Ruth P, Kamm S, Nau U, Pfeifer A, Hofmann F
Institut für Pharmakologie und Toxikologie, Technischen Universität München, Germany.
Biol Chem. 1996 Jul-Aug;377(7-8):513-20. doi: 10.1515/bchm3.1996.377.7-8.513.
Synthetic peptides corresponding to the active domain of the heat-stable inhibitor protein PKI are very potent inhibitors of cAMP-dependent protein kinase, but are extremely weak inhibitors of cGMP-dependent protein kinase. In this study, we tried to confer PKI sensitivity to cGMP kinase by site-directed mutagenesis. The molecular requirements for high affinity inhibition by PKI were deduced from the crystal structure of the cAMP kinase/PKI complex. A prominent site of interaction are residues Tyr235 and Phe239 in the catalytic subunit, which from a sandwich-like structure with Phe10 of the PKI(5-24) peptide. To increase the sensitivity for PKI, the cGMP kinase codons at the corresponding sites, Ser555 and Ser559, were changed to Tyr and Phe. The mutant cGMP kinase was stimulated half maximally by cGMP at 3-fold higher concentrations (240 nM) than the wild type (77 nM). Wild type and mutant cGMP kinase did not differ significantly in their Km and Vmax for three different substrate peptides. The PKI(5-24) peptide inhibited phosphotransferase activity of the mutant cGMP kinase with higher potency than that of wild type, with Ki values of 42 +/- .3 microM and 160 +/- .7 microM, respectively. The increased affinity of the mutant cGMP kinase was specific for the PKI(5-24) peptide. Mutation of the essential Phe10 in the PKI(5-24) sequence to an Ala yielded a peptide that inhibited mutant and wild type cGMP kinase with similar potency, with Ki values of 160 +/- 11 and 169 +/- 27 microM, respectively. These results suggest that the mutations Ser555Tyr and Ser559Phe are required, but not sufficient, for high affinity inhibition of cGMP kinase by PKI.
与热稳定抑制蛋白PKI活性结构域相对应的合成肽是cAMP依赖性蛋白激酶的强效抑制剂，但对cGMP依赖性蛋白激酶的抑制作用极弱。在本研究中，我们试图通过定点诱变使cGMP激酶对PKI敏感。从cAMP激酶/PKI复合物的晶体结构推导出PKI进行高亲和力抑制的分子要求。一个突出的相互作用位点是催化亚基中的Tyr235和Phe239残基，它们与PKI(5 - 24)肽的Phe10形成类似三明治的结构。为了提高对PKI的敏感性，将相应位点（Ser555和Ser559）的cGMP激酶密码子分别改为Tyr和Phe。突变型cGMP激酶被cGMP半最大激活时的浓度（240 nM）比野生型（77 nM）高3倍。野生型和突变型cGMP激酶对三种不同底物肽的Km和Vmax没有显著差异。PKI(5 - 24)肽抑制突变型cGMP激酶磷酸转移酶活性的效力高于野生型，其Ki值分别为42±0.3 microM和160±0.7 microM。突变型cGMP激酶增加的亲和力对PKI(5 - 24)肽具有特异性。PKI(5 - 24)序列中必需的Phe10突变为Ala后产生的肽抑制突变型和野生型cGMP激酶的效力相似，Ki值分别为160±11和169±27 microM。这些结果表明，Ser555Tyr和Ser559Phe突变是PKI对cGMP激酶进行高亲和力抑制所必需的，但并不充分。
