Mechanistic studies of the yeast polyamine oxidase Fms1: kinetic mechanism, substrate specificity, and pH dependence.

Polyamines are essential for the growth and function of normal cells. The common polyamines putrescine, spermidine, and spermine are widely distributed in biological systems, not only as free bases but also as alkylated or acylated conjugates with sugars, steroids, phospholipids, fatty acids, and peptides. Because polyamines interact with multiple macromolecules, they affect a variety of cellular processes, such as cell growth, differentiation, and death (1-3). As a result, the biosynthetic pathway for formation of polyamines has been a target for the development of anti-cancer drugs (4). In comparison, the enzymes involved in polyamine catabolism have received much less study (5). In mammalian cells, catabolism of spermine can be initiated by the action of one of two enzymes. Acetylation of spermine by spermidine/spermine N1-acetyltransferase forms N1-acetylspermine (Scheme 2). This can be exported from the cell or oxidized by the peroxisomal flavoprotein polyamine oxidase (PAO) to spermidine and N-acetyl-3-aminopropanal (6). A further round of acetylation and oxidation by the same enzymes yields putrescine and 3-aminopropanal. Alternatively, the inducible flavoenzyme spermine oxidase (SMO) can oxidize spermine directly to spermidine and 3-aminopropanal (7-9). Saccharomyces cerevisiae also contains a flavoenzyme, Fms1, capable of oxidizing spermine and N1-acetylspermine (Scheme 1) (10, 11). The 3-aminopropanal produced by Fms1 is oxidized further to β-alanine, a precursor of pantothenic acid (10, 11). Spermidine is required for formation of the modified amino acid hypusine in yeast eIF5A (12) and helps protect the yeast cell from oxidative stress (13). Plant cells also contain a flavoenzyme capable of oxidizing polyamines (14). In contrast to the animal enzymes, plant polyamine oxidases oxidize the endo CN bond, forming 1,3-diaminopropane and N-(3-aminopropyl)-4-aminobutanal from spermine. Scheme 1 Scheme 2 The mammalian enzymes PAO and SMO differ by 2-3 orders of magnitude in their relative preferences for N1-acetylspermine versus spermine (8, 15-17), in line with their roles in the cell. In addition, the pH optimum of PAO is more basic than that of SMO, consistent with the higher pH within peroxisomes than in the cytoplasm (18); the kcat/Km values for spermine and N1-acetylspermine as substrates for PAO have pH maxima of 10-10.5, while the kcat/Km value with spermine as substrate for SMO is highest at pH 8.3 (16, 17). This difference reflects the preferences of these two enzymes for different forms of the substrate. PAO is active on the singly-charged forms of polyamines, while SMO prefers polyamines with three positive charges. The structural basis for the differences is not known, since no structures are available of a mammalian PAO or SMO. Structures are available of Fms1 (19) and maize polyamine oxidase (20). These show that these two proteins have similar overall structures to the monoamine oxidase (MAO) family of flavoproteins; this family also includes MAO A and B, the lysine-specific demethylase LSD1, and L-amino acid oxidases. While the sequence identities between either mammalian enzyme and Fms1 or maize PAO are only about 20%, this is enough to assign both to the MAO structural family (21, 22). Since Fms1 catalyzes the same reaction as PAO and SMO, its structure serves as the best current model for understanding the structural basis for the different specificities of PAO and SMO. However, to date there has been little mechanistic study of Fms1 to guide interpretation of the relationship of structure to specificity. We describe here mechanistic studies of Fms1 using both steady-state and rapid-reaction kinetic methods and analysis of the effects of pH on the kinetic parameters. The results provide insight into the mechanism of amine oxidation by Fms1 and the basis for the differences in substrate specificity among Fms1, PAO, and SMO.