Ionization states of the complex formed between 2-benzyl-3-phosphonopropionic acid and carboxypeptidase A.
Ionization states of the complex formed between 2-benzyl-3-phosphonopropionic acid and carboxypeptidase A.
复制标题
2-苄基-3-膦酰基丙酸和羧肽酶 A 之间形成的复合物的电离态。
DOI：
10.1042/bj2540847
复制
发表时间：
1988
期刊：
影响因子：
--
通讯作者：
Galardy,RE
中科院分区：
文献类型：
--
作者：
Goli,UB;Grobelny,D;Galardy,RE
通过观察2-苄基琥珀酸酯的两种膦酸类似物2-DL-2-苄基-3-膦酰基丙酸（抑制剂I）和2-DL-2-苄基-3-（-O-乙基膦酰基）丙酸（抑制剂II）在pH 5 - 10范围内游离和与羧肽酶A结合时的~（31）P共振，研究了它们与羧肽酶A的结合。I通过配位与活性位点Zn（II）的结合将I的最高pKa值从7.66（+/-0.10）降低至6.71（+/-0.17）。在研究的pH范围内，II上没有任何质子的滴定。酶结合抑制剂II也没有滴定超过pH范围6.17-7.60。以N-（-2-（呋喃丙烯酰基）-L-苯丙氨酰-L-苯丙氨酸为底物，研究了I和II的表观抑制常数随pH的变化关系。pKa值为5.90（+/- 0.06）和9.79（+/- 0.14）的两个酶官能团必须质子化以结合抑制剂I，pKa值为6.29（+/- 0.10）和9.19（+/- 0.15）的两个基团必须质子化以结合抑制剂II。在6.71至7.66的pH范围内，抑制剂I以更质子化的形式与酶的复合物中的酶结合，并且抑制剂以比在该pH下的主要未连接形式更少质子化的形式与酶结合。2920-2927]对L-2-（1-羧基-2-苯乙基）-4-苯基偶氮苯酚在接近4个单位的pH范围内的结合作出了类似的发现。抑制剂I的双阴离子形式（外消旋）的真实抑制常数计算为54.0（+/- 5.9）nM，三阴离子形式的真实抑制常数计算为5.92（+/- 0.65）nM。完全电离的II（外消旋）的真实抑制常数计算为79.8（+/- 6.4）nM。
The binding to carboxypeptidase A of two phosphonic acid analogues of 2-benzylsuccinate, 2-DL-2-benzyl-3-phosphonopropionic acid (inhibitor I) and 2-DL-2-benzyl-3-(-O-ethylphosphono)propionic acid (inhibitor II) was studied by observing their 31P resonances when free and bound to the enzyme in the range of pH from 5 to 10. The binding of I by co-ordination to the active-site Zn(II) lowered the highest pKa of I from a value of 7.66(+/- 0.10) to a value of 6.71(+/- 0.17). No titration of any protons on II occurred over the pH range studied. The enzyme-bound inhibitor II also did not titrate over the pH range 6.17-7.60. The pH-dependencies of the apparent inhibition constants for I and II were also investigated by using N-(-2-(furanacryloyl)-L-phenylalanyl-L-phenylalanine as substrate. Two enzymic functional groups with pKa values of 5.90(+/- 0.06) and 9.79(+/- 0.14) must be protonated for binding of inhibitor I, and two groups with pKa values of 6.29(+/- 0.10) and 9.19(+/- 0.15) for binding of inhibitor II. Over the pH range from 6.71 to 7.66, inhibitor I binds to the enzyme in a complex of the enzyme in a more protonated form, and the inhibitor in a less protonated form than the predominant unligated forms at this pH. Mock & Tsay [(1986) Biochemistry 25, 2920-2927] made a similar finding for the binding of L-2-(1-carboxy-2-phenylethyl)-4-phenylazophenol over a pH range of nearly 4 units. The true inhibition constant for the dianionic form of inhibitor I (racemic) was calculated to be 54.0(+/- 5.9) nM and that of the trianionic form to be 5.92(+/- 0.65) nM. The true inhibition constant of the fully ionized II (racemic) was calculated to be 79.8(+/- 6.4) nM.