Kinetic analyses of mutations in the glycine-rich loop of cAMP-dependent protein kinase
Kinetic analyses of mutations in the glycine-rich loop of cAMP-dependent protein kinase
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DOI：
10.1021/bi972987w
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发表时间：
1998-05-26
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：
Grant, BD;Hemmer, W;Taylor, SS
camp依赖性蛋白激酶催化(C)亚基富甘氨酸环(Leu-Gly(50)-Thr-Gly（52)-Ser- ph - gly (55)-Arg-Val）中的保守甘氨酸分别突变为Ser （G50S， G52S和G55S）。这些突变的影响在这里用稳态和预稳态动力学方法进行了评估。G50S和G52S对ATP的表观亲和力降低了约10倍，而Gly55的取代对核苷酸结合没有影响。与ATP相比，只有50位突变干扰了ADP的结合。这三个突变使磷酸化转移率降低了7-300倍。综合数据表明，G50和G52是环中催化作用最关键的残基，由于磷酸化转移速率的较大下降(29 vs 1.6 s(-1)，而野生型C为500 s(-1))，位置52的替换最为极端。令人惊讶的是，虽然没有一个环甘氨酸与底物直接接触，但所有三个突变都将对Kemptide的亲和力降低了大约10倍。无法将净产物释放的速率常数与ADP的解离常数联系起来，意味着其他步骤可能限制三元产物络合物的分解。观察到G52S (a)选择性地影响ATP结合，(b)在不直接与核苷酸或肽接触的情况下显著降低磷酸化转移率，这意味着该残基在环中起结构性作用，最有可能是通过定位Ser53的主酰胺以与ATP的γ -磷酸接触。突变蛋白的能量最小化模型与观察到的每个突变的动力学结果一致。该模型预测只有Gly52突变才会干扰主链和ATP之间观察到的氢键。
The conserved glycines in the glycine-rich loop (Leu-Gly(50)-Thr-Gly(52)-Ser-Phe-Gly(55)-Arg-Val) of the catalytic (C) subunit of cAMP-dependent protein kinase were each mutated to Ser (G50S, G52S, and G55S). The effects of these mutations were assessed here using both steady-state and presteady-state kinetic methods. While G50S and G52S reduced the apparent affinity for ATP by approximately 10-fold, substitution at Gly55 had no effect on nucleotide binding. In contrast to ATP, only mutation at position 50 interfered with ADP binding. These three mutations lowered the rate of phosphoryl transfer by 7-300-fold. The combined data indicate that G50 and G52 are the most critical residues in the loop for catalysis, with replacement at position 52 being the most extreme owing to a larger decrease in the rate of phosphoryl transfer (29 vs 1.6 s(-1) in contrast to 500 s(-1) for wild-type C). Surprisingly, all three mutations lowered the affinity for Kemptide by approximately 10-fold, although none of the loop glycines makes direct contact with the substrate. The inability to correlate the rate constant for net product release with the dissociation constant for ADP implies that other steps may limit the decomposition of the ternary product complex. The observations that G52S (a) selectively affects ATP binding and (b) significantly lowers the rate of phosphoryl transfer without making direct contact with either the nucleotide or the peptide imply that this residue serves a structural role in the loop, most likely by positioning the backbone amide of Ser53 for contacting the gamma-phosphate of ATP. Energy minimized models of the mutant proteins are consistent with the observed kinetic consequences of each mutation. The models predict that only mutation of Gly52 will interfere with the observed hydrogen bonding between the backbone and ATP.