Mechanistic studies of the yeast polyamine oxidase Fms1

Kinetic mechanism, substrate specificity, and pH dependence

Mariya S. Adachi, Jason M. Torres, Paul F Fitzpatrick

Research output: Contribution to journalArticle

11 Citations (Scopus)

Abstract

The flavoprotein oxidase Fms1 from Saccharomyces cerevisiae catalyzes the oxidation of spermine and N1-acetylspermine to yield spermidine and 3-aminopropanal or N-acetyl-3-aminopropanal. The kinetic mechanism of the enzyme has been determined with both substrates. The initial velocity patterns are ping-pong, consistent with reduction being kinetically irreversible. Reduction of Fms1 by either substrate is biphasic. The rate constant for the rapid phase varies with the substrate concentration, with limiting rates for reduction of the enzyme of 126 and 1410 s-1 and apparent Kd values of 24.3 and 484 μM for spermine and N1-acetylspermine, respectively. The rapid phase is followed by a concentration-independent phase that is slower than turnover. The reaction of the reduced enzyme with oxygen is monophasic, with a rate constant of 402 mM-1 s-1 with spermine at 25 °C and 204 mM-1 s-1 with N1-acetylspermine at 4 °C and pH 9.0. This step is followed by rate-limiting product dissociation. The kcat/Kamine-pH profiles are bell-shaped, with an average pKa value of 9.3 with spermine and pKa values of 8.3 and 9.6 with N1-acetylspermine. Both profiles are consistent with the active forms of substrates having two charged nitrogens. The pH profiles for the rate constant for flavin reduction show pKa values of 8.3 and 7.2 for spermine and N1-acetylspermine, respectively, for groups that must be unprotonated; these pKa values are assigned to the substrate N4. The kcat/K O2-pH profiles show pKa values of 7.5 for spermine and 6.8 for N 1-acetylspermine. With both substrates, the kcat value decreases when a single residue is protonated.

Original languageEnglish (US)
Pages (from-to)10440-10448
Number of pages9
JournalBiochemistry
Volume49
Issue number49
DOIs
StatePublished - Dec 14 2010

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Spermine
Substrate Specificity
Yeast
Yeasts
Kinetics
Substrates
Rate constants
Enzymes
Flavoproteins
Spermidine
Saccharomyces cerevisiae
polyamine oxidase
Oxidoreductases
Nitrogen
Oxygen
Oxidation

ASJC Scopus subject areas

  • Biochemistry

Cite this

Mechanistic studies of the yeast polyamine oxidase Fms1 : Kinetic mechanism, substrate specificity, and pH dependence. / Adachi, Mariya S.; Torres, Jason M.; Fitzpatrick, Paul F.

In: Biochemistry, Vol. 49, No. 49, 14.12.2010, p. 10440-10448.

Research output: Contribution to journalArticle

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abstract = "The flavoprotein oxidase Fms1 from Saccharomyces cerevisiae catalyzes the oxidation of spermine and N1-acetylspermine to yield spermidine and 3-aminopropanal or N-acetyl-3-aminopropanal. The kinetic mechanism of the enzyme has been determined with both substrates. The initial velocity patterns are ping-pong, consistent with reduction being kinetically irreversible. Reduction of Fms1 by either substrate is biphasic. The rate constant for the rapid phase varies with the substrate concentration, with limiting rates for reduction of the enzyme of 126 and 1410 s-1 and apparent Kd values of 24.3 and 484 μM for spermine and N1-acetylspermine, respectively. The rapid phase is followed by a concentration-independent phase that is slower than turnover. The reaction of the reduced enzyme with oxygen is monophasic, with a rate constant of 402 mM-1 s-1 with spermine at 25 °C and 204 mM-1 s-1 with N1-acetylspermine at 4 °C and pH 9.0. This step is followed by rate-limiting product dissociation. The kcat/Kamine-pH profiles are bell-shaped, with an average pKa value of 9.3 with spermine and pKa values of 8.3 and 9.6 with N1-acetylspermine. Both profiles are consistent with the active forms of substrates having two charged nitrogens. The pH profiles for the rate constant for flavin reduction show pKa values of 8.3 and 7.2 for spermine and N1-acetylspermine, respectively, for groups that must be unprotonated; these pKa values are assigned to the substrate N4. The kcat/K O2-pH profiles show pKa values of 7.5 for spermine and 6.8 for N 1-acetylspermine. With both substrates, the kcat value decreases when a single residue is protonated.",
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