The polyamines, putrescine, spermidine, and spermine, are major polybasic compounds in all living cells. These amines are important for many systems related to growth and differentiation. For many years we have been studying how these polyamines are synthesized, how their biosynthesis and degradation are regulated, their physiologic functions, how they act in vivo, and the structure of the various biosynthetic enzymes. For this purpose we have constructed null mutants in each of the biosynthetic steps in both Escherichia coli and in Saccharomyces cerevisiae, and have prepared overexpression systems for the biosynthetic enzymes. During the current year we have applied this background tpwards comparable studies on mutants of the fission yeast, S, pombe. In particular we have been interested in the effect of polyamine deprivation on cell-cycle progression. We have also continued our studies on the involvement of post-translational proteolysis in the in vivo regulation of ornithine decarboxylase (the first enzyme in the polyamine biosynthetic pathway), and have shown that a newly synthesized enzyme is induced to effect this regulation. We have also continued our studies on one of the enzymes in the biosynthetic pathway; namely, S-adenosylmethionine decarboxylase. The structure of S-adenosylmethionine decarboxylase is of particular interest since it is formed as a proenzyme and is post-translationally cleaved to form a pyruvoyl group that is essential for enzymatic activity. By mass spectrometric techniques (carried out by Drs. Sonja Hess and Lewis Pannell) we have shown an unexpected in vivo modification of the enzyme, resulting from a substitution on a specific cysteine residue by a three carbon moiety derived from S-adenosylmethionine. As part of these studies we have shown in in vitro experiments that the product of the reaction, """"""""decarboxylated S-adenosylmethionine"""""""", reacts covalently with the enzyme. We have also prepared numerous mutants by site-specific mutagenesis, and have used plasmids containing these mutants to study the structural requirements for processing of the proenzyme
