García J J, Gómez-Puyou A, de Gómez-Puyou M T
Instituto de Fisiología Celular, Universidad Nacional Autónoma de México, México, D. F. México.
J Bioenerg Biomembr. 1997 Feb;29(1):61-70. doi: 10.1023/a:1022463822929.
Some of the characteristics of unisite hydrolysis of [gamma 32P]ATP as well as the changes that occur on the transition to multisite catalysis were further studied. It was found that a fraction of [gamma 32P]ATP bound at the catalytic sites of F1 under unisite conditions undergoes both hydrolysis and release induced by medium nucleotides upon addition of millimolar concentrations of ADP or ATP. The fraction of [gamma 32P]ATP that undergoes release is similar to the fraction that undergoes hydrolytic cleavage, indicating that the rates of the release and hydrolytic reactions of bound [gamma 32P]ATP are in the same range. As part of studies on the mechanisms through which trifluoperazine inhibits ATP hydrolysis, its effect on unisite hydrolysis of [gamma 32P]ATP was also studied. Trifluoperazine diminishes the rate of unisite hydrolysis by 30-40%. The inhibition is accompanied by a nearly tenfold increase in the ratio of [gamma 32P]ATP/32Pi bound at the catalytic site and a 50% diminution in the rate of 32Pi release from the enzyme into the media. Trifluoperazine also induces heterogeneity of the three catalytic sites of F1 in the sense that in a fraction of F1 molecules, the high-affinity catalytic site has a turnover rate lower than the other two. Trifluoperazine does not modify the release of previously bound [gamma 32P]ATP induced by medium nucleotides. The latter indicates that hindrances in the release of Pi do not necesarily accompany alterations in the release of ATP even though both species lie in the same site.
对[γ-32P]ATP单位点水解的一些特性以及向多位点催化转变时发生的变化进行了进一步研究。发现在单位点条件下，结合在F1催化位点的一部分[γ-32P]ATP在加入毫摩尔浓度的ADP或ATP后，会经历由介质核苷酸诱导的水解和释放。发生释放的[γ-32P]ATP部分与发生水解裂解的部分相似，这表明结合的[γ-32P]ATP的释放速率和水解反应速率在同一范围内。作为对三氟拉嗪抑制ATP水解机制研究的一部分，还研究了其对[γ-32P]ATP单位点水解的影响。三氟拉嗪使单位点水解速率降低30 - 40%。这种抑制伴随着催化位点结合的[γ-32P]ATP/32Pi比率增加近10倍，以及32Pi从酶释放到介质中的速率降低50%。三氟拉嗪还在一定意义上诱导了F1三个催化位点的异质性，即在一部分F1分子中，高亲和力催化位点的周转速率低于其他两个位点。三氟拉嗪不会改变介质核苷酸诱导的先前结合的[γ-32P]ATP的释放。后者表明，即使Pi和ATP都位于同一位点，Pi释放的阻碍不一定伴随着ATP释放的改变。
