Effect of lysine methylation and other ATPase modulators on the active site of myosin subfragment 1.
Effect of lysine methylation and other ATPase modulators on the active site of myosin subfragment 1.
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DOI：
10.1073/pnas.91.18.8665
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发表时间：
1994-08
影响因子：
11.1
通讯作者：
D. Bivin;K. Ue;M. Khoroshev;M. Morales
中科院分区：
文献类型：
--
作者：
D. Bivin;K. Ue;M. Khoroshev;M. Morales
肌凝蛋白亚片段1 （S-1）的许多不同修饰增加（调节）其atp酶活性，包括该颗粒与肌动蛋白的相互作用；除了这些修饰外，最近还对S-1进行了广泛的赖氨酸修饰，这似乎是使其结晶以进行结构分析的先决条件。在这项研究中，我们首先从动力学上建立了肌球蛋白S-1酶的各种处理诱导的atp酶调节，并且我们还测量了S-1活性位点的两个特性——该位点结合酶核苷酸产物的亲和力（荧光模拟物）和荧光猝灭剂对结合的ADP产物的访问——以努力获得调节机制。通过用Ca2+取代正常的Mg2+助催化剂或用Cl-取代正常的羧酸阴离子来实现的调节似乎是由于被调节的酶更松散地保持产物。在其他说明性调节（赖氨酸甲基化，或Cys-707的烷基化，或从中性pH到pH 9.2的转变）中，核苷酸产物亲和性和对猝灭剂的获取确实发生了变化，但不是以产物抑制解除的模式来解释的。赖氨酸甲基化导致核苷酸产物结合较弱。
Many and diverse modifications of the myosin subfragment 1 (S-1) increase (modulate) its ATPase activity, including interaction of this particle with actin; a recent addition to these modifications is the extensive lysine modification of S-1 that seems prerequisite to crystallizing it for structure analysis. In this study we first established kinetically the ATPase modulations induced by various treatments of the myosin S-1 enzyme, and we also measured two properties of the S-1 active site--the affinity with which the site binds (a fluorescent analog of) the enzymatic nucleotide product and the access that a fluorescence quencher has to the bound ADP product--in an effort to get at the mechanism of modulation. Modulations achieved by substituting Ca2+ for the normal Mg2+ cocatalyst or by substituting Cl- for the normal carboxylate anion seem due to the product being held more loosely by the modulated enzyme. In other illustrative modulations (lysine methylation, or alkylation of Cys-707, or transition from neutral pH to pH 9.2) nucleotide product affinity and access to quencher do change, but not in a pattern explained simply by a lifting of product inhibition. Lysine methylation results in weaker binding of nucleotide product.