Mechanism of propylamine-transfer reactions. Kinetic and inhibition studies on spermidine synthase from Escherichia coli.

Spermidine synthase (EC 2.5.1.16) purified from Escherichia coli has been subjected to a kinetic analysis including initial velocity and substrate analogs inhibition studies. Evidence is reported for a ping-pong mechanism, indicating that a propylaminated form of the enzyme is an obligatory intermediate in the reaction mechanism. S-Adenosyl(5')-3-methylthiopropylamine exerts a competitive substrate inhibition by combining with the improper stable enzyme form, while putrescine does not show any inhibitory effect. In order to investigate the substrate binding sites, new sulfonium-deaminated analogs of S-adenosyl(5')-3-methylthiopropylamine have been synthesized and assayed as substrates and as inhibitors of the reaction. The replacement of the amino group of adenine, or propylamine moiety of the sulfonium compound by the hydroxyl group, or both, resulted in a complete loss of activity as substrate. On the other hand, the deaminated analogs exert a competitive inhibition with respect to putrescine. On the basis of these results and in analogy with methyltransfer reactions, three recognition sites for S-adenosyl(5')-3-methylthiopropylamine on propylamine transfer enzymes are proposed.