p1. The transient-state and steady-state phases of the reaction between iEscherichia coli/i alkaline phosphatase and 4-methylumbelliferyl phosphate were investigated by using a fluorimetric stopped-flow technique. 2. At low substrate concentration (5μm) in the pH range 3·8–6·3 there was an initial rapid liberation of up to 1mole of 4-methylumbelliferone/mole of enzyme. 3. At very low substrate concentration (0·1μm) in the pH range 4·9–5·9 an initial lag in 4-methylumbelliferone production was observed, from which values for ik/isub+1/sub and ik/isub?1/sub could be obtained. 4. The pH profiles for the rates of phosphorylation and dephosphorylation are quite different, and it is postulated that an ionizing group which determines the conformation during the phosphorylation step is not involved in the dephosphorylation step. 5. The binding constants for substrate and Psubi/sub are similar throughout the pH range 4–8. The ionization of substrate or Psubi/sub appeared to have no marked effect on the binding./p
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机译:> 1。利用荧光停止流技术研究了大肠杆菌碱性磷酸酶和4-甲基伞形磷酸酯之间反应的过渡态和稳态。 2.在pH范围3·8-6·3下,低底物浓度(5μm)时,最初的快速释放速率为1摩尔4-甲基伞形酮/每摩尔酶。 3.在pH范围4·9-5·9的底物浓度极低(0·1μm)时,观察到4-甲基伞形酮生产的初始滞后,从中得出 k i> +的值可以获得1 sub>和 k i> ?1 sub>。 4.用于磷酸化和去磷酸化的速率的pH曲线完全不同,并且假定在磷酸化步骤中不涉及确定磷酸化步骤中的构象的电离基团。 5.在4-8的pH范围内,底物和P i sub>的结合常数相似。底物或P i sub>的电离似乎对结合没有明显影响。 p>
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