Muneyuki E, Hisabori T, Sasayama T, Mochizuki K, Yoshida M
Research Laboratory of Resources Utilization, Tokyo Institute of Technology, Yokohama.
J Biochem. 1996 Nov;120(5):940-5. doi: 10.1093/oxfordjournals.jbchem.a021510.
The interactions of substoichiometric TNP-ATP and F1-ATPase from Escherichia coli (EF1) were examined and compared with those in the case of mitochondrial F1-ATPase (MF1) and F1-ATPase from thermophilic Bacillus PS3 (TF1). EF1 hydrolyzed substoichiometric TNP-ATP faster than TF1 or MF1, although some 20% of the TNP-ATP remained unhydrolyzed even in the presence of excess chase ATP. The affinity of the catalytic site of EF1 for the product, TNP-ADP, was weaker than that of TF1 or MF1, and the TNP-ADP was readily released upon addition of excess ATP. The amplitude of the difference absorption spectrum induced by binding of TNP-AT(D)P to EF1 was smaller than that of MF1 or TF1 under similar experimental conditions. When an excess amount of TNP-ATP was added to EF1 and the change of the difference spectrum was measured, the shape of the difference spectrum of the ATP-replaceable fraction was very similar to that in the case of binding of TNP-ATP to the isolated beta subunit of TF1, indicating that the rapidly replaceable fraction of bound TNP-ATP was actually at the catalytic site and most of the non-replaceable portion was bound at noncatalytic sites. Weaker affinity of the catalytic site for TNP-ATP may account for the heterogeneous binding and hydrolysis under the conditions described in this paper.
研究了亚化学计量的TNP - ATP与大肠杆菌F1 - ATP酶（EF1）之间的相互作用，并将其与线粒体F1 - ATP酶（MF1）和嗜热芽孢杆菌PS3的F1 - ATP酶（TF1）的情况进行了比较。EF1水解亚化学计量的TNP - ATP的速度比TF1或MF1快，尽管即使在存在过量追踪ATP的情况下，仍有约20%的TNP - ATP未被水解。EF1催化位点对产物TNP - ADP的亲和力比TF1或MF1弱，并且在加入过量ATP后，TNP - ADP很容易释放。在类似实验条件下，TNP - AT(D)P与EF1结合诱导的差异吸收光谱的幅度小于MF1或TF1。当向EF1中加入过量的TNP - ATP并测量差异光谱的变化时，ATP可置换部分的差异光谱形状与TNP - ATP与TF1分离的β亚基结合时的情况非常相似，表明结合的TNP - ATP的快速可置换部分实际上位于催化位点，而大部分不可置换部分则结合在非催化位点。催化位点对TNP - ATP的较弱亲和力可能解释了本文所述条件下的异质结合和水解。
