Moore Michael J, Adams Joseph A, Taylor Susan S
Howard Hughes Medical Institute, the Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla 92093-0654, USA.
J Biol Chem. 2003 Mar 21;278(12):10613-8. doi: 10.1074/jbc.M210807200. Epub 2002 Dec 23.
For optimal activity the catalytic subunit of cAMP-dependent protein kinase requires a phosphate on Thr-197. This phosphate anchors the activation loop in the proper conformation and contributes to catalytic efficiency by enhancing the phosphoryl transfer rate and increasing the affinity for ATP (1). The crystal structure of the catalytic subunit bound to ATP, and the inhibitor peptide, IP20, highlights the contacts made by the Thr-197 phosphate as well as the role adjacent residues play in contacting the substrate peptide. Glu-203 and Tyr-204 interact with arginines in the consensus sequence of PKA substrates at the P-6 and P-2 positions, respectively. To assess the contribution that each residue makes to peptide recognition, the kinetic properties of three mutant proteins (E203A, Y204A, and Y204F) were monitored using multiple peptide substrates. The canonical peptide substrate, Kemptide, as well as a longer 9-residue peptide and corresponding peptides with alanine substitutions at the P-6 and P-2 positions were used. While the effect of Glu-203 is more localized to the P-6 site, Tyr-204 contributes to global peptide recognition. An aromatic hydrophobic residue is essential for optimal peptide recognition and is conserved throughout the protein kinase family.
为实现最佳活性，环磷酸腺苷（cAMP）依赖性蛋白激酶的催化亚基需要苏氨酸-197（Thr-197）上的一个磷酸基团。该磷酸基团将激活环固定在适当的构象中，并通过提高磷酰基转移速率和增加对ATP的亲和力来提高催化效率（1）。与ATP及抑制剂肽IP20结合的催化亚基的晶体结构突出了Thr-197磷酸基团形成的接触以及相邻残基在与底物肽接触中所起的作用。谷氨酸-203（Glu-203）和酪氨酸-204（Tyr-204）分别与PKA底物共有序列中P-6和P-2位置的精氨酸相互作用。为评估每个残基对肽识别的贡献，使用多种肽底物监测了三种突变蛋白（E203A、Y204A和Y204F）的动力学特性。使用了典型的肽底物肯普肽（Kemptide），以及一个更长的9残基肽和在P-6和P-2位置具有丙氨酸替代的相应肽。虽然Glu-203的作用更局限于P-6位点，但Tyr-204有助于整体肽识别。一个芳香族疏水残基对于最佳肽识别至关重要，并且在整个蛋白激酶家族中保守。
