Amino acid residues necessary for putrescine stimulation of human S-adenosylmethionine decarboxylase proenzyme processing and catalytic activity

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Abstract

Human S-adenosylmethionine (AdoMet) decarboxylase is synthesized as a 38-kDa proenzyme that is autocatalytically cleaved, forming the covalently attached pyruvate cofactor and the two subunits (67 and 267 amino acid residues) of the mature enzyme. Both the cleavage reaction and the catalytic activity of the mature enzyme are stimulated by putrescine. Using site-specific mutagenesis and in vitro transcription followed by translation of the resultant RNA in a cell-free system, we have examined the importance of several amino acid residues on the processing reaction rate and catalytic activity and on stimulation of each of these by putrescine. Changing Cys82 to Ala decreased the stimulatory effect of putrescine on processing and completely eliminated catalytic activity, indicating a probable role in the active site of the enzyme, whereas changing Cys49 or Cys226 to Ala had minimal effects on processing and activity or the putrescine stimulation of either. Since Cys49 is the only cysteine residue in the smaller subunit, this indicates that disulfide-bond formation between the two subunits cannot be necessary for maintenance of the conformation for proenzyme processing or catalytic activity. Changing Glu8 or Glu11 to Gln produced an AdoMet decarboxylase that processed, but was catalytically inactive. Mutation at Glu11 also completely eliminated the putrescine stimulation of proenzyme processing, as did double mutation at Glu8 and Glu11, whereas the single mutation at Glu8 had no effect on the putrescine stimulation of proenzyme processing. Changing Glu15, Glu61, Glu67, Glu247, or Glu247 and Glu249 to Gln had a minimal effect on processing and activity or putrescine stimulation of either. These results demonstrate a role for the smaller subunit in the catalytic activity of the mature AdoMet decarboxylase enzyme and show that Glu8, Glu11, and Cys82 are essential for catalytic activity, with Glu11 also being essential for the putrescine stimulation of AdoMet decarboxylase proenzyme processing.

Original languageEnglish (US)
Pages (from-to)18502-18506
Number of pages5
JournalJournal of Biological Chemistry
Volume266
Issue number28
StatePublished - Nov 22 1991

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Adenosylmethionine Decarboxylase
Enzyme Precursors
Putrescine
Catalyst activity
Amino Acids
Processing
Enzymes
Mutation
Catalytic Domain
Mutagenesis
Cell-Free System
Transcription
Site-Directed Mutagenesis
Pyruvic Acid
Disulfides
Reaction rates
Cysteine
Conformations
Maintenance
RNA

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

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title = "Amino acid residues necessary for putrescine stimulation of human S-adenosylmethionine decarboxylase proenzyme processing and catalytic activity",
abstract = "Human S-adenosylmethionine (AdoMet) decarboxylase is synthesized as a 38-kDa proenzyme that is autocatalytically cleaved, forming the covalently attached pyruvate cofactor and the two subunits (67 and 267 amino acid residues) of the mature enzyme. Both the cleavage reaction and the catalytic activity of the mature enzyme are stimulated by putrescine. Using site-specific mutagenesis and in vitro transcription followed by translation of the resultant RNA in a cell-free system, we have examined the importance of several amino acid residues on the processing reaction rate and catalytic activity and on stimulation of each of these by putrescine. Changing Cys82 to Ala decreased the stimulatory effect of putrescine on processing and completely eliminated catalytic activity, indicating a probable role in the active site of the enzyme, whereas changing Cys49 or Cys226 to Ala had minimal effects on processing and activity or the putrescine stimulation of either. Since Cys49 is the only cysteine residue in the smaller subunit, this indicates that disulfide-bond formation between the two subunits cannot be necessary for maintenance of the conformation for proenzyme processing or catalytic activity. Changing Glu8 or Glu11 to Gln produced an AdoMet decarboxylase that processed, but was catalytically inactive. Mutation at Glu11 also completely eliminated the putrescine stimulation of proenzyme processing, as did double mutation at Glu8 and Glu11, whereas the single mutation at Glu8 had no effect on the putrescine stimulation of proenzyme processing. Changing Glu15, Glu61, Glu67, Glu247, or Glu247 and Glu249 to Gln had a minimal effect on processing and activity or putrescine stimulation of either. These results demonstrate a role for the smaller subunit in the catalytic activity of the mature AdoMet decarboxylase enzyme and show that Glu8, Glu11, and Cys82 are essential for catalytic activity, with Glu11 also being essential for the putrescine stimulation of AdoMet decarboxylase proenzyme processing.",
author = "Bruce Stanley and Anthony Pegg",
year = "1991",
month = "11",
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language = "English (US)",
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journal = "Journal of Biological Chemistry",
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T1 - Amino acid residues necessary for putrescine stimulation of human S-adenosylmethionine decarboxylase proenzyme processing and catalytic activity

AU - Stanley, Bruce

AU - Pegg, Anthony

PY - 1991/11/22

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N2 - Human S-adenosylmethionine (AdoMet) decarboxylase is synthesized as a 38-kDa proenzyme that is autocatalytically cleaved, forming the covalently attached pyruvate cofactor and the two subunits (67 and 267 amino acid residues) of the mature enzyme. Both the cleavage reaction and the catalytic activity of the mature enzyme are stimulated by putrescine. Using site-specific mutagenesis and in vitro transcription followed by translation of the resultant RNA in a cell-free system, we have examined the importance of several amino acid residues on the processing reaction rate and catalytic activity and on stimulation of each of these by putrescine. Changing Cys82 to Ala decreased the stimulatory effect of putrescine on processing and completely eliminated catalytic activity, indicating a probable role in the active site of the enzyme, whereas changing Cys49 or Cys226 to Ala had minimal effects on processing and activity or the putrescine stimulation of either. Since Cys49 is the only cysteine residue in the smaller subunit, this indicates that disulfide-bond formation between the two subunits cannot be necessary for maintenance of the conformation for proenzyme processing or catalytic activity. Changing Glu8 or Glu11 to Gln produced an AdoMet decarboxylase that processed, but was catalytically inactive. Mutation at Glu11 also completely eliminated the putrescine stimulation of proenzyme processing, as did double mutation at Glu8 and Glu11, whereas the single mutation at Glu8 had no effect on the putrescine stimulation of proenzyme processing. Changing Glu15, Glu61, Glu67, Glu247, or Glu247 and Glu249 to Gln had a minimal effect on processing and activity or putrescine stimulation of either. These results demonstrate a role for the smaller subunit in the catalytic activity of the mature AdoMet decarboxylase enzyme and show that Glu8, Glu11, and Cys82 are essential for catalytic activity, with Glu11 also being essential for the putrescine stimulation of AdoMet decarboxylase proenzyme processing.

AB - Human S-adenosylmethionine (AdoMet) decarboxylase is synthesized as a 38-kDa proenzyme that is autocatalytically cleaved, forming the covalently attached pyruvate cofactor and the two subunits (67 and 267 amino acid residues) of the mature enzyme. Both the cleavage reaction and the catalytic activity of the mature enzyme are stimulated by putrescine. Using site-specific mutagenesis and in vitro transcription followed by translation of the resultant RNA in a cell-free system, we have examined the importance of several amino acid residues on the processing reaction rate and catalytic activity and on stimulation of each of these by putrescine. Changing Cys82 to Ala decreased the stimulatory effect of putrescine on processing and completely eliminated catalytic activity, indicating a probable role in the active site of the enzyme, whereas changing Cys49 or Cys226 to Ala had minimal effects on processing and activity or the putrescine stimulation of either. Since Cys49 is the only cysteine residue in the smaller subunit, this indicates that disulfide-bond formation between the two subunits cannot be necessary for maintenance of the conformation for proenzyme processing or catalytic activity. Changing Glu8 or Glu11 to Gln produced an AdoMet decarboxylase that processed, but was catalytically inactive. Mutation at Glu11 also completely eliminated the putrescine stimulation of proenzyme processing, as did double mutation at Glu8 and Glu11, whereas the single mutation at Glu8 had no effect on the putrescine stimulation of proenzyme processing. Changing Glu15, Glu61, Glu67, Glu247, or Glu247 and Glu249 to Gln had a minimal effect on processing and activity or putrescine stimulation of either. These results demonstrate a role for the smaller subunit in the catalytic activity of the mature AdoMet decarboxylase enzyme and show that Glu8, Glu11, and Cys82 are essential for catalytic activity, with Glu11 also being essential for the putrescine stimulation of AdoMet decarboxylase proenzyme processing.

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