THE CRYSTAL-STRUCTURE OF A PHOSPHORYLASE-KINASE PEPTIDE SUBSTRATE COMPLEX - KINASE SUBSTRATE RECOGNITION

The structure of a truncated form of the gamma-subunit of phosphorylas e kinase (PHK gamma(t)) has been solved in a ternary complex with a no n-hydrolysable ATP analogue (adenylyl imidodiphosphate, AMPPNP) and a heptapeptide substrate related in sequence to both the natural substra te and to the optimal peptide substrate, Kinetic characterization of t he phosphotransfer reaction confirms the peptide to be a good substrat e, and the structure allows identification of key features responsible for its high affinity, Unexpectedly, the substrate peptide forms a sh ort anti-parallel beta-sheet with the kinase activation segment, the r egion which in other kinases plays an important role in regulation of enzyme activity, This anchoring of the main chain of the substrate pep tide at a fixed distance from the gamma-phosphate of ATP explains the selectivity of PHK for serine/threonine over tyrosine as a substrate, The catalytic core of PHK exists as a dimer in crystals of the ternary complex, and the relevance of this phenomenon to its in vivo recognit ion of dimeric glycogen phosphorylase b is considered.