Partial activation of muscle phosphorylase by replacement of serine 14 with acidic residues at the site of regulatory phosphorylation
Partial activation of muscle phosphorylase by replacement of serine 14 with acidic residues at the site of regulatory phosphorylation
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DOI：
10.1021/bi9704820
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发表时间：
1997-07-01
期刊：
影响因子：
2.9
通讯作者：
Fletterick, RJ
中科院分区：
文献类型：
--
作者：
Buchbinder, JL;Luong, CBH;Fletterick, RJ
糖原磷酸化酶在残基Ser14处的磷酸化触发构象转变，从而激活该酶。蛋白质的N末端，响应磷酸化，折叠成3(10)螺旋，并从它在蛋白质表面上靠近一簇酸性残基的位置移动到二聚体界面上的位置，在那里一对精氨酸残基与磷酸丝氨酸形成带电氢键。定点突变用于用天冬氨酸和谷氨酸残基取代Ser14，这些残基是磷酸丝氨酸的类似物，可能参与与二聚体界面上的精氨酸侧链的离子相互作用。对突变体的动力学分析表明，在调节磷酸化的位置用酸性残基取代Ser14可以部分激活该酶。S14D突变体的V-max增加了1.6倍，AMP的表观解离常数降低了10倍，而葡萄糖-1-磷酸的S-0.5降低了3倍。S14E突变体表现出类似的行为，表现出Vmax增加了2.2倍，AMP的表观解离常数降低了6倍，而S-0.5对葡萄糖L磷酸的解离常数降低了2倍。突变能够增强AMP与葡萄糖L磷酸的结合并提高催化活性，这表明在第14位引入羧酸侧链促进了亚基界面N末端的对接，并伴随着酶激活构象的稳定。与天然酶一样，这两个突变体只有在激活剂AMP存在的情况下才显示出显著的活性。由于磷酸化部位的电荷和离子相互作用的几何形状的不同，可能无法实现与酶的共价磷酸化类似的完全激活。
Phosphorylation of glycogen phosphorylase at residue Ser14 triggers a conformational transition that activates the enzyme. The N-terminus of the protein, in response to phosphorylation, folds into a 3(10) helix and moves from its location near a cluster of acidic residues on the protein surface to a site at the dimer interface where a pair of arginine residues form charged hydrogen bonds with the phosphoserine. Site-directed mutagenesis was used to replace Ser14 with Asp and Glu residues, analogs of the phosphoserine, that might be expected to participate in ionic interactions with the arginine side chains at the dimer interface. Kinetic analysis of the mutants indicates that substitution of an acidic residue in place of Ser14 at the site of regulatory phosphorylation partially activates the enzyme. The S14D mutant shows a 1.6-fold increase in V-max, a 10-fold decrease in the apparent dissociation constant for AMP, and a 3-fold decrease in the S-0.5 for glucose 1-phosphate. The S14E mutant behaves similarly, showing a 2.2-fold increase in V-max, a 6-fold decrease in the apparent dissociation constant for AMP, and a 2-fold decrease in the S-0.5 for glucose l-phosphate. The ability of the mutations to enhance binding of AMP and glucose l-phosphate and to raise catalytic activity suggests that the introduction of a carboxylate side chain at position 14 promotes docking of the N-terminus at the subunit interface and concomitant stabilization of the activated conformation of the enzyme. Like the native enzyme, both mutants show significant activity only in the presence of the activator, AMP. Full activation, analogous to that provided by covalent phosphorylation of the enzyme, likely is not achieved because of differences in the charge and the geometry of ionic interactions at the phosphorylation site.