AFFINITY LABELING OF THE ATP-BINDING SITE OF TYPE-II CALMODULIN-DEPENDENT PROTEIN-KINASE BY 5'P-FLUOROSULFONYLBENZOYL ADENOSINE
AFFINITY LABELING OF THE ATP-BINDING SITE OF TYPE-II CALMODULIN-DEPENDENT PROTEIN-KINASE BY 5'P-FLUOROSULFONYLBENZOYL ADENOSINE
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DOI：
10.1016/0003-9861(88)90052-5
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发表时间：
1988-12-01
影响因子：
3.9
通讯作者：
KWIATKOWSKI, AP
中科院分区：
文献类型：
--
作者：
KING, MM;SHELL, DJ;KWIATKOWSKI, AP
用5“-对氟磺酰苄基腺苷（FSBA）修饰II型钙调蛋白依赖性蛋白激酶，导致该酶的时间依赖性失活。反应遵循准一级动力学，并表现出非线性依赖于试剂浓度。失活率是敏感的Mg 2+和钙调素诱导的构象变化的酶。然而，这些配体的增强作用是不加和的;事实上，Mg 2+刺激的失活反应与FSBA（Kinact = 2.4 mM; kmax = 0.12 min-1）的动力学参数几乎不受同时加入钙调蛋白（Kinact = 1.5 mM; kmax = 0.086 min-1）。Mg 2 +-ADP可保护FSBA的失活，这与催化位点的修饰一致。在不存在（Kd = 590 μ M）和存在（Kd = 68 μ M）钙调蛋白的情况下Mg 2 +-ADP的保护作用的分析表明，调节蛋白与酶的结合增加了蛋白激酶对核苷酸的亲和力。FSBA修饰导致Tyr和Lys残基的标记，但仅Lys的标记被Mg 2 +-ADP降低，这与保守的Lys残基在核苷酸与蛋白激酶结合中是重要的假设一致。然而，失活反应的动力学结果表明，该赖氨酸不参与介导钙调素促进的Mg 2 +-核苷酸复合物的酶的亲和力的增加。
Modification of the type II calmodulin-dependent protein kinase by 5''-p-fluorosulfonylbenzyl adenosine (FSBA) resulted in a time-dependent inactivation of the enzyme. The reaction followed pseudo-first-order kinetics and showed a nonlinear dependence on reagent concentration. The rate of inactivation was sensitive to Mg2+- and calmodulin-induced conformational changes on the enzyme. However, the enhancing effects of these ligands were not additive; indeed, the kinetic parameters of the Mg2+-stimulated inactivation reaction with FSBA (Kinact = 2.4 mM; kmax = 0.12 min-1) were almost unaffected by the simultaneous addition of calmodulin (Kinact = 1.5 mM; kmax = 0.086 min-1). Protection from inactivation by FSBA was provided by Mg2+-ADP which is consistent with modification of the catalytic site. An analysis of the protective effect of Mg2+-ADP in the absence (Kd = 590 .mu.M) and presence of (Kd = 68 .mu.M) of calmodulin demonstrated that binding of the modulator protein to the enzyme increases the affinity of the protein kinase for nucleotides. Modification by FSBA resulted in labeling of both Tyr and Lys residues but only labeling of Lys was decreased by Mg2+-ADP which is consistent with the hypothesis that a conserved Lys residue is important in nucleotide binding to the protein kinase. However, the kinetic results of the inactivation reaction suggest that this Lys is not involved in mediating the calmodulin-promoted increase in the affinity of the enzyme for Mg2+-nucleotide complexes.