Lowe E D, Noble M E, Skamnaki V T, Oikonomakos N G, Owen D J, Johnson L N
Laboratory of Molecular Biophysics and Oxford Centre for Molecular Sciences, University of Oxford, Rex Richards Building, South Parks Road, Oxford OX1 3QU, UK.
EMBO J. 1997 Nov 17;16(22):6646-58. doi: 10.1093/emboj/16.22.6646.
The structure of a truncated form of the gamma-subunit of phosphorylase kinase (PHKgammat) has been solved in a ternary complex with a non-hydrolysable ATP analogue (adenylyl imidodiphosphate, AMPPNP) and a heptapeptide substrate related in sequence to both the natural substrate and to the optimal peptide substrate. Kinetic characterization of the phosphotransfer reaction confirms the peptide to be a good substrate, and the structure allows identification of key features responsible for its high affinity. Unexpectedly, the substrate peptide forms a short anti-parallel beta-sheet with the kinase activation segment, the region which in other kinases plays an important role in regulation of enzyme activity. This anchoring of the main chain of the substrate peptide 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 recognition of dimeric glycogen phosphorylase b is considered.
磷酸化酶激酶γ亚基（PHKgammat）截短形式的结构已在与一种不可水解的ATP类似物（腺苷酰亚胺二磷酸，AMPPNP）和一种与天然底物及最佳肽底物序列相关的七肽底物形成的三元复合物中得到解析。磷酸转移反应的动力学特征证实该肽是一种良好的底物，并且该结构使得能够鉴定出导致其具有高亲和力的关键特征。出乎意料的是，底物肽与激酶激活片段形成了一个短的反平行β折叠，在其他激酶中该区域在酶活性调节中起重要作用。底物肽主链在距ATP的γ磷酸基团固定距离处的这种锚定解释了PHK对丝氨酸/苏氨酸作为底物的选择性高于酪氨酸。在三元复合物晶体中，PHK的催化核心以二聚体形式存在，并探讨了这种现象与其在体内对二聚体糖原磷酸化酶b的识别的相关性。
