Mechanistic Studies of Mouse Polyamine Oxidase with N1,N12-Bisethylspermine as a Substrate

Abstract

In mammalian cells, the flavoprotein polyamine oxidase catalyzes a key step in the catabolism of polyamines, the oxidation of N1-acetylspermine and N1-acetylspermidine to spermidine and putrescine, respectively. The mechanism of the mouse enzyme has been studied with N1,N12-bisethylspermine (BESPM) as a substrate. At pH 10, the pH optimum, the limiting rate of reduction of the flavin in the absence of oxygen is comparable to the k cat value for turnover, establishing reduction as rate-limiting. Oxidation of the reduced enzyme is a simple second-order reaction. No intermediates are seen in the reductive or oxidative half-reactions. The k cat value decreases below a pK a of 9.0. The k cat /K m value for BESPM exhibits a bell-shaped pH profile, with pK a values of 9.8 and 10.8. These pK a values are assigned to the substrate nitrogens. The rate constant for the reaction of the reduced enzyme with oxygen is not affected by a pH between 7.5 and 10. Active site residue Tyr430 is conserved in the homologous protein monoamine oxidase. Mutation of this residue to phenylalanine results in a 6-fold decrease in the k cat value and the k cat /K m value for oxygen due to a comparable decrease in the rate constant for flavin reduction. This moderate change is not consistent with this residue forming a tyrosyl radical during catalysis.The polyamines spermine, spermidine, and putrescine are ubiquitous in cells. Higher concentrations are found in rapidly growing cells (1-3), and compounds which deplete polyamines from cells inhibit cell growth (2). These observations have led to the general conclusion that polyamines are essential for cell growth, although their specific role in the cell is still a matter of discussion. Consequently, a variety of polyamine analogues have been examined as anticancer drugs (4-7); a number of clinical trials are underway, and analogues with cytotoxic potential have been developed (8,9). The metabolic pathways for synthesis and degradation of polyamines are generally conserved (1). In mammals, the biosynthetic pathway involves decarboxylation of ornithine to putrescine by ornithine decarboxylase, extension of putrescine to spermidine by spermidine synthase using decarboxylated S-adenosylmethionine as the propylamine donor, and a subsequent extension of spermidine with another propylamine moiety to form spermine catalyzed by the enzyme spermine synthase. Depletion of spermine and spermidine from the cell involves the action of two enzymes: spermidine/spermine N1-acetyltransferase converts spermine and spermidine to the respective N1-acetylated compound, and polyamine oxidase oxidizes N1-acetylspermidine and N1-acetylspermine to putrescine and spermidine, respectively, and 3-acetamidopropanal (Scheme 1). While polyamine oxidase can also oxidize spermine, the enzyme strongly prefers N1-acetylated polyamines as substrates (10). Instead, the related enzyme spermine oxidase is responsible for direct oxidation of † This work was supported in part by grants from the NIH (R01 GM58698) and The Welch Fo...

Mechanism of the Flavoprotein l-Hydroxynicotine Oxidase: Kinetic Mechanism, Substrate Specificity, Reaction Product, and Roles of Active-Site Residues

“…24 In mouse polyamine oxidase the decrease is ~3-fold. 25 In other members of the family this residue is replaced with phenylalanine, tryptophan or nonaromatic residues in the wild-type enzyme. This variability and the frequent observation of small effects when the tyrosine is mutated to phenylalanine suggests that the major role of this residue in this enzyme family is to maintain the active site shape, with the size of the residue being the most important feature.…”

13C kinetic isotope effects on the reaction of a flavin amine oxidase determined from whole molecule isotope effects

“…PAO was expressed in E. coli and purified as previously reported [19]. The enzyme concentration was determined using a ε 458 value of 10,400 M −1 cm −1 .…”

Mechanistic studies of the role of a conserved histidine in a mammalian polyamine oxidase

“…The H64A, H64N and H64Q mutations were introduced into the pET28b-based expression vector for mouse PAO [18] using the QuikChange site-directed mutagenesis kit (Stratagene) and the mutagenic primers 5′-CATTGGATC GCT GGTCCAGCCAGGACAACCCAG-3′, 5′-GGGCGCGCATTGGATC CAG GGTCCAAGCC-3′ and 5′-GCGCGCATTGGATC AAT GGTCCAAGCCAG-3′, for the H64A, H64Q and H64N mutations (in bold), respectively. The DNA sequence of the entire gene was determined to ensure that no unwanted mutations occurred.…”

“…The DNA sequence of the entire gene was determined to ensure that no unwanted mutations occurred. All enzymes were purified using the protocol developed for the wild -type enzyme [18] and stored with 10% glycerol at −80 °C. Enzyme concentrations were determined using an ε 458 value of 10,400 M −1 cm −1 .…”