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 language | English |
|---|---|
| Pages (from-to) | 16725-16729 |
| Number of pages | 5 |
| Journal | Journal of Biological Chemistry |
| Volume | 268 |
| Issue number | 22 |
| Publication status | Published - 1993 Jan 1 |
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All Science Journal Classification (ASJC) codes
- Biochemistry
- Molecular Biology
- Cell Biology
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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 journal › Article
TY - JOUR
T1 - Stereochemistry specifies the regiochemistry of phosphorylation in two cAMP-dependent protein kinase substrates
AU - Kwon, Y. G.
AU - Srinivasan, J.
AU - Mendelow, M.
AU - Pluskey, S.
AU - Lawrence, D. S.
PY - 1993/1/1
Y1 - 1993/1/1
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.
AB - 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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M3 - Article
VL - 268
SP - 16725
EP - 16729
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
SN - 0021-9258
IS - 22
ER -