Bünning P, Riordan J F
Biochemistry. 1983 Jan 4;22(1):110-6. doi: 10.1021/bi00270a016.
The angiotensin converting enzyme catalyzed hydrolysis of furanacryloyl-Phe-Gly-Gly is activated by monovalent anions in the order C1- greater than Br- greater than F- greater than NO3- greater than CH3COO-. In the alkaline pH region, increasing anion concentrations decrease the KM but do not change the kcat. This behavior is characteristic of an ordered bireactant mechanism in which the anion binds to the enzyme prior to the substrate. At acidic pH values, however, the anion activation is a result of both a decrease in KM and an increase in kcat, implying a bireactant mechanism in which anion and substrate bind randomly. For both the ordered and the bireactant mechanisms the anion serves as an essential activator. The effect of chloride on enzyme activity was studied over the pH range 5-10 under kcat/KM conditions and demonstrates that the apparent chloride binding constant increases from 3.3 mM at pH 6.0 to 190 mM at pH 9.0. The kcat vs. pH profile exhibits two pK values of 5.6 and 9.6, while the variation of KM with pH is characterized by a pK of 8.9 and a 2-fold increase between pH 6.5 and 7.5. The chloride activation of the hydrolysis of furanacryloyl-Phe-Gly-Gly is compared with that of the physiological substrates angiotensin I and bradykinin.
血管紧张素转换酶催化的呋喃丙烯酰 - 苯丙氨酸 - 甘氨酸 - 甘氨酸水解反应被单价阴离子激活，其激活顺序为：氯离子＞溴离子＞氟离子＞硝酸根离子＞醋酸根离子。在碱性pH区域，阴离子浓度增加会降低米氏常数（KM），但不改变催化常数（kcat）。这种行为是有序双反应物机制的特征，即阴离子在底物之前与酶结合。然而，在酸性pH值下，阴离子激活是由于KM降低和kcat增加，这意味着阴离子和底物随机结合的双反应物机制。对于有序和双反应物机制，阴离子都是必需的激活剂。在kcat/KM条件下，研究了氯离子在pH 5 - 10范围内对酶活性的影响，结果表明，表观氯离子结合常数从pH 6.0时的3.3 mM增加到pH 9.0时的190 mM。kcat与pH的关系曲线呈现出5.6和9.6两个pK值，而KM随pH的变化特征是pK为8.9，且在pH 6.5至7.5之间增加了2倍。将呋喃丙烯酰 - 苯丙氨酸 - 甘氨酸 - 甘氨酸水解反应的氯离子激活作用与生理底物血管紧张素I和缓激肽的激活作用进行了比较。
