Larsen K S, Zhang K, Auld D S
European Molecular Biology Laboratory, Grenoble Outstation, France.
J Inorg Biochem. 1996 Nov 15;64(3):149-62. doi: 10.1016/0162-0134(96)00037-2.
The binding of D-phenylalanine, D-Phe, to both zinc and cobalt carboxypeptidase A, ZnCPD and CoCPD, has been investigated by a combination of kinetic and spectroscopic techniques. Kinetic studies of the ZnCPD catalyzed hydrolysis of dansyl-Gly-Ala-L-Phe indicate that D-Phe inhibition occurs through a two-site sequential competitive inhibition mode with Ki values of 45 microM and 11.6 mM at pH 8.4, 1 M NaCl, 25 degrees C. Spectral titration of CoCPD under the same conditions indicates a very strong binding mode of D-Phe (KD < 100 microM) that only slightly perturbs the visible cobalt electronic transitions. However, the conversion of CoCPD.D-Phe into a CoCPD.D-Phe2 (KD, 1.13 mM) is accompanied by a very strong spectral perturbation resulting in a complex that is characterized by Amax values of 506 nm (epsilon = 27 M-1 cm-1) and 605 nm (epsilon = 17 M-1 cm-1) and a shoulder at 530 nm (epsilon = 23 M-1 cm-1). The spectral properties of this ternary complex differ markedly from that of the CoCPD.L-Phe.N3-ternary complex. X-ray absorption fine structure, XAFS, studies indicate that these differences are likely due to a more regular tetrahedral coordination sphere for the ternary azide complexes compared to an octahedral coordination geometry for the Zn and CoCPD.D-Phe2 complexes.
通过动力学和光谱技术相结合的方法，研究了D-苯丙氨酸（D-Phe）与锌羧肽酶A（ZnCPD）和钴羧肽酶A（CoCPD）的结合情况。对ZnCPD催化的丹磺酰甘氨酰丙氨酰-L-苯丙氨酸水解反应的动力学研究表明，在pH 8.4、1 M氯化钠、25℃条件下，D-Phe抑制作用通过双位点顺序竞争性抑制模式发生，其抑制常数Ki值分别为45 μM和11.6 mM。在相同条件下对CoCPD进行光谱滴定，结果表明D-Phe具有很强的结合模式（解离常数KD < 100 μM），仅对可见钴电子跃迁产生轻微扰动。然而，CoCPD·D-Phe转化为CoCPD·D-Phe2（解离常数KD为1.13 mM）时伴随着非常强烈的光谱扰动，形成的复合物的特征吸收峰波长分别为506 nm（摩尔吸光系数ε = 27 M-1·cm-1）、605 nm（ε = 17 M-1·cm-1），在530 nm处有一个肩峰（ε = 23 M-1·cm-1）。该三元复合物的光谱性质与CoCPD·L-Phe·N3-三元复合物明显不同。X射线吸收精细结构（XAFS）研究表明，这些差异可能是由于与Zn和CoCPD·D-Phe2复合物的八面体配位几何结构相比，三元叠氮复合物具有更规则的四面体配位球结构。
