Pre-steady-state and steady-state kinetic analysis of the low molecular weight phosphotyrosyl protein phosphatase from bovine heart.
Pre-steady-state and steady-state kinetic analysis of the low molecular weight phosphotyrosyl protein phosphatase from bovine heart.
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DOI：
10.1016/s0021-9258(18)52325-5
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发表时间：
1991-01
期刊：
影响因子：
--
通讯作者：
Zhong Yin Zhang;R. L. Vanetten
中科院分区：
文献类型：
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作者：
Zhong Yin Zhang;R. L. Vanetten
对牛心低分子量（酸性）磷酸酪氨酰蛋白磷酸酶催化磷酸对硝基苯酯水解的全时程进行了详细的解析。爆发滴定动力学首次证明了这类酶。在pH 7.0，4.5摄氏度，一个短暂的前稳态的对硝基苯酚的“突发”形成的速率常数为48秒-1。爆发是有效的化学计量和对应于一个单一的酶活性位点/分子。爆发之后是缓慢的稳态营业额的磷酸酶中间体的速率常数为1.2 s-1。产物抑制研究表明，一个有序的单双动力学水解计划。对几种底物进行的分配实验揭示了恒定的产物比。Vmax是恒定的，这些基板，和水解的总体速率大大增加，在醇受体的存在下。检测到无机磷酸盐和水之间的酶催化18 O交换，并且在pH 5.0，37 ℃下发生kcat = 4.47 x 10（-3）s-1。这些结果都是一致的存在的磷酸酶中间体的催化途径和中间体的分解是限速步骤。真实米氏结合常数Ks = 6.0 mM，表观Km = 0.38 mM，磷酸化速率常数（k2 = 540 s-1）和去磷酸化速率常数（k3 = 36.5 s-1）在磷酸受体存在下，在pH 5.0和37 ℃下，用对硝基苯基磷酸盐在稳态条件下测定。在pH5.0和7.0时，酶催化水解的活化能分别为13.6和14.1kcal/mol。酶催化介质中磷酸根与水交换18 O的活化能为20.2kcal/mol。利用已有的平衡常数和速率常数，绘制了酶催化反应的能量图。
The complete time course of the hydrolysis of p-nitrophenyl phosphate catalyzed by the low molecular weight (acid) phosphotyrosyl protein phosphatase from bovine heart was elucidated and analyzed in detail. Burst titration kinetics were demonstrated for the first time with this class of enzyme. At pH 7.0, 4.5 degrees C, a transient pre-steady-state “burst” of p-nitrophenol was formed with a rate constant of 48 s-1. The burst was effectively stoichiometric and corresponded to a single enzyme active site/molecule. The burst was followed by a slow steady-state turnover of the phosphoenzyme intermediate with a rate constant of 1.2 s-1. Product inhibition studies indicated an ordered uni-bi kinetic scheme for the hydrolysis. Partition experiments conducted for several substrates revealed a constant product ratio. Vmax was constant for these substrates, and the overall rate of hydrolysis was increased greatly in the presence of alcohol acceptors. An enzyme-catalyzed 18O exchange between inorganic phosphate and water was detected and occurred with kcat = 4.47 x 10(-3) s-1 at pH 5.0, 37 degrees C. These results were all consistent with the existence of a phosphoenzyme intermediate in the catalytic pathway and with the breakdown of the intermediate being the rate-limiting step. The true Michaelis binding constant Ks = 6.0 mM, the apparent Km = 0.38 mM, and the rate constants for phosphorylation (k2 = 540 s-1) and dephosphorylation (k3 = 36.5 s-1) were determined under steady-state conditions with p-nitrophenyl phosphate at pH 5.0 and 37 degrees C in the presence of phosphate acceptors. The energies of activation for the enzyme-catalyzed hydrolysis at pH 5.0 and 7.0 were 13.6 and 14.1 kcal/mol, respectively. The activation energy for the enzyme-catalyzed medium 18O exchange between phosphate and water was 20.2 kcal/mol. Using the available equilibrium and rate constants, an energetic diagram was constructed for the enzyme-catalyzed reaction.