Coan C, DiCarlo R
Department of Physiology, School of Dentistry, University of the Pacific, San Francisco, California 94115.
J Biol Chem. 1990 Apr 5;265(10):5376-84.
Diethyl pyrocarbonate was used to modify histidyl residues on the sarcoplasmic reticulum ATPase. Difference spectra of the N-carbethoxyhistidyl derivative indicated that most all the histidyl residues on the enzyme had been modified. These residues could be divided into two populations on the basis of their reaction rate with the reagent. It could then be shown that enzyme inhibition followed modification of the slower reacting population. Reversal with hydroxylamine verified that the loss of activity was due specifically to histidyl modification. Using [32P]ATP as a substrate it was further determined that the modified ATPase could form a phosphoenzyme intermediate, but that the hydrolysis of this intermediate was inhibited. Size exclusion chromatography was used to obtain equilibrium binding curves for high affinity Ca2+ sites on the enzyme. With the normal ATPase a cooperative binding curve for two Ca2+ with a Hill coefficient of 1.8 was observed. With the modified ATPase binding to two independent sites was observed; however, the dissociation constants remained the same as in the cooperative mechanism (K1 = 14 microM; K2 = 0.5 microM). That is, modification had eliminated cooperativity without changing the site specific binding affinities. E-P formation was then shown to follow binding to the higher affinity of the two sites. This would be the second site to bind Ca2+ in a sequential, cooperative mechanism. A model is suggested in which the binding of Ca2+ to an initial site allows for the binding of a second Ca2+ to an occluded site, this second site being responsible for enzyme activation. Modification apparently allows the binding properties of both sites to be observed independently.
焦碳酸二乙酯用于修饰肌浆网ATP酶上的组氨酸残基。N - 乙氧羰基组氨酸衍生物的差光谱表明该酶上几乎所有的组氨酸残基都已被修饰。根据这些残基与试剂的反应速率，可将它们分为两类。结果表明，酶的抑制作用是随着反应较慢的那一类残基的修饰而发生的。用羟胺进行的逆转实验证实了活性的丧失是由于组氨酸残基的特异性修饰。以[32P]ATP作为底物进一步确定，修饰后的ATP酶能够形成磷酸化酶中间体，但该中间体的水解受到抑制。采用尺寸排阻色谱法获得了该酶上高亲和力Ca2+位点的平衡结合曲线。对于正常的ATP酶，观察到两个Ca2+的协同结合曲线，希尔系数为1.8。对于修饰后的ATP酶，观察到其与两个独立位点的结合；然而，解离常数与协同机制中的相同（K1 = 14 microM；K2 = 0.5 microM）。也就是说，修饰消除了协同性，而没有改变位点特异性结合亲和力。然后表明E - P的形成是在与两个位点中亲和力较高的位点结合之后发生的。在顺序协同机制中，这将是第二个结合Ca2+的位点。提出了一个模型，其中Ca2+与初始位点的结合允许第二个Ca2+与一个封闭位点结合，这个第二个位点负责酶的激活。修饰显然使两个位点的结合特性能够独立观察到。
