Senear D F, Betts G, Bernhard S A
Biochemistry. 1985 Nov 19;24(24):6789-98. doi: 10.1021/bi00345a010.
The hydrolysis of beta-(2-furyl)acryloyl phosphate (FAP), catalyzed by the Na+/K+-ATPase, is faster than the catalyzed hydrolysis of ATP. This is due to catalyzed hydrolysis of the pseudosubstrate by K+-dependent states of the enzyme, thus bypassing the Na+-dependent enzyme states that are required and are rate limiting in ATP hydrolysis. Unlike ATP, FAP is a positive effector of the E2 state. A study of FAP hydrolysis permits a detailed analysis of later steps in the overall ion translocation-ATP hydrolysis pathway. During the steady state of FAP hydrolysis in the presence of K+, substantial phosphoryl-enzyme is formed, as is indicated by the covalent incorporation of 32P from [32P]FAP. A comparison of the phosphoryl-enzyme yield with the rate of overall hydrolysis reveals that at 25 degrees C the phosphoryl-enzyme formed is all kinetically competent. Both the yield of phosphoryl-enzyme and the rate of overall hydrolysis of FAP are [K+] dependent. The transition E1 in equilibrium E2 is also [K+] dependent, but the rate of transition is differently affected by [K+] than are the above-mentioned two processes. Two distinct roles for K+ are indicated, as an effector of the E1-E2 equilibrium and as a "catalyst" in the hydrolysis of the E2-P. In contrast to the results at 25 degrees C, a virtually stoichiometric yield of phosphoryl-enzyme occurs at 0 degree C in the presence of Na+ and the absence of K+. At lower concentrations of K+ and in the presence of Na+, the hydrolysis of FAP at 0 degree C proceeds substantially through the E1-E2 pathway characteristic of ATP hydrolysis. The selectivity of FAP for the E2-K+-dependent pathway is due to the thermal inactivation of E1 at 25 degrees C in the absence of ATP or ATP analogues, even at high concentrations of Na+. These results emphasize the existence of multiple functional "E1" and "E2" states in the overall ATPase-ion translocation pathway.
由钠钾ATP酶催化的β-(2-呋喃基)丙烯酰磷酸(FAP)水解速度比催化的ATP水解速度快。这是由于该酶的钾离子依赖状态催化了假底物的水解，从而绕过了ATP水解中所需的、限速的钠离子依赖酶状态。与ATP不同，FAP是E2状态的正效应物。对FAP水解的研究有助于对整个离子转运-ATP水解途径的后续步骤进行详细分析。在有钾离子存在的情况下，FAP水解的稳态过程中会形成大量的磷酰化酶，这可通过[32P]FAP中32P的共价掺入来表明。将磷酰化酶的产量与总水解速率进行比较发现，在25℃时形成的磷酰化酶在动力学上都是有效的。FAP的磷酰化酶产量和总水解速率均依赖于[K+]。平衡态E2中的E1转变也依赖于[K+]，但转变速率受[K+]的影响与上述两个过程不同。钾离子有两个不同的作用，一是作为E1-E2平衡的效应物，二是作为E2-P水解的“催化剂”。与25℃时的结果相反，在0℃、有钠离子存在且无钾离子的情况下，磷酰化酶的产量几乎是化学计量的。在较低的钾离子浓度和有钠离子存在的情况下，0℃时FAP的水解基本上通过ATP水解特有的E1-E2途径进行。FAP对E2-钾离子依赖途径的选择性是由于在25℃时，即使在高浓度钠离子存在的情况下，没有ATP或ATP类似物时E1也会发生热失活。这些结果强调了在整个ATP酶-离子转运途径中存在多种功能性的“E1”和“E2”状态。
