Effect of mutations at active site residues on the activity of ornithine decarboxylase and its inhibition by active site-directed irreversible inhibitors

C. S. Coleman, B. A. Stanley, A. E. Pegg

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Abstract

Mouse ornithine decarboxylase (ODC) and mutants changing residues thought to be involved at the active site were expressed in Escherichia coli, purified to homogeneity by affinity chromatography on a pyridoxamine 5'- phosphate-agarose affinity column, and tested for their kinetic properties and their inactivation by enzyme-activated irreversible inhibitors. All of the mutant enzymes were expressed at comparable levels to the wild type protein (2-4% of the total soluble protein), all bound to the affinity column, and there were only small differences in the apparent K(m) values for L-ornithine providing strong evidence that the mutations did not lead to any gross changes in the protein structure. The mutation K69A led to a change in the spectrum of the enzyme and a 550-fold decrease in the k(cat)/K(m) (specificity constant) value. These results are consistent with lysine 69 being the residue that forms a Schiff base with the pyridoxal 5'-phosphate co-factor. Mutation C70S did not greatly affect the activity despite its proximity to this lysine but increased the K(m) about 2-fold. In contrast, the mutation C360A greatly reduced the specificity constant (by 26-fold) despite a 2-fold decrease in the K(m), suggesting that this cysteine residue is critically involved at the active site. Although cysteine 360 is known to be the major site of binding of the inhibitor, α-difluoromethylornithine (DFMO), the C360A mutant was still sensitive to inhibition by this drug. However, the kinetics of inactivation were altered, the partition ratio was 10 times greater, and the labeled adduct formed by reaction with [5- 14C]DFMO was removed from the protein under some denaturing conditions. This adduct was found to occur at lysine 69. The K69A mutant was also sensitive to DFMO with a lower partition ratio than the wild type enzyme. These results indicate that inactivation of ODC by DFMO can occur via interaction with either of two separate residues that form essential parts of the active site. This renders it unlikely that resistant mutants will arise from changes in the enzyme structure. In contrast to the results with DFMO, the C360A mutant ODC was completely resistant to inactivation by (R,R)-δ- methyl-α-acetylenicputrescine and was much less sensitive than the wild type enzyme to α-monofluoromethyldehydromethylornithine, showing that the reactive species formed from these inhibitors either cannot be formed by this mutant or are unable to react with lysine 69. Finally, the well known, extreme reliance of mammalian ODC on the presence of thiol-reducing agents to maintain activity is probably explained by the critical role of cysteine 360, since the C360A mutant was much less sensitive to inactivation by incubation in the absence of dithiothreitol.

Original languageEnglish (US)
Pages (from-to)24572-24579
Number of pages8
JournalJournal of Biological Chemistry
Volume268
Issue number33
StatePublished - Jan 1 1993

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Ornithine Decarboxylase
Eflornithine
Catalytic Domain
Mutation
Lysine
Enzymes
Cysteine
Proteins
Pyridoxamine
Affinity chromatography
Kinetics
Pyridoxal Phosphate
Ornithine
Schiff Bases
Dithiothreitol
Reducing Agents
Affinity Chromatography
Sulfhydryl Compounds
Sepharose
Escherichia coli

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

@article{ae85ee1fb11c4ad0abc6e522f2e84c2f,
title = "Effect of mutations at active site residues on the activity of ornithine decarboxylase and its inhibition by active site-directed irreversible inhibitors",
abstract = "Mouse ornithine decarboxylase (ODC) and mutants changing residues thought to be involved at the active site were expressed in Escherichia coli, purified to homogeneity by affinity chromatography on a pyridoxamine 5'- phosphate-agarose affinity column, and tested for their kinetic properties and their inactivation by enzyme-activated irreversible inhibitors. All of the mutant enzymes were expressed at comparable levels to the wild type protein (2-4{\%} of the total soluble protein), all bound to the affinity column, and there were only small differences in the apparent K(m) values for L-ornithine providing strong evidence that the mutations did not lead to any gross changes in the protein structure. The mutation K69A led to a change in the spectrum of the enzyme and a 550-fold decrease in the k(cat)/K(m) (specificity constant) value. These results are consistent with lysine 69 being the residue that forms a Schiff base with the pyridoxal 5'-phosphate co-factor. Mutation C70S did not greatly affect the activity despite its proximity to this lysine but increased the K(m) about 2-fold. In contrast, the mutation C360A greatly reduced the specificity constant (by 26-fold) despite a 2-fold decrease in the K(m), suggesting that this cysteine residue is critically involved at the active site. Although cysteine 360 is known to be the major site of binding of the inhibitor, α-difluoromethylornithine (DFMO), the C360A mutant was still sensitive to inhibition by this drug. However, the kinetics of inactivation were altered, the partition ratio was 10 times greater, and the labeled adduct formed by reaction with [5- 14C]DFMO was removed from the protein under some denaturing conditions. This adduct was found to occur at lysine 69. The K69A mutant was also sensitive to DFMO with a lower partition ratio than the wild type enzyme. These results indicate that inactivation of ODC by DFMO can occur via interaction with either of two separate residues that form essential parts of the active site. This renders it unlikely that resistant mutants will arise from changes in the enzyme structure. In contrast to the results with DFMO, the C360A mutant ODC was completely resistant to inactivation by (R,R)-δ- methyl-α-acetylenicputrescine and was much less sensitive than the wild type enzyme to α-monofluoromethyldehydromethylornithine, showing that the reactive species formed from these inhibitors either cannot be formed by this mutant or are unable to react with lysine 69. Finally, the well known, extreme reliance of mammalian ODC on the presence of thiol-reducing agents to maintain activity is probably explained by the critical role of cysteine 360, since the C360A mutant was much less sensitive to inactivation by incubation in the absence of dithiothreitol.",
author = "Coleman, {C. S.} and Stanley, {B. A.} and Pegg, {A. E.}",
year = "1993",
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pages = "24572--24579",
journal = "Journal of Biological Chemistry",
issn = "0021-9258",
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TY - JOUR

T1 - Effect of mutations at active site residues on the activity of ornithine decarboxylase and its inhibition by active site-directed irreversible inhibitors

AU - Coleman, C. S.

AU - Stanley, B. A.

AU - Pegg, A. E.

PY - 1993/1/1

Y1 - 1993/1/1

N2 - Mouse ornithine decarboxylase (ODC) and mutants changing residues thought to be involved at the active site were expressed in Escherichia coli, purified to homogeneity by affinity chromatography on a pyridoxamine 5'- phosphate-agarose affinity column, and tested for their kinetic properties and their inactivation by enzyme-activated irreversible inhibitors. All of the mutant enzymes were expressed at comparable levels to the wild type protein (2-4% of the total soluble protein), all bound to the affinity column, and there were only small differences in the apparent K(m) values for L-ornithine providing strong evidence that the mutations did not lead to any gross changes in the protein structure. The mutation K69A led to a change in the spectrum of the enzyme and a 550-fold decrease in the k(cat)/K(m) (specificity constant) value. These results are consistent with lysine 69 being the residue that forms a Schiff base with the pyridoxal 5'-phosphate co-factor. Mutation C70S did not greatly affect the activity despite its proximity to this lysine but increased the K(m) about 2-fold. In contrast, the mutation C360A greatly reduced the specificity constant (by 26-fold) despite a 2-fold decrease in the K(m), suggesting that this cysteine residue is critically involved at the active site. Although cysteine 360 is known to be the major site of binding of the inhibitor, α-difluoromethylornithine (DFMO), the C360A mutant was still sensitive to inhibition by this drug. However, the kinetics of inactivation were altered, the partition ratio was 10 times greater, and the labeled adduct formed by reaction with [5- 14C]DFMO was removed from the protein under some denaturing conditions. This adduct was found to occur at lysine 69. The K69A mutant was also sensitive to DFMO with a lower partition ratio than the wild type enzyme. These results indicate that inactivation of ODC by DFMO can occur via interaction with either of two separate residues that form essential parts of the active site. This renders it unlikely that resistant mutants will arise from changes in the enzyme structure. In contrast to the results with DFMO, the C360A mutant ODC was completely resistant to inactivation by (R,R)-δ- methyl-α-acetylenicputrescine and was much less sensitive than the wild type enzyme to α-monofluoromethyldehydromethylornithine, showing that the reactive species formed from these inhibitors either cannot be formed by this mutant or are unable to react with lysine 69. Finally, the well known, extreme reliance of mammalian ODC on the presence of thiol-reducing agents to maintain activity is probably explained by the critical role of cysteine 360, since the C360A mutant was much less sensitive to inactivation by incubation in the absence of dithiothreitol.

AB - Mouse ornithine decarboxylase (ODC) and mutants changing residues thought to be involved at the active site were expressed in Escherichia coli, purified to homogeneity by affinity chromatography on a pyridoxamine 5'- phosphate-agarose affinity column, and tested for their kinetic properties and their inactivation by enzyme-activated irreversible inhibitors. All of the mutant enzymes were expressed at comparable levels to the wild type protein (2-4% of the total soluble protein), all bound to the affinity column, and there were only small differences in the apparent K(m) values for L-ornithine providing strong evidence that the mutations did not lead to any gross changes in the protein structure. The mutation K69A led to a change in the spectrum of the enzyme and a 550-fold decrease in the k(cat)/K(m) (specificity constant) value. These results are consistent with lysine 69 being the residue that forms a Schiff base with the pyridoxal 5'-phosphate co-factor. Mutation C70S did not greatly affect the activity despite its proximity to this lysine but increased the K(m) about 2-fold. In contrast, the mutation C360A greatly reduced the specificity constant (by 26-fold) despite a 2-fold decrease in the K(m), suggesting that this cysteine residue is critically involved at the active site. Although cysteine 360 is known to be the major site of binding of the inhibitor, α-difluoromethylornithine (DFMO), the C360A mutant was still sensitive to inhibition by this drug. However, the kinetics of inactivation were altered, the partition ratio was 10 times greater, and the labeled adduct formed by reaction with [5- 14C]DFMO was removed from the protein under some denaturing conditions. This adduct was found to occur at lysine 69. The K69A mutant was also sensitive to DFMO with a lower partition ratio than the wild type enzyme. These results indicate that inactivation of ODC by DFMO can occur via interaction with either of two separate residues that form essential parts of the active site. This renders it unlikely that resistant mutants will arise from changes in the enzyme structure. In contrast to the results with DFMO, the C360A mutant ODC was completely resistant to inactivation by (R,R)-δ- methyl-α-acetylenicputrescine and was much less sensitive than the wild type enzyme to α-monofluoromethyldehydromethylornithine, showing that the reactive species formed from these inhibitors either cannot be formed by this mutant or are unable to react with lysine 69. Finally, the well known, extreme reliance of mammalian ODC on the presence of thiol-reducing agents to maintain activity is probably explained by the critical role of cysteine 360, since the C360A mutant was much less sensitive to inactivation by incubation in the absence of dithiothreitol.

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