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.
|Number of pages||5|
|Journal||Journal of Biological Chemistry|
|Publication status||Published - 1993 Jan 1|
All Science Journal Classification (ASJC) codes
- Molecular Biology
- Cell Biology