PRIMARY-STRUCTURE REQUIREMENTS FOR INHIBITION BY THE HEAT-STABLE INHIBITOR OF THE CAMP-DEPENDENT PROTEIN-KINASE
PRIMARY-STRUCTURE REQUIREMENTS FOR INHIBITION BY THE HEAT-STABLE INHIBITOR OF THE CAMP-DEPENDENT PROTEIN-KINASE
复制标题
DOI：
10.1073/pnas.83.6.1613
复制
发表时间：
1986-03-01
影响因子：
11.1
通讯作者：
KREBS, EG
中科院分区：
文献类型：
--
作者：
SCOTT, JD;GLACCUM, MB;KREBS, EG
最近测定了cAMP依赖性蛋白激酶（PKI）的热稳定抑制剂的氨基酸序列[Scott，J.D.，Fischer，E.H.，Takio，K.，德迈耶，J.G.和Krebs，E.G.（1985）Proc，Natl. Acad. Sci. USA 82，5732-5736]。早期的报告[斯科特，J.D.，Fischer，E. H、Demaille，J. G.和Krebs，E.G.（1985）Proc.Natl. Acad. Sci. USA 82，4379-4383]显示PKI的抑制结构域的至少一部分位于从残基11延伸至残基30的20个残基的区段中：Ile-Ala-Ser-Gly-Arg-Thr-Gly-Arg-Arg-Asn-Ala-Ile-His-Asp-Ile-Leu-Val-Ser-Ser-Ala。在本研究中，我们进一步映射的抑制区域的PKI添加或删除残基在两端的这个肽和特定的氨基酸取代。结果表明（i）第25-30位残基的缺失没有改变抑制活性，但向氨基末端添加残基使抑制效力增加高达150倍（Ki 4.8 nM），达到接近PKI的水平;（ii）丙氨酸-21被丝氨酸取代，将抑制剂转化为具有相对低亲和力的底物（Km 280 μ M）;（iii）用磷酸丝氨酸或α-丙氨酸取代丙氨酸-21氨基丁酸分别降低了120和20倍的抑制活性;（iv）丝氨酸-13的取代基本上没有影响，而苏氨酸-16的取代降低了抑制活性。抑制效力的最大降低发生在位置18和19处的取代基上。
The amino acid sequence of the heat-stable inhibitor of the cAMP-dependent protein kinase (PKI) was determined recently [Scott, J. D., Fischer, E.H., Takio, K., Demaille, J.G. and Krebs, E.G. (1985) Proc, Natl. Acad. Sci. USA 82, 5732-5736]. An earlier report [Scott, J. D., Fischer, E. H., Demaille, J. G. and Krebs, E.G. (1985) Proc. Natl. Acad. Sci. USA 82, 4379-4383] showed that at least part of the inhibitory domain of PKI is located in a 20-residue segment extending from residue 11 to residue 30: Ile-Ala-Ser-Gly-Arg-Thr-Gly-Arg-Arg-Asn-Ala-Ile-His-Asp-Ile-Leu-Val-Ser-Ser-Ala. In the present study, we further mapped the inhibitory region of PKI by addition or deletion of residues at both ends of this peptide and by substitutions for specific amino acids. The result show that (i) deletion of residues 25-30 did not change inhibitory activity but addition of residues toward the amino terminus increased the inhibitory potency up to 150-fold (Ki 4.8 nM), to a level approaching that of PKI; (ii) replacement of alanine-21 by serine converted the inhibitor into a substrate having a relatively low affinity (Km 280 .mu.M) for the enzyme; (iii) replacement of alanine-21 by phosphoserine or .alpha.-aminobutyric acid decreased inhibitory activity by a factor of 120 and 20, respectively; (iv) replacement of serine-13 had essentially no effect, whereas substitution of threonine-16 decreased inhibitory activity. The greatest decreases of inhibitory potency occurred with replacements of the arginines in positions 18 and 19.