Serine-53 at the tip of the glycine-rich loop of cAMP-dependent protein kinase: Role in catalysis, P-site specificity, and interaction with inhibitors
Serine-53 at the tip of the glycine-rich loop of cAMP-dependent protein kinase: Role in catalysis, P-site specificity, and interaction with inhibitors
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DOI：
10.1021/bi992800w
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发表时间：
2000-07-18
期刊：
影响因子：
2.9
通讯作者：
Taylor, SS
中科院分区：
文献类型：
--
作者：
Aimes, RT;Hemmer, W;Taylor, SS
富含甘氨酸的环是保守蛋白激酶催化核心中最重要的基序之一，它包含整个核苷酸，具有很强的移动性，并且对占据活性位点的物质非常敏感。在camp依赖性蛋白激酶（cAPK）中的三个保守甘氨酸[G(50)TG(52)SFG(55)]中，Gly（52）对催化作用最为重要，因为它允许环端Ser（53）的主酰胺与ATP的γ -磷酸形成氢键[Grant， b.d. et al. (1998) Biochemistry 37, 7708]。催化亚基：ATP:PKI(5-24)（热稳定蛋白激酶抑制剂）三元配合物在封闭构象中的结构模型表明，Ser53也可能通过PKI中p位羰基和Ser53侧链羟基之间的氢键来稳定肽底物-酶复合物[Bossemeyer， D. et al. (1993) EMBO J. 12,849]。为了解决Ser53侧链在催化、抑制和p位点特异性方面的重要性，用苏氨酸、甘氨酸和脯氨酸代替了Ser53。去除侧链（即突变为甘氨酸）对肽底物（LRRASLG）的稳态磷酸化或与生理抑制剂（包括i型和ii型调节亚基和PKI）的相互作用没有影响。然而，这种突变确实影响了P-sire的特异性；甘氨酸突变体可以更容易地磷酸化肽底物中的p位点苏氨酸（比野生型好5-6倍）。脯氨酸突变体对肽和ATP的k（cat）和k -m都发生了催化性改变，对调控亚基和PKI的敏感性也发生了改变。空间约束和受限的灵活性可以解释这些影响。这些综合结果表明，虽然Sers的主链酰胺可能是有效催化所必需的，但侧链却不是。
The glycine-rich loop, one of the most important motifs in the conserved protein kinase catalytic core, embraces the entire nucleotide, is very mobile, and is exquisitely sensitive to what occupies the active site deft. Of the three conserved glycines [G(50)TG(52)SFG(55) in cAMP-dependent protein kinase (cAPK)], Gly(52) is the most important for catalysis because it allows the backbone amide of Ser(53) at the tip of the loop to hydrogen bond to the gamma-phosphate of ATP [Grant, B. D. et al. (1998) Biochemistry 37, 7708]. The structural model of the catalytic subunit:ATP:PKI(5-24) (heat-stable protein kinase inhibitor) ternary complex in the closed conformation suggests that Ser53 also might be essential for stabilization of the peptide substrate-enzyme complex via a hydrogen bond between the P-site carbonyl in PKI and the Ser53 side-chain hydroxyl [Bossemeyer, D. et al. (1993) EMBO J. 12, 849]. To address the importance of the Ser53 side chain in catalysis, inhibition, and P-site specificity, Ser53 was replaced with threonine, glycine, and proline. Removal of the side chain (i.e., mutation to glycine) had no effect on the steady-state phosphorylation of a peptide substrate (LRRASLG) or on the interaction with physiological inhibitors, including the type-I and -II regulatory subunits and PKI. However, this mutation did affect the P-sire specificity; the glycine mutant can more readily phosphorylate a P-site threonine in a peptide substrate (5-6-fold better than wild-type). The proline mutant is compromised catalytically with altered k(cat) and K-m for both peptide and ATP and with altered sensitivity to both regulatory subunits and PKI. Steric constraints as well as restricted flexibility could account for these effects. These combined results demonstrate that while the backbone amide of Sers' may be required for efficient catalysis, the side chain is not.