Goli U B, Galardy R E
Biochemistry. 1986 Nov 4;25(22):7136-42. doi: 10.1021/bi00370a056.
Five phosphorus-containing inhibitors of angiotensin converting enzyme were found to exhibit slow, tight-binding kinetics by using furanacryloyl-L-phenylalanylglycylglycine as substrate at pH 7.50 and T = 25 degrees C. Two of the inhibitors, (O-ethylphospho)-Ala-Pro (2) and (O-isopropylphospho)-Ala-Pro (3), are found to follow at minimum a two-step mechanism of binding (mechanism B) to the enzyme. This mechanism consists of an initial fast formation of a weaker enzyme-inhibitor complex (Ki = 130 nM for 2 and 180 nM for 3) followed by a slow reversible isomerization to a tighter complex with measurable forward (K3) and reverse (k4) rate constants (k3 = 4.5 X 10(-2) s-1 for 2 and 5.4 X 10(-2) s-1 for 3; k4 = 9.2 X 10(-3) s-1 for 2 and 3.5 X 10(-3) s-1 for 3). For the remaining three inhibitors, phospho-Ala-Pro (1), (O-benzyl-phospho)-Ala-Pro (4), and (P-phenethylphosphono)-Ala-Pro (5), a one-step binding mechanism (mechanism A) is observed under the conditions of the experiment. The second-order rate constants k1 (M-1 s-1) for the binding of these inhibitors to converting enzyme are found to have values more than 3 orders of magnitude lower than the diffusion-controlled limit for a bimolecular reaction involving the enzyme, viz., 3.9 X 10(5) for 1, 2.2 X 10(5) for 4, and 4.8 X 10(5) for 5.(ABSTRACT TRUNCATED AT 250 WORDS)
在pH 7.50和T = 25摄氏度条件下，以呋喃丙烯酰-L-苯丙氨酰甘氨酰甘氨酸为底物，发现五种含磷的血管紧张素转换酶抑制剂呈现出缓慢、紧密结合的动力学特性。其中两种抑制剂，(O-乙基磷酰基)-丙氨酰-脯氨酸(2)和(O-异丙基磷酰基)-丙氨酰-脯氨酸(3)，被发现至少遵循与该酶结合的两步机制(机制B)。该机制包括最初快速形成较弱的酶-抑制剂复合物(2的Ki = 130 nM，3的Ki = 180 nM)，随后缓慢可逆异构化为具有可测量的正向(K3)和反向(k4)速率常数的更紧密复合物(k3 = 4.5×10−2 s−1，2的k4 = 9.2×10−3 s−1；3的k3 = 5.4×10−2 s−1，3的k4 = 3.5×10−3 s−1)。对于其余三种抑制剂，磷酰基-丙氨酰-脯氨酸(1)、(O-苄基磷酰基)-丙氨酰-脯氨酸(4)和(P-苯乙基膦酰基)-丙氨酰-脯氨酸(5)，在实验条件下观察到一步结合机制(机制A)。发现这些抑制剂与转换酶结合的二级速率常数k1 (M−1 s−1)的值比涉及该酶的双分子反应的扩散控制极限低3个数量级以上，即1的为3.9×105，4的为2.2×105，5的为4.8×105。(摘要截短于250字)
