Stereochemistry specifies the regiochemistry of phosphorylation in two cAMP-dependent protein kinase substrates

Y. G. Kwon, J. Srinivasan, M. Mendelow, S. Pluskey, D. S. Lawrence

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Abstract

The substrate specificity of the cAMP-dependent protein kinase has been assessed with peptides bearing threoninol diastereomers. The threoninol residue contains both a primary alcohol and a secondary alcohol, either of which may serve as a site of phosphorylation. The enzyme-catalyzed phosphorylation of Gly-Arg-Thr-Gly-Arg-Arg-Asn-(2R,3R)-threoninol furnishes a K(m) of 498 ± 39 μM and a V(max) of 7.8 ± 0.2 μmol/min-mg, whereas the phosphorylation of Gly-Arg-Thr-Gly-Arg-Arg-Asn-(2S,3S)-threoninol provides a K(m) of 16.3 ± 0.8 μM and a V(max) of 16.0 ± 0.4 μmol/min-mg. Mass spectral analysis of the phosphopeptide reaction products revealed that each species is phosphorylated only once. 1H-coupled 31P NMR experiments unequivocally demonstrated that the (2R,3R)-isomer is specifically phosphorylated at the secondary alcohol, whereas the (2S,3S)-isomer is exclusively phosphorylated at the primary alcohol. This regiospecificity appears to be a consequence of the stereochemistry at C-2 in the threoninol residues. The structural attributes of the protein kinase that appear to be responsible for the observed differentiation between the C-2 stereoisomers is discussed.

Original languageEnglish
Pages (from-to)16725-16729
Number of pages5
JournalJournal of Biological Chemistry
Volume268
Issue number22
Publication statusPublished - 1993 Jan 1

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Stereochemistry
Phosphorylation
Cyclic AMP-Dependent Protein Kinases
Alcohols
Substrates
Isomers
Bearings (structural)
Phosphopeptides
Stereoisomerism
Substrate Specificity
Reaction products
Spectrum analysis
Protein Kinases
Nuclear magnetic resonance
threoninol
Peptides
Enzymes
Experiments

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

Kwon, Y. G. ; Srinivasan, J. ; Mendelow, M. ; Pluskey, S. ; Lawrence, D. S. / Stereochemistry specifies the regiochemistry of phosphorylation in two cAMP-dependent protein kinase substrates. In: Journal of Biological Chemistry. 1993 ; Vol. 268, No. 22. pp. 16725-16729.
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Kwon, YG, Srinivasan, J, Mendelow, M, Pluskey, S & Lawrence, DS 1993, 'Stereochemistry specifies the regiochemistry of phosphorylation in two cAMP-dependent protein kinase substrates', Journal of Biological Chemistry, vol. 268, no. 22, pp. 16725-16729.

Stereochemistry specifies the regiochemistry of phosphorylation in two cAMP-dependent protein kinase substrates. / Kwon, Y. G.; Srinivasan, J.; Mendelow, M.; Pluskey, S.; Lawrence, D. S.

In: Journal of Biological Chemistry, Vol. 268, No. 22, 01.01.1993, p. 16725-16729.

Research output: Contribution to journalArticle

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T1 - Stereochemistry specifies the regiochemistry of phosphorylation in two cAMP-dependent protein kinase substrates

AU - Kwon, Y. G.

AU - Srinivasan, J.

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AU - Lawrence, D. S.

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N2 - The substrate specificity of the cAMP-dependent protein kinase has been assessed with peptides bearing threoninol diastereomers. The threoninol residue contains both a primary alcohol and a secondary alcohol, either of which may serve as a site of phosphorylation. The enzyme-catalyzed phosphorylation of Gly-Arg-Thr-Gly-Arg-Arg-Asn-(2R,3R)-threoninol furnishes a K(m) of 498 ± 39 μM and a V(max) of 7.8 ± 0.2 μmol/min-mg, whereas the phosphorylation of Gly-Arg-Thr-Gly-Arg-Arg-Asn-(2S,3S)-threoninol provides a K(m) of 16.3 ± 0.8 μM and a V(max) of 16.0 ± 0.4 μmol/min-mg. Mass spectral analysis of the phosphopeptide reaction products revealed that each species is phosphorylated only once. 1H-coupled 31P NMR experiments unequivocally demonstrated that the (2R,3R)-isomer is specifically phosphorylated at the secondary alcohol, whereas the (2S,3S)-isomer is exclusively phosphorylated at the primary alcohol. This regiospecificity appears to be a consequence of the stereochemistry at C-2 in the threoninol residues. The structural attributes of the protein kinase that appear to be responsible for the observed differentiation between the C-2 stereoisomers is discussed.

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