Carreira J, Muñoz E
Mol Cell Biochem. 1975 Nov 14;9(2):85-95. doi: 10.1007/BF01732200.
Basal and trypsin-stimulated adenosine triphosphatase activities of Escherichia coli K 12 have been characterized at pH 7.5 in the membrane-bound state and in a soluble form of the enzyme. The saturation curve for Mg2+/ATP = 1/2 was hyperbolic with the membrane-bound enzyme and sigmoidal with the soluble enzyme. Trypsin did not modify the shape of the curves. The kinetic parameters were for the membrane-bound ATPase: apparent Km = 2.5 mM, Vmax (minus trypsin) = 1.6 mumol-min-1-mg protein-1, Vmax (plus trypsin) = 2.44 mumol-min-1-mg protein-1; for the soluble ATPase: [S0.5] = 1.2 mM, Vmax (-trypsin) = 4 mumol-min-1-mg protein-1; Vmax (+ trypsin) = 6.6 mumol-min-1-mg protein-1. Hill plot analysis showed a single slope for the membrane-bound ATPase (n = 0.92) but two slopes were obtained for the soluble enzyme (n = 0.98 and 1.87). It may suggest the existence of an initial positive cooperativity at low substrate concentrations followed by a lack of cooperativity at high ATP concentrations. Excess of free ATP and Mg2+ inhibited the ATPase but excess of Mg/ATP (1/2) did not. Saturation for ATP at constant Mg2+ concentration (4 mM) showed two sites (groups) with different Kms: at low ATP the values were 0.38 and 1.4 mM for the membrane-bound and soluble enzyme; at high ATP concentrations they were 17 and 20 mM, respectively. Mg2+ saturation at constant ATP (8 mM) revealed michealian kinetics for the membrane-bound ATPase and sigmoid one for the protein in soluble state. When the ATPase was assayed in presence of trypsin we obtained higher Km values for Mg2+. These results might suggest that trypsin stimulates E. coli ATPase by acting on some site(s) involved in Mg2+ binding. Adenosine diphosphate and inorganic phosphate (Pi) act as competitive inhibitors of Escherichia coli ATPase. The Ki values for Pi were 1.6 +/- 0.1 mM for the membrane-bound ATPase and 1.3 +/- 0.1 mM for the enzyme in soluble form, the Ki values for ADP being 1.7 mM and 0.75 mM for the membrane-bound and soluble ATPase, respectively. Hill plots of the activity of the soluble enzyme in presence of ADP showed that ADP decreased the interaction coefficient at ATP concentrations below its Km value. Trypsin did not modify the mechanism of inhibition or the inhibition constants. Dicyclohexylcarbodiimide (0.4 mM) inhibited the membrane-bound enzyme by 60-70% but concentrations 100 times higher did not affect the residual activity nor the soluble ATPase. This inhibition was independent of trypsin. Sodium azide (20 muM) inhibited both states of E. coli ATPase by 50%. Concentrations 25-fold higher were required for complete inhibition. Ouabain, atebrin and oligomycin did not affect the bacterial ATPase.
已对大肠杆菌K12的基础和胰蛋白酶刺激的三磷酸腺苷酶活性进行了表征，该活性是在pH 7.5条件下处于膜结合状态和酶的可溶形式时测定的。Mg2+/ATP = 1/2时的饱和曲线，对于膜结合酶是双曲线型，对于可溶酶是S型。胰蛋白酶未改变曲线形状。膜结合ATP酶的动力学参数为：表观Km = 2.5 mM，Vmax（减去胰蛋白酶）= 1.6 μmol·min-1·mg蛋白-1，Vmax（加上胰蛋白酶）= 2.44 μmol·min-1·mg蛋白-1；可溶ATP酶的动力学参数为：[S0.5] = 1.2 mM，Vmax（-胰蛋白酶）= 4 μmol·min-1·mg蛋白-1；Vmax（+胰蛋白酶）= 6.6 μmol·min-1·mg蛋白-1。希尔图分析表明，膜结合ATP酶的斜率单一（n = 0.92），而可溶酶得到两个斜率（n = 0.98和1.87）。这可能表明在低底物浓度下存在初始正协同性，而在高ATP浓度下缺乏协同性。过量的游离ATP和Mg2+抑制ATP酶，但过量的Mg/ATP（1/2）则不然。在恒定Mg2+浓度（4 mM）下对ATP的饱和显示有两个具有不同Km值的位点（组）：在低ATP时，膜结合酶和可溶酶的值分别为0.38和1.4 mM；在高ATP浓度下，它们分别为17和20 mM。在恒定ATP（8 mM）下对Mg2+的饱和显示，膜结合ATP酶呈米氏动力学，可溶状态的蛋白呈S型动力学。当在胰蛋白酶存在下测定ATP酶时，我们得到Mg2+的Km值更高。这些结果可能表明，胰蛋白酶通过作用于参与Mg2+结合的某些位点来刺激大肠杆菌ATP酶。二磷酸腺苷和无机磷酸（Pi）作为大肠杆菌ATP酶的竞争性抑制剂。膜结合ATP酶的Pi的Ki值为1.6±0.1 mM，可溶形式酶的Pi的Ki值为1.3±0.1 mM，膜结合和可溶ATP酶的ADP的Ki值分别为1.7 mM和0.75 mM。在ADP存在下可溶酶活性的希尔图表明，ADP在ATP浓度低于其Km值时降低了相互作用系数。胰蛋白酶未改变抑制机制或抑制常数。二环己基碳二亚胺（0.4 mM）使膜结合酶抑制60 - 70%，但浓度高100倍时既不影响残余活性，也不影响可溶ATP酶。这种抑制与胰蛋白酶无关。叠氮化钠（20 μM）使大肠杆菌ATP酶的两种状态均抑制50%。完全抑制需要高25倍的浓度。哇巴因、阿的平和寡霉素不影响细菌ATP酶。
