Wagner P D, Yount R G
Biochemistry. 1975 Nov 18;14(23):5156-62. doi: 10.1021/bi00694a021.
A purine disulfide analog of ATP, 6,6'-dithiobis(inosinyl imidodiphosphate), forms mixed disulfide bonds between the 6 thiol group on the purine ring and certain key cysteines on myosin, heavy meromyosin, and subfragment one. The EDTA ATPase activities of myosin and heavy meromyosin were completely inactivated when 4 mol of thiopurine nucleotide was bound. When similarly inactivated, subfragment one, depending on its method of preparation, incorporated either 1 or 2 mol of thiopurine nucleotide. Modification of a single cysteine on subfragment one resulted in an inhibition of both the Ca2+ and the EDTA ATPase activities, but the latter always to a greater extent. Modification of two cysteines per head of heavy meromyosin had the same effect suggesting that the active sites were not blocked by the thiopurine nucleotides. Direct evidence for this suggestion was provided by equilibrium dialysis experiments. Heavy meromyosin and subfragment one bound 1.9 and 0.8 mol of [8-3H]adenylyl imidodiphosphate per mol of enzyme, respectively, with an average dissociation constant of 5 X 10(-7) M. Heavy meromyosin with four thiopurine nucleotides bound or subfragment one with two thiopurine nucleotides bound retained 65-80% of these tight adenylyl imidodiphosphate binding sites confirming the above suggestion. Thus previous work assuming reaction of thiopurine nucleotide analogs at the active site of myosin must be reevaluated. Ultracentrifugation studies showed that heavy meromyosin which had incorporated four thiopurine nucleotides did not bind to F-actin while subfragment one with one thiopurine nucleotide bound interacted only very weakly with F-actin. Thus reaction of 6,6'-dithiobis(inosinyl imidodiphosphate) at nucleotide binding sites other than the active sites reduces the rate of ATP hydrolysis and inhibits actin binding. It is suggested that these second sites may function as regulatory sites on myosin.
一种ATP的嘌呤二硫化物类似物，即6,6'-二硫代双（肌苷酰亚氨基二磷酸），在嘌呤环上的6位巯基与肌球蛋白、重酶解肌球蛋白及亚片段1上的某些关键半胱氨酸之间形成混合二硫键。当结合4摩尔硫代嘌呤核苷酸时，肌球蛋白和重酶解肌球蛋白的EDTA ATP酶活性完全失活。同样失活时，根据制备方法不同，亚片段1结合1或2摩尔硫代嘌呤核苷酸。亚片段1上单个半胱氨酸的修饰导致Ca2+和EDTA ATP酶活性均受到抑制，但后者受到的抑制程度总是更大。重酶解肌球蛋白每个头部两个半胱氨酸的修饰产生相同效果，表明活性位点未被硫代嘌呤核苷酸阻断。平衡透析实验为这一推测提供了直接证据。重酶解肌球蛋白和亚片段1每摩尔酶分别结合1.9和0.8摩尔[8-3H]腺苷酰亚氨基二磷酸，平均解离常数为5×10(-7)M。结合四个硫代嘌呤核苷酸的重酶解肌球蛋白或结合两个硫代嘌呤核苷酸的亚片段1保留了65-80%的这些紧密的腺苷酰亚氨基二磷酸结合位点，证实了上述推测。因此，先前关于硫代嘌呤核苷酸类似物在肌球蛋白活性位点反应的工作必须重新评估。超速离心研究表明，结合了四个硫代嘌呤核苷酸的重酶解肌球蛋白不与F-肌动蛋白结合，而结合一个硫代嘌呤核苷酸的亚片段1与F-肌动蛋白的相互作用非常弱。因此，6,6'-二硫代双（肌苷酰亚氨基二磷酸）在活性位点以外的核苷酸结合位点的反应降低了ATP水解速率并抑制了肌动蛋白结合。有人认为这些第二位点可能作为肌球蛋白上的调节位点发挥作用。
