International Journal of Inorganic ChemistryVolume 2011 (2011), Article ID 395418, 4 pagesdoi:10.1155/2011/395418

Research Article

Improvement of Aminopeptidase Activity of Dizinc(II) Complexes by Increasing Substrate Accessibility

Md. Jamil Hossain, Akinobu Wada, Yasuhiro Igarashi, Kei-ichiro Aimono, Keisuke Suzuki, Katsuya Tone, and Hiroshi Sakiyama

Department of Material and Biological Chemistry, Faculty of Science, Yamagata University, Kojirakawa, Yamagata 990-8560, Japan

Received 22 November 2010; Accepted 2 March 2011

Academic Editor: Rabindranath Mukherjee

Copyright © 2011 Md. Jamil Hossain et al. Md. Jamil Hossain et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract.

A new dizinc(II) complex, [Zn₂(bhmp)(MeCO₂)₂]BPh₄ [(bhmp)⁻: 2,6-bis[bis(2-hydroxyethyl)aminomethyl]-4-methylphenolate anion], performs aminopeptidase activity to hydrolyze L-leucine-p-nitroanilide. As compared with a related dizinc(II) complex [Zn₂(bomp)(MeCO₂)₂]BPh₄ [(bomp)⁻: 2,6-bis[bis(2-methoxyethyl)aminomethyl]-4-methylphenolate anion], the activity of the present bhmp complex was about 80 times greater than that of the bomp complex. This is mainly because the substrate accessibility was improved by changing the terminal methoxy groups to hydroxyl groups.

1. Introduction

Aminopeptidases are exopeptidases that remove the N-terminal amino acid from a protein [1–5]. It is interesting that most of the well-characterized aminopeptidases contain dinuclear metal cores at their active sites, while carboxypeptidases, which remove the C-terminal amino acid, do not have dinuclear cores. The well-characterized aminopeptidases are leucine aminopeptidase (LAP, EC 3.4.11.1) [1], methionine aminopeptidase (MAP, EC 3.4.11.18) [2], aminopeptidase from Aeromonas proteolytica (AAP, EC 3.4.11.10) [3], Streptomyces griseus aminopeptidase (SGAP, EC 3.4.11.-) [4], and proline aminopeptidase (PAP, EC 3.4.11.9) [5]. LAP, AAP, and SGAP contain dizinc(II) cores at their active sites, while MAP contains a dicobalt(II) core and PAP contains a dimanganese(II) core.

With the intention of finding a minimum functional unit of aminopeptidase, Sakiyama and coworkers developed a dizinc(II) complex [Zn(bomp)(MeCO₂)₂]BPh₄ as the first functional model of aminopeptidase [6] [(bomp)⁻: 2,6-bis[bis(2-methoxyethyl)aminomethyl]-4-methylphenolate anion]; later, the aminopeptidase activity was improved by the introduction of stronger electron-withdrawing p-substituents [7] (10 times for the chloro-substituted complex [Zn₂(bocp)(MeCO₂)₂]BPh₄ and 250 times for the nitro-substituted complex [Zn₂(bonp)(MeCO₂)₂]BPh₄ [(bocp)⁻: 4-chloro-2,6-bis[bis(2-methoxyethyl)aminomethyl]phenolate anion; (bonp)⁻: 2,6-bis[bis(2-methoxyethyl)aminomethyl]-4-nitrophenolate anion]). From the kinetic studies, the substrate was proved to be incorporated within the dizinc center [7].

On the other hand, we found that the substrate accessibility of the bomp complexes was not good because of the steric hindrance of the terminal methoxy groups [7]. Therefore, in the present study, a new dizinc(II) complex, [Zn₂(bhmp)(MeCO₂)₂]BPh₄ (1), has been synthesized using a dinucleating ligand, bhmp⁻ [(bhmp)⁻: 2,6-bis[bis(2-hydroxyethyl)aminomethyl]-4-methylphenolate anion] [8], in which the methoxy groups of the bomp ligand are substituted into less-hindered hydroxyl groups, and the aminopeptidase activity of the complex was examined (see Scheme 1).

Figure 1: A /[substrate] versus [complex] plot in the hydrolysis of l-leucine-p-nitroanilide by 1 at nominal pH 8.

3.2. Effect of pH on the Aminopeptidase Function

The above procedure was also carried out at nominal pH’s varying from 7 to 10. At nominal pH 7, the activity was too small to determine the rate constant, but in the nominal pH range from 8 to 10, the second-order rate equation was found to be valid. The versus nominal pH plot for 1 is shown in Figure 2. The plot is sigmoidal around the nominal pH 9.5, and the data could be fitted using (2) with parameters  dm³ mo1⁻¹ s⁻¹ and , where is the rate constant for the deprotonated form.

Figure 2: versus nominal pH plots for the bhmp complex 1 (O) and the previous bomp complex (∆). The curves are drawn by (2) using the parameters in the text.

The result indicates that the reaction was promoted by the deprotonated form of the complex, which will be discussed in Section 3.3. In the case of the previous bomp complex, the data was reexamined using (2), and the parameters were determined as  dm³ mo1⁻¹ s⁻¹ and . The activity () of the present bhmp complex 1 was about 80 times greater than that of the bomp complex although the value at nominal pH 8 was about only 2 times greater. This is because the pKa value of 1 is slightly larger than that of the bomp complex.

3.3. Some Considerations about the Active Species

According to the crystal structure of related cobalt(II) complex [Co₂(bhmp)(MeCO₂)₂]BPh₄ [8], the coordination geometry around each zinc(II) ion is saturated, and the two zinc(II) ions are bridged by two acetate ions. However, the dissociation of the acetate ions occurs rather easily in an aqueous solution, affording vacant coordination sites for substrate incorporation. Indeed, in the cases of related cobalt(II) and nickel(II) complexes, [Co₂(bhmp)(MeCO₂)₂]ClO₄ and [Ni₂(bhmp)(MeCO₂)₂]ClO₄, the dominant species in an aqueous solution were identified as [M₂(bhmp)(H₂O)₄]²⁺ and [M₂(bhmp)(MeCO₂)(H₂O)₂]⁺ (M CoII, NiII) by analyzing the electronic spectra [10]. When one or two water molecules of [Zn₂(bhmp)(H₂O)₄]²⁺ are exchanged with the substrate and a remaining water molecule is deprotonated, a nucleophilic Zn-OH moiety is thought to attack the carbonyl carbon of the bound substrate.

4. Conclusion

For the purpose of improving the substrate accessibility, a new dizinc(II) complex, [Zn₂(bhmp)(MeCO₂)₂]BPh₄ (1), was synthesized, and its aminopeptidase activity was investigated. As compared with the previous dizinc(II) complex, [Zn₂(bomp)(MeCO₂)₂]BPh₄, the activity of 1 was about 80 times greater. Routine kinetic results have been deposited as supplementary material at 10.1155/2011/395418.

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