Bivin D B, Ue K, Khoroshev M, Morales M
Physiology Department, University of the Pacific, San Francisco, CA 94115.
Proc Natl Acad Sci U S A. 1994 Aug 30;91(18):8665-9. doi: 10.1073/pnas.91.18.8665.
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.
肌球蛋白亚片段1（S-1）存在多种不同的修饰，这些修饰会增强（调节）其ATP酶活性，包括该颗粒与肌动蛋白的相互作用；这些修饰中最近增加的一项是S-1的广泛赖氨酸修饰，这似乎是将其结晶以进行结构分析的先决条件。在本研究中，我们首先从动力学角度确定了对肌球蛋白S-1酶进行各种处理所诱导的ATP酶调节，并且我们还测量了S-1活性位点的两个特性——该位点与酶促核苷酸产物（的荧光类似物）结合的亲和力以及荧光猝灭剂接近结合的ADP产物的程度——以便探究调节机制。通过用Ca2+替代正常的Mg2+助催化剂或用Cl-替代正常的羧酸根阴离子所实现的调节，似乎是由于调节后的酶对产物的结合更松散。在其他示例性调节（赖氨酸甲基化、半胱氨酸-707的烷基化或从中性pH转变为pH 9.2）中，核苷酸产物亲和力和对猝灭剂的可及性确实会发生变化，但并非以简单地解除产物抑制所能解释的模式变化。赖氨酸甲基化导致核苷酸产物的结合变弱。
