CHARACTERIZATION OF AN INHIBITORY METAL-BINDING SITE IN CARBOXYPEPTIDASE-A
CHARACTERIZATION OF AN INHIBITORY METAL-BINDING SITE IN CARBOXYPEPTIDASE-A
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DOI：
10.1021/bi00224a007
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发表时间：
1991-03-12
期刊：
影响因子：
2.9
通讯作者：
AULD, DS
中科院分区：
文献类型：
--
作者：
LARSEN, KS;AULD, DS
使用荧光肽底物 Dns-Gly-Ala-Phe 在停流条件下检查金属离子抑制牛羧肽酶 A ([CPD)Zn]) 催化的特异性。 该酶被 Zn(II)、Pb(II) 和 Cd(II) 竞争性抑制，在 pH 7.5、25 摄氏度的 0.5 M NaCl 中，表观 K(I) 值为 2.4 X 10(-5)、4.8 X 10(-5) 和 1.1 X 10(-2) M。 k(cat)/K(m) 值 7.3 X 10(6) M-1 s-1 在 1 X 10(-4) M Mn(II) 或 Cu(II) 以及 1 X 10(-2) M Co(II)、Ni(II)、Hg(II) 或 Pt(IV) 时受影响小于 10%。 Zn(II) 和 Pb(II) 是互斥的抑制剂。 先前对 Zn(II) 抑制的 pH 依赖性的研究 [Larsen, K. S., & Auld, D. S. (1989) Biochemistry 28, 9620] 表明 [(CPD)Zn] 被一氢氧化锌复合物 ZnOH+ 选择性抑制，并且配体 LH 在酶的抑制位点电离 (pK(LH) 5.8) 对其具有约束力。本研究可以进一步定义这种抑制性锌位点。 可电离配体 (LH) 被指定为 Glu-270，因为该残基的特定化学修饰使 [(CPD)Zn] 对 Zn(II) 和 Pb(II) 的结合亲和力分别降低了 60 倍和 200 倍以上。 Glu-270 配位的金属氢氧化物和催化金属离子之间的桥联相互作用与 Zn(II) 和 Pb(II) 诱导钴羧肽酶 A ([CPD)Co]) 电子吸收光谱扰动的能力有关。 瞬态 [(CPD)Co].Zn 和稳定 [(CPD)Co].Pb 二金属酶物种的光谱特征均在于 560 nm 处的最大值 (DELTA-epsilon-560 = +60 M-1 cm-1) 以及 520 和 585 nm 处的肩峰。 [(CPD)Co].Pb 与叠氮化物络合物的光谱滴定表明，Pb(II) 阻止叠氮化物与催化 Co(II) 离子结合，为抑制性金属一氢氧化物与催化金属离子结合的观点提供了进一步的支持。 抑制位点的显着金属离子特异性可以部分地通过所检查的金属离子的一羟基复合物的稳定性来解释。 基于活性位点的电荷考虑，金属一氢氧化物络合物可能还携带来自支持电解质的一个阴离子（Cl-）；中性络合物 Zn(OH)Cl 和 Pb(OH)Cl 的 K(I) 值约为 5 x 10(-7) M。如果抑制性金属一氢氧化物络合物被多齿配体电荷中和，则可以预期更低的抑制常数。 这种抑制复合物对于锌酶的调节和/或毒理学过程可能非常重要。
The specificity of metal ion inhibition of bovine carboxypeptidase A ([CPD)Zn]) catalysis is examined under stopped-flow conditions with the use of the fluorescent peptide substrate Dns-Gly-Ala-Phe. The enzyme is inhibited competitively by Zn(II), Pb(II), and Cd(II) with apparent K(I) values of 2.4 X 10(-5), 4.8 X 10(-5), and 1.1 X 10(-2) M in 0.5 M NaCl at pH 7.5 and 25-degrees-C. The k(cat)/K(m) value, 7.3 X 10(6) M-1 s-1, is affected less than 10% at 1 X 10(-4) M Mn(II) or Cu(II) and at 1 X 10(-2) M Co(II), Ni(II), Hg(II), or Pt(IV). Zn(II) and Pb(II) are mutually exclusive inhibitors. Previous studies of the pH dependence of Zn(II) inhibition [Larsen, K. S., & Auld, D. S. (1989) Biochemistry 28, 9620] indicated that [(CPD)Zn] is selectively inhibited by a zinc monohydroxide complex, ZnOH+, and that ionization of a ligand, LH, in the enzyme's inhibitory site (pK(LH) 5.8) is obligatory for its binding. The present study allows further definition of this inhibitory zinc site. The ionizable ligand (LH) is assigned to Glu-270, since specific chemical modification of this residue decreases the binding affinity of [(CPD)Zn] for Zn(II) and Pb(II) by more than 60- and 200-fold, respectively. A bridging interaction between the Glu-270-coordinated metal hydroxide and the catalytic metal ion is implicated from the ability of Zn(II) and Pb(II) to induce a perturbation in the electronic absorption spectrum of cobalt carboxypeptidase A ([CPD)Co]). The spectra of the transient [(CPD)Co].Zn and stable [(CPD)Co].Pb dimetallic enzyme species are both characterized by a maximum at 560 nm (DELTA-epsilon-560 = +60 M-1 cm-1) and shoulders at 520 and 585 nm. Spectral titrations of the [(CPD)Co].Pb complex with azide show that Pb(II) prevents azide binding to the catalytic Co(II) ion, providing further support for the idea that the inhibitory metal monohydroxide binds to the catalytic metal ion. The marked metal ion specificity of the inhibitory site can in part be accounted for by the stabilities of monohydroxide complexes of the metal ions examined. On the basis of charge considerations in the active site, the metal monohydroxide complex likely also carries one anion (Cl-) from the supporting electrolyte; the K(I) values for the neutral complexes Zn(OH)Cl and Pb(OH)Cl are approximately 5 x 10(-7) M. If the inhibitory metal monohydroxide complex is charge neutralized by a multidentate ligand, an even lower inhibition constant can be anticipated. Such inhibitory complexes could be very important to regulatory and/or toxicological processes of zinc enzymes.