Abstract
Polyamine oxidases are peroxisomal flavoproteins that catalyze the oxidation of an endo carbon nitrogen bond of N1-acetylspermine in the catabolism of polyamines. While no structure has been reported for a mammalian polyamine oxidase, sequence alignments of polyamine oxidizing flavoproteins identify a conserved histidine residue. Based on the structure of a yeast polyamine oxidase, Saccharomyces cerevisiae Fms1, this residue has been proposed to hydrogen bond to the reactive nitrogen in the polyamine substrate. The corresponding histidine in mouse polyamine oxidase, His64, has been mutated to glutamine, asparagine, and alanine to determine if this residue plays a similar role in the mammalian enzymes. The kinetics of the mutant enzymes were examined with N1-acetylspermine and the slow substrates spermine and N,N′-dibenzyl- 1,4-diaminobutane. On average the mutations result in a decrease of ∼15-fold in the rate constant for amine oxidation. Rapid-reaction kinetic analyses established that amine oxidation is rate-limiting with spermine as substrate for the wild-type and mutant enzymes and for the H64N enzyme with N1-acetylspermine as substrate. The kcat/KO 2 value was unaffected by the mutations with N1-acetylspermine as substrate, but decreased ∼55-fold with the two slower substrates. The results are consistent with this residue assisting in properly positioning the amine substrate for oxidation.
| Original language | English (US) |
|---|---|
| Pages (from-to) | 45-49 |
| Number of pages | 5 |
| Journal | Archives of Biochemistry and Biophysics |
| Volume | 528 |
| Issue number | 1 |
| DOIs | |
| State | Published - Dec 1 2012 |
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Keywords
- Enzyme kinetics
- Flavoenzymes
- Mutagenesis
- Oxidase
- Polyamine
ASJC Scopus subject areas
- Biochemistry
- Biophysics
- Molecular Biology
Cite this
Mechanistic studies of the role of a conserved histidine in a mammalian polyamine oxidase. / Tormos, José R.; Henderson Pozzi, Michelle; Fitzpatrick, Paul F.
In: Archives of Biochemistry and Biophysics, Vol. 528, No. 1, 01.12.2012, p. 45-49.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Mechanistic studies of the role of a conserved histidine in a mammalian polyamine oxidase
AU - Tormos, José R.
AU - Henderson Pozzi, Michelle
AU - Fitzpatrick, Paul F
PY - 2012/12/1
Y1 - 2012/12/1
N2 - Polyamine oxidases are peroxisomal flavoproteins that catalyze the oxidation of an endo carbon nitrogen bond of N1-acetylspermine in the catabolism of polyamines. While no structure has been reported for a mammalian polyamine oxidase, sequence alignments of polyamine oxidizing flavoproteins identify a conserved histidine residue. Based on the structure of a yeast polyamine oxidase, Saccharomyces cerevisiae Fms1, this residue has been proposed to hydrogen bond to the reactive nitrogen in the polyamine substrate. The corresponding histidine in mouse polyamine oxidase, His64, has been mutated to glutamine, asparagine, and alanine to determine if this residue plays a similar role in the mammalian enzymes. The kinetics of the mutant enzymes were examined with N1-acetylspermine and the slow substrates spermine and N,N′-dibenzyl- 1,4-diaminobutane. On average the mutations result in a decrease of ∼15-fold in the rate constant for amine oxidation. Rapid-reaction kinetic analyses established that amine oxidation is rate-limiting with spermine as substrate for the wild-type and mutant enzymes and for the H64N enzyme with N1-acetylspermine as substrate. The kcat/KO 2 value was unaffected by the mutations with N1-acetylspermine as substrate, but decreased ∼55-fold with the two slower substrates. The results are consistent with this residue assisting in properly positioning the amine substrate for oxidation.
AB - Polyamine oxidases are peroxisomal flavoproteins that catalyze the oxidation of an endo carbon nitrogen bond of N1-acetylspermine in the catabolism of polyamines. While no structure has been reported for a mammalian polyamine oxidase, sequence alignments of polyamine oxidizing flavoproteins identify a conserved histidine residue. Based on the structure of a yeast polyamine oxidase, Saccharomyces cerevisiae Fms1, this residue has been proposed to hydrogen bond to the reactive nitrogen in the polyamine substrate. The corresponding histidine in mouse polyamine oxidase, His64, has been mutated to glutamine, asparagine, and alanine to determine if this residue plays a similar role in the mammalian enzymes. The kinetics of the mutant enzymes were examined with N1-acetylspermine and the slow substrates spermine and N,N′-dibenzyl- 1,4-diaminobutane. On average the mutations result in a decrease of ∼15-fold in the rate constant for amine oxidation. Rapid-reaction kinetic analyses established that amine oxidation is rate-limiting with spermine as substrate for the wild-type and mutant enzymes and for the H64N enzyme with N1-acetylspermine as substrate. The kcat/KO 2 value was unaffected by the mutations with N1-acetylspermine as substrate, but decreased ∼55-fold with the two slower substrates. The results are consistent with this residue assisting in properly positioning the amine substrate for oxidation.
KW - Enzyme kinetics
KW - Flavoenzymes
KW - Mutagenesis
KW - Oxidase
KW - Polyamine
UR - http://www.scopus.com/inward/record.url?scp=84866518900&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=84866518900&partnerID=8YFLogxK
U2 - 10.1016/j.abb.2012.08.007
DO - 10.1016/j.abb.2012.08.007
M3 - Article
C2 - 22959971
AN - SCOPUS:84866518900
VL - 528
SP - 45
EP - 49
JO - Archives of Biochemistry and Biophysics
JF - Archives of Biochemistry and Biophysics
SN - 0003-9861
IS - 1
ER -