Role of the glycine triad in the ATP-binding site of cAMP-dependent protein kinase
Role of the glycine triad in the ATP-binding site of cAMP-dependent protein kinase
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DOI：
10.1074/jbc.272.27.16946
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发表时间：
1997-07-04
影响因子：
4.8
通讯作者：
Taylor, SS
中科院分区：
文献类型：
--
作者：
Hemmer, W;McGlone, M;Taylor, SS
ATP结合位点的富含甘氨酸的环是蛋白激酶中最保守的序列基序之一。cAMP依赖性蛋白激酶（cAPK）催化（C）亚基中的每个保守甘氨酸（Gly-50、Gly-52和Gly-SS）都被Ser和/或Ala取代。活性突变体蛋白在大肠杆菌中表达，纯化至表观均一性，分离成磷酸异构体，并进行表征。替换Gly-55对稳态动力学参数的影响极小，而替换Gly-50或Gly-52对K-m和k（cat）值的影响较大。预测富含甘氨酸的环的末端对于催化的重要性，Gly-50的取代导致K-m（ATP）降低5-8倍，K-m（肽）增加8-12倍，k（cat）降低3-5倍。C（G52 S）的K-m（ATP）和K-m（肽）值分别增加了8倍和18倍，k（cat）降低了6倍。与催化效率相反，ATP酶：C（G50 S）和C（G52 S）的速率增加了一个数量级以上，每个突变体的热稳定性略有增加，未磷酸化的C（G52 S）以及几种异构体在一个单一位点Ser-338磷酸化。所有这些磷酸化缺陷的突变体表现出浴酶活性和热稳定性的大幅下降，与在Thr-197缺失的磷酸盐。这些结果与晶体结构，各自的突变蛋白的模型，和蛋白激酶家族内的甘氨酸的保守性相关。
A glycine-rich loop in the ATP-binding site is one of the most highly conserved sequence motifs in protein kinases, Each conserved glycine (Gly-50, Gly-52, and Gly-SS) in the catalytic (C) subunit of cAMP-dependent protein kinase (cAPK) was replaced with Ser and/or Ala. Active mutant proteins were expressed in Escherichia coli, purified to apparent homogeneity, separated into phosphoisoforms, and characterized, Replacing Gly-55 had minimal effects on steady-state kinetic parameters, whereas replacement of either Gly-50 or Gly-52 had major effects on both K-m and k(cat) values consistent. with the prediction of the importance of the tip of the glycine-rich loop for catalysis, Substitution of Gly-50 caused a 5-8-fold reduction in K-m(ATP), an 8-12-fold increase in K-m(peptide), and a 3-5-fold drop in k(cat). The K-m(ATP) and K-m(peptide) values of C(G52S) were increased 8- and 18-fold, respectively, and the k(cat) was decreased 6-fold. In contrast to catalytic efficiency, the ATPase: rates of C(G50S) and C(G52S) were increased by more than an order of magnitude, The thermostability of each mutant was slightly increased, Unphosphorylated C(G52S) was characterized as well as several isoforms phosphorylated at a single site, Ser-338. All of these phosphorylation-defective mutants displayed a substantial decrease in bath enzymatic activity and thermal stability that correlated with the missing phosphate at Thr-197. These results are correlated with the crystal structure, models of the respective mutant proteins, and conservation of the Glys within the protein kinase family.