Stereochemistry specifies the regiochemistry of phosphorylation in two cAMP-dependent protein kinase substrates.
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 Km of 498 +/- 39 microM and a Vmax of 7.8 +/- 0.2 mumol/min-mg, whereas the phosphorylation of Gly-Arg-Thr-Gly-Arg-Arg-Asn-(2S,3S)-threoninol provides a Km of 16.3 +/- 0.8 microM and a Vmax of 16.0 +/- 0.4 mumol/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.
Kwon
Y G
YG
Department of Chemistry, State University of New York, Buffalo 14214.
Srinivasan
J
J
Mendelow
M
M
Pluskey
S
S
Lawrence
D S
DS
D000595:Amino Acid Sequence; D001665:Binding Sites; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D010766:Phosphorylation; D011494:Protein Kinases / Q000737:chemistry / Q000378:metabolism*; D013237:Stereoisomerism; D013379:Substrate Specificity
D011494:Protein Kinases