Menon A S, Goldberg A L
Department of Physiology and Biophysics, Harvard Medical School, Boston, Massachusetts 02115.
J Biol Chem. 1987 Nov 5;262(31):14921-8.
A critical enzyme in protein breakdown in Escherichia coli is the ATP-hydrolyzing protease La, the lon gene product. In order to clarify the role of ATP in proteolysis, we studied ATP and ADP binding to this enzyme using rapid gel filtration to separate free from bound ligands. In the presence of Mg2+ or Mn2+ and 10 microM ATP, two molecules of ATP were bound to the tetrameric enzyme, while at 100 microM ATP (or higher), four ATP molecules were bound, both at 0 and 37 degrees C. Protease La thus has two high affinity sites (S0.5 less than 10(-7) M) for ATP and two lower affinity sites (S0.5 = 12-15 microM). Binding was reversible. In the absence of a divalent ion, ATP bound to only two sites. However, much lower Mg2+ concentrations (50 microM) were required for maximal ATPase binding than for maximal proteolytic and ATPase activity (2 mM). Decavanadate, which is a potent inhibitor of proteolysis, also blocked ATP binding, but orthovanadate had neither effect. Different ATP analogs bind to these sites in distinct ways. Adenyl-5'-yl imidodiphosphate binds to only one high affinity site, while adenyl-5'-yl methylene monophosphonate binds to two. Nevertheless, both non-metabolizable analogs can activate oligopeptide hydrolysis as well as ATP. Although binding of a single nucleotide can activate peptide hydrolysis, occupancy of all four sites appears necessary for maximal protein breakdown. The ATP molecules on all four sites are hydrolyzed rapidly. The Pi is released, but ADP remains on the enzyme. ADP binds to the same four sites, but this process does not require divalent ions. Protease La shows higher affinity for ADP than for ATP. Therefore, in vivo, ADP should inhibit ATP binding and protease La function.
大肠杆菌中蛋白质分解的一种关键酶是水解ATP的蛋白酶La，即lon基因产物。为了阐明ATP在蛋白水解中的作用，我们利用快速凝胶过滤法分离游离配体和结合配体，研究了ATP和ADP与该酶的结合情况。在Mg2+或Mn2+以及10微摩尔ATP存在的情况下，两个ATP分子与四聚体酶结合，而在100微摩尔ATP（或更高浓度）时，在0和37摄氏度下均有四个ATP分子结合。因此，蛋白酶La对ATP有两个高亲和力位点（半数饱和浓度小于10^-7 M）和两个低亲和力位点（半数饱和浓度=12-15微摩尔）。结合是可逆的。在没有二价离子的情况下，ATP仅结合到两个位点。然而，与最大蛋白水解和ATP酶活性（2毫摩尔）相比，最大ATP酶结合所需的Mg2+浓度要低得多（50微摩尔）。十钒酸盐是蛋白水解的有效抑制剂，也能阻断ATP结合，但正钒酸盐没有作用。不同的ATP类似物以不同的方式结合到这些位点。腺苷-5'-亚氨二磷酸仅结合到一个高亲和力位点，而腺苷-5'-亚甲基单磷酸结合到两个位点。然而，这两种不可代谢的类似物都能激活寡肽水解以及ATP。虽然单个核苷酸的结合可以激活肽水解，但似乎需要占据所有四个位点才能实现最大程度的蛋白质分解。所有四个位点上的ATP分子都能迅速水解。磷酸根离子被释放，但ADP仍留在酶上。ADP结合到相同的四个位点，但这个过程不需要二价离子。蛋白酶La对ADP的亲和力高于对ATP的亲和力。因此，在体内，ADP应该会抑制ATP结合和蛋白酶La的功能。
