Magnetic Circular Dichroism and Cobalt(II) Binding Equilibrium Studies of Escherichia coli Methionyl Aminopeptidase

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• 作者：James A. Larrabee ; Chin Hin Leung ; Rhonda L. Moore ; Thun Thamrong-nawasawat ; and Benjamin S. H. Wessler
• 刊名：Journal of the American Chemical Society
• 出版年：2004
• 出版时间：October 6, 2004
• 年：2004
• 卷：126
• 期：39
• 页码：12316 - 12324
• 全文大小：157K
• 年卷期：v.126,no.39(October 6, 2004)
• ISSN：1520-5126

文摘

Equilibrium dialysis of methionyl aminopeptidase from Escherichia coli (EcMetAP) monitored byatomic absorption spectrometry and magnetic circular dichroism (MCD) shows that the enzyme binds upto 1.1 ± 0.1 equiv of Co²⁺ in the metal concentration range likely to be found in vivo. The dissociationconstant, K_d, is estimated to be between 2.5 and 4.0 M. Analysis of the temperature and magnetizationbehavior of the two major peaks in the MCD spectrum at 495 and 567 nm suggests that these transitionsarise from Co²⁺ with different ground states. Ligand field calculations using AOMX are used to assign the495 nm peak to Co²⁺ in the 6-coordinate binding site and the 567 nm peak to Co²⁺ in the 5-coordinate site.This is further supported by the fact that the binding affinity of the Co²⁺ associated with the 567 nm peakis enhanced when the pH is increased from 7.5 to 9.0, consistent with having an imidazole ligand from ahistidine amino acid residue. On the basis of the MCD intensities, it is estimated that, when the 5-coordinatesite is fully occupied, 0.1 equiv of cobalt is in the 6-coordinate site. Even when the cobalt concentration isvery low, there is a small fraction of binuclear sites in EcMetAP formed through cooperative binding betweenthe 5- and 6-coordinate Co²⁺ ions. The magnetization behavior of the 6-coordinate Co²⁺ MCD peak isconsistent with an isolated pseudo-Kramer doublet ground state, suggesting that the cobalt ions in thebinuclear sites are not magnetically coupled.