enzyme (specific activity 2.05 units/mg protein), is homogeneous by criteria of gel electrophoresis, gel filtration, isoelectric focusing and ultracentrifugation analysis. The molecular mass of the native enzyme was estimated to be about 110 kDa by gel permeation and ultracentrifugation analysis. The protein migrates on SDS/polyacrylamide gel electrophoresis as a single band of 35 kDa, suggesting that the enzyme is a trimer composed by identical subunits. An optimum pH of 7.5 and an acidic isoelectric point of 5.3 have been calculated. The optimum temperature was 90°C and no loss of activity is observable even after exposure of the purified enzyme to 100°C for 1 h. No reducing agents are required for enzymatic activity. Substrate specificity towards the amine acceptors is rather broad in that 1,3-diaminopropane (Km= 1675 μM), putrescine (Km= 3850 μM), sym-norspermidine (Km= 954 μM) and spermidine (Km= 1539 μM) are recognized as substrates. Conversely S-adenosyl(5′)-3-methylthiopropylamine is the only propylamine donor (Km= 7.9 μM) and the deamination of the sulfonium compound prevents the recognition by the enzyme. The reaction is irreversible and initial-rate kinetic studies indicate that the propylamine transfer is operated through a sequential mechanism. 5′-Methylthioadenosine, a product of the reaction, acts as a powerful competitive inhibitor with a Ki of 3.7 μM. Enzyme-substrate binding sites have been investigated with the aid of several substrate analogs and products. Among the compounds assayed, 5′-methylthiotubercidin, S-adenosyl(5′)-3-thiopropylamine and S-adenosyl-3-thio-1,8-diaminooctane are the most active inhibitors.