Greene L E, Zinner R, Naficy S, Eisenberg E
Laboratory of Cell Biology, NHLBI, National Institutes of Health, Bethesda, Maryland 20892.
J Biol Chem. 1995 Feb 17;270(7):2967-73. doi: 10.1074/jbc.270.7.2967.
In previous work we found that bovine brain hsp70 has a single binding site for nucleotide, and that, with ATP at this site, the rates of association and dissociation of clathrin from hsp70 are fast, whereas with ADP at this site, these rates are unmeasurably slow. In the present study we show, first, that peptide C, cytochrome c peptide, and RNase S peptide bind competitively with clathrin, suggesting that they bind to the same site on hsp70, although RNase S peptide binds an order of magnitude more weakly than peptide C and cytochrome c peptide. Second, we show that, with ADP bound to hsp70, as occurs with clathrin, the rate constant for dissociation of peptide markedly decreases compared to the rate constant observed in ATP. In contrast, ADP only slightly decreases the rate of association of peptide. Based on these data we propose a model in which substrates of hsp70 bind to and dissociate from the ATP form of the enzyme, while, following ATP hydrolysis, they are locked onto the ATP form of the enzyme, unable to dissociate until ADP is released and ATP rebinds.
在之前的工作中，我们发现牛脑热休克蛋白70（hsp70）有一个单一的核苷酸结合位点，并且在此位点结合ATP时，网格蛋白与hsp70的结合和解离速率很快，而在此位点结合ADP时，这些速率慢到无法测量。在本研究中，我们首先表明，肽C、细胞色素c肽和核糖核酸酶S肽与网格蛋白竞争性结合，这表明它们结合到hsp70上的同一位点，尽管核糖核酸酶S肽的结合强度比肽C和细胞色素c肽弱一个数量级。其次，我们表明，与网格蛋白结合时一样，hsp70结合ADP时，肽解离的速率常数与在ATP存在时观察到的速率常数相比显著降低。相反，ADP仅略微降低肽的结合速率。基于这些数据，我们提出了一个模型，其中hsp70的底物与酶的ATP形式结合并从其解离，而在ATP水解后，它们被锁定在酶的ADP形式上，直到ADP释放且ATP重新结合才能够解离。
