Taylor S S, Zheng J, Radzio-Andzelm E, Knighton D R, Ten Eyck L F, Sowadski J M, Herberg F W, Yonemoto W M
Department of Chemistry, University of California, La Jolla 92093-0654.
Philos Trans R Soc Lond B Biol Sci. 1993 Jun 29;340(1293):315-24. doi: 10.1098/rstb.1993.0073.
The structure of the recombinant mouse catalytic subunit of cAMP-dependent protein kinase is reviewed with particular emphasis on the overall features and specific amino acids that are shared by all members of the eukaryotic protein kinase family. The crystal structure of a ternary complex containing both MgATP and a twenty-residue inhibitor peptide defines the precise role of the conserved residues that are clustered at the active site. In addition to catalysing the post-translational modification of other proteins, the catalytic subunit is itself subject to covalent modifications. It is a phosphoprotein and is also myristylated at its amino terminus. The enzyme when crystallized in the presence of detergent shows a detergent molecule bound to an acyl pocket that is presumably occupied by the myristyl moiety in the mammalian enzyme. When expressed in E. coli, the catalytic subunit is autophosphorylated at four sites. Two stable phosphates at Ser338 and Thr197 interact with multiple protein side chains thus explaining why they are inaccessible to phosphatases. Although all substrates and inhibitors of the catalytic subunit share a general minimum consensus sequence, the high affinity binding of protein inhibitors such as the regulatory subunits and the heat stable protein kinase inhibitors require additional determinants that lie beyond the consensus site. These two physiological inhibitors of the catalytic subunit appear to use different sites to achieve high-affinity binding.
本文综述了重组小鼠环磷酸腺苷依赖性蛋白激酶催化亚基的结构，特别强调了真核蛋白激酶家族所有成员共有的整体特征和特定氨基酸。包含MgATP和一个20个残基的抑制肽的三元复合物的晶体结构确定了聚集在活性位点的保守残基的确切作用。除了催化其他蛋白质的翻译后修饰外，催化亚基本身也会发生共价修饰。它是一种磷蛋白，其氨基末端也被肉豆蔻酰化。当在去污剂存在下结晶时，该酶显示出一个与酰基口袋结合的去污剂分子，该口袋在哺乳动物酶中可能被肉豆蔻酰部分占据。当在大肠杆菌中表达时，催化亚基在四个位点发生自磷酸化。Ser338和Thr197处的两个稳定磷酸盐与多个蛋白质侧链相互作用，从而解释了为什么它们对磷酸酶不可接近。尽管催化亚基的所有底物和抑制剂都共享一个一般的最小共有序列，但蛋白质抑制剂如调节亚基和热稳定蛋白激酶抑制剂的高亲和力结合需要共有位点之外的额外决定因素。催化亚基的这两种生理抑制剂似乎使用不同的位点来实现高亲和力结合。
