Dual catalytic role of the metal ion in nickel-assisted peptide bond hydrolysis

Abstract

In our previous research we demonstrated the sequence specific peptide bond hydrolysis of the R₁-(Ser/Thr)-Xaa-His-Zaa-R₂ in the presence of Ni(II) ions. The molecular mechanism of this reaction includes an N–O acyl shift of the R₁ group from the Ser/Thr amine to the side chain hydroxyl group of this amino acid. The proposed role of the Ni(II) ion is to establish favorable geometry of the reacting groups. In this work we aimed to find out whether the crucial step of this reaction – the formation of the intermediate ester – is reversible. For this purpose we synthesized the test peptide Ac-QAASSHEQA-am, isolated and purified its intermediate ester under acidic conditions, and reacted it, alone, or in the presence of Ni(II) or Cu(II) ions at pH 8.2. We found that in the absence of either metal ion the ester was quickly and quantitatively (irreversibly) rearranged to the original peptide. Such reaction was prevented by either metal ion. Using Cu(II) ions as CD spectroscopic probe we showed that the metal binding structures of the ester and the final amine are practically identical. Molecular calculations of Ni(II) complexes indicated the presence of steric strain in the substrate, distorting the complex structure from planarity, and the absence of steric strain in the reaction products. These results demonstrated the dual catalytic role of the Ni(II) ion in this mechanism. Ni(II) facilitates the acyl shift by setting the peptide geometry, and prevents the reversal of the acyl shift, by stabilizing subsequent reaction products.

Introduction

Nickel and its compounds are toxic to humans, via inhalatory, dermal and alimentary routes. Contact allergy and respiratory cancers are two major health hazards related to nickel exposure, under environmental and occupational conditions, respectively. While it has become clear that Ni(II) ions are the ultimate toxic species, the exact molecular mechanisms of toxicity are the subject of dispute [1], [2], [3], [4], [5], [6], [7], [8], [9].

Nickel(II) dependent peptide bond hydrolysis (in brief, nickel-assisted hydrolysis) is a promising molecular concept for several aspects of nickel toxicity. This sequence specific reaction may impair function of important intracellular proteins, such as zinc finger transcription factors, at the same time yielding stable and reactive Ni(II) complexes, capable of damaging DNA and proteins [10], [11], [12], [13], [14], [15], [16]. Peptides susceptible to nickel hydrolysis have a general sequence R₁-(Ser/Thr)-Xaa-His-Zaa-R₂ (where Xaa and Zaa are any amino acids except of Pro and Cys as Xaa, and Cys as Zaa, and R₁ and R₂ are nonspecific N-terminal and C-terminal peptide sequences). The hydrolysis occurs, with an absolute selectivity, at the peptide bond preceding Ser/Thr. Scheme 1 illustrates key steps of the reaction [13], [14]. The reaction is enabled by the formation of a square-planar complex in which the Ni(II) ion is bonded by the imidazole nitrogen of the His residue and three preceding amide nitrogens. The first crucial step of the reaction is the N–O acyl shift of the carbonyl group of to hydrolysable R₁-Ser/Thr peptide bond to the Ser/Thr hydroxyl group. This step follows an apparent 1st order kinetic regime (k₁ in Scheme 1). The resulting ester intermediate product (IP) is unstable in water solution, hydrolyzing into two peptides, also according to the apparent 1st order kinetics (k₂ in Scheme 1). The C-terminal peptide product of this reaction step remains bonded to the Ni(II) ion. Therefore, the overall reaction is stoichiometric (1:1), rather than catalytic, with respect to Ni(II). The reaction rate is strongly dependent on the spatial orientations of side chains of amino acids belonging to the Ni(II) coordination site [17], and is strongly (up to a factor of 200) enhanced in the presence of bulky residues C-terminal to the His residue [18]. These facts confirm the notion that crucial role of the Ni(II) ion is largely geometrical. The square-planar coordination mode induces strain on the hydrolysable peptide bond, modulated by sterical crowding of the above mentioned side chains.

In the course of our studies we noted that the stability of IP varies significantly among various peptide sequences studied. In all cases the overall reaction was irreversible, resulting in the full conversion of the substrate into final products. This was assured by the irreversible character of the 2nd step of the reaction, the well-known acid- or base-catalyzed ester hydrolysis [19]. Nevertheless, the data we collected so far did not provide information on the reversibility of the 1st step, the IP formation. This issue can be quite important for many purposes, including the practical application of the reaction for purification of recombinant proteins [20].

We decided to study this issue using a peptide of the sequence Ac-QAASSHEQA-am (Ac- denotes the N-terminal acetylation, and -am the C-terminal amidation of the peptide chain), which yields a long-lived and easy to extract IP. This peptide was chosen on the basis of our current research on hydrolysis of filaggrin, a protein involved in the process of outer skin keratinization [21].