Tagaya M, Hayakawa Y, Fukui T
Institute of Scientific and Industrial Research, Osaka University, Japan.
J Biol Chem. 1988 Jul 25;263(21):10219-23.
To reveal the structure of the ATP-binding site(s) in rabbit muscle phosphorylase kinase, we modified the enzyme with adenosine polyphosphopyridoxals. Adenosine tri- and tetraphosphopyridoxals at micromolar concentrations effectively inactivated the enzyme in a time-dependent manner. Inactivation of the enzyme was accelerated by the addition of Ca2+ and Mg2+. Protection from inactivation was afforded by adenylyl beta,gamma-imidodiphosphate and ADP. In reversible inhibition kinetics, adenosine polyphosphopyridoxals as well as their reduced compounds (adenosine polyphosphopyridoxines) competed with ATP. These results suggest that adenosine polyphosphopyridoxals bind to the ATP-binding site(s) in phosphorylase kinase. When phosphorylase kinase was incubated with adenosine triphosphopyridoxal in the presence of Ca2+ and Mg2+, incorporation of the label into alpha, beta, and gamma subunits was observed. In the absence of both cations, larger amounts of the label were incorporated into all the subunits. Structural study on adenosine triphosphopyridoxal-modified sites in the gamma subunit (having a catalytic site) revealed that Lys-151 is mainly labeled. Based on the results of the present and other studies, it is suggested that the site around Lys-151 is involved in recognition of the substrate protein.
为揭示兔肌肉磷酸化酶激酶中ATP结合位点的结构，我们用腺苷多磷酸吡哆醛修饰了该酶。微摩尔浓度的三磷酸腺苷和四磷酸腺苷吡哆醛能以时间依赖的方式有效使该酶失活。添加Ca2+和Mg2+可加速酶的失活。腺苷β,γ-亚氨二磷酸和ADP可保护酶不被失活。在可逆抑制动力学中，腺苷多磷酸吡哆醛及其还原产物（腺苷多磷酸吡哆辛）与ATP竞争。这些结果表明腺苷多磷酸吡哆醛与磷酸化酶激酶中的ATP结合位点结合。当磷酸化酶激酶在Ca2+和Mg2+存在的情况下与三磷酸腺苷吡哆醛一起孵育时，观察到标记物掺入α、β和γ亚基。在两种阳离子都不存在的情况下，更多的标记物掺入所有亚基。对γ亚基（具有催化位点）中三磷酸腺苷吡哆醛修饰位点的结构研究表明，主要标记的是Lys-151。根据本研究及其他研究结果，提示Lys-151周围的位点参与底物蛋白的识别。
