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首页> 外文期刊>Biochemistry >Serine-53 at the Tip of the Glycine-Rich Loop of cAMP-Dependent Protein Kinase: Role in Catalysis, P-Site Specificity, and Interaction wiht Inhibitors
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Serine-53 at the Tip of the Glycine-Rich Loop of cAMP-Dependent Protein Kinase: Role in Catalysis, P-Site Specificity, and Interaction wiht Inhibitors

机译：丝氨酸53的cAMP依赖性蛋白激酶的富含甘氨酸的环的尖端：在催化，P站点特异性和与抑制剂的相互作用中的作用。

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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 thea ctive site cleft. Of the three conserved glycines [G~50TG~52SFG~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 protien kinase inhibitor) ternary complex in the closed conformation suggests that Ser~53 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 Ser~53 side-chain hydroxyl [Bossemeyer, D.et al (1993) EMBO J. 12,849]. To address the importance of the Ser~53 side chain in catalysis, inhibition, and P-site specificity, Ser~53 was replaced with threonine, glycine, and proline. Removal of the side chain (i.e., mutation to glycine) had no efffect on the steady-state phosphoryltaion of a petpdie substrate (LRRASLG) or on the interaction wiht physiological inhibitors, including the type-I and-II regulatory subunits and PKI. However, this mutation did affect the P-site 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 peptdie and ATP and with altered sensitivity to both regulatory subunits and PHI. Steric constraints as well as restricted flexibility could account for these effects. These combined results demonstrate that while the backbone amide of Ser~53 may be required for efficient catalysis, the side chain is not.
机译：富含甘氨酸的环是保守的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，非常易移动，并且对占据治疗位点的缝隙非常敏感。在三个保守的甘氨酸[cAMP依赖性蛋白激酶（cAPK）中的G〜50TG〜52SFG〜55]中，Gly〜52对于催化最重要，因为它允许环末端的Ser〜53主链酰胺氢键与ATP的＃γ＃-磷酸[Grant，BDet al。 （1998）Biochemistry 37，7708]。催化亚基的结构模型：ATP：PKI_（5-24）（热稳定的蛋白激酶抑制剂）三元复合物呈封闭构象，表明Ser〜53可能对于通过多肽稳定肽底物-酶复合物也是必不可少的。 PKI中P-位羰基与Ser〜53侧链羟基之间的氢键[Bossemeyer，D.et al（1993）EMBO J.12,849]。为了解决Ser〜53侧链在催化，抑制和P位特异性方面的重要性，将Ser〜53替换为苏氨酸，甘氨酸和脯氨酸。侧链的去除（即突变为甘氨酸）对petpdie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位点的特异性。甘氨酸突变体可以更容易地使肽底物中的P位苏氨酸磷酸化（比野生型好5-6倍）。脯氨酸突变体的催化作用是肽和ATP的k_cat和K-m均发生变化，并且对调节亚基和PHI的敏感性均发生变化。严格的限制以及有限的灵活性可能会造成这些影响。这些结合的结果表明，虽然Ser〜53的骨架酰胺可能需要有效的催化作用，但侧链却不是。

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