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Artificial Metalloenzymes: Equlibrium, structural and kinetic study of low molecular weight metal complexes with diverse catalytic effects

Final Activity Report Summary - METALLOENZYME MODELS (Artificial Metalloenzymes: Equlibrium, structural and kinetic study of low molecular weight metal complexes with diverse catalytic effects)

Recent biochemical studies pointed out the presence of relatively short, histidine-rich subunits with no fixed structure in a great number of proteins and enzymes that bind metal ions independently from other parts of the molecule, while having a great importance for the given function. Our aim was to use these metal binding sequences to develop artificial enzymes. These fragments without any modifications could be suitable for the binding of metal ions exclusively by side chain donor groups thus they can mimic the active centres of metalloproteins/enzymes and provide catalytic functions. During the first year of the project, five original multihistidine ligands were synthesised.

Two of the ligands contain the -HXHXH- sequence which is a well conserved fragment in zinc transporters, the assumed Zn2+ binding site in piruvate carboxylases and one of the Cu2+ binding sites in amiloid precursor protein. We have incorporated positively charged lysine residues into the fragments to prevent the aggregation of Zn2+-peptide complexes and to promote the binding of the species to the negatively charged DNA/RNA molecules. We have thoroughly characterised the Cu2+ and Zn2+ complexes of one of the ligands, Ac-HKHKH-NH2. In case of Zn2+, water soluble mixed hydroxo complexes were detected even above pH 9 which is likely related to the positively charged lysine e-NH3+ groups that increase the solubility and hinder the aggregation. The hydroxo mixed complexes may provide excellent possibility to develop artificial nucleases. In the presence of Cu2+ a species with the exclusive binding of the imidazole groups of the ligand predominated at pH ~ 6. This complex mimics the active centre of some type II copper proteins. The binding ability of the above mentioned complexes to Calf Thymus DNA have also been monitored and a very high affinity, based on metal ion coordination, was proved. These findings could be essential to achieve nuclease activity.

The N-terminal sequence of the Cu,Zn-SOD of Haemophilus ducreyi, HGDHMHNHDTK- was also synthesised. It is believed to play a fundamental role in the uptake of Cu2+, supporting the efficiency of protection against the reactive superoxide radical. The metal ion interaction of the ligand has been studied in details. The outstanding copper(II) sequestering ability of this peptide sequence in the physiological pH-range supports the chaperon-like role of this N-terminal fragment in the Cu,Zn-SOD of H. ducreyi. According to our results, amide coordination does not occur in the species being predominant between pH 6-8, which is very promising to develop functional models of copper-containing oxidases. In case of Zn2+ the formation of species with high stability was observed, too, suggesting, that this fragment mayalso participate in zinc(II)-chaperoning. The mixed hydroxo complex formed between pH 7-8 may be promising for the development of artificial nucleases.

Two fragments, Ac-(HHPHG)x-NH2 (x=1,2) from the histidine rich region (HRR) of Histidine-rich glycoprotein were synthesized. The HRR region is involved in the control of many biological functions where its strong interaction with different metal ions plays a significant role. Very recent results described the metal ion dependent antiangiogenic effect of peptide fragments related to the HRR. The metal ion coordination of the two fragments will be studied in the next year.

The Cu2+ complexes of a triamino-triol ligand have also been investigated. Equilibrium studies indicated a very complicated complex speciation. A bimetallic species formed around pH~8 showed an outstanding catalytic activity and selectivity for the hydrolysis of phosphomonoesters which is a very unique phenomenon amongst transition-metal based model complexes.

The characterisation of the metal complexes of the studied ligands may hopefully assist to explore the biological functions of the modelled metalloproteins/enzymes in more detail. In case of efficient models further derivatisation of the compounds could be achieved, which may lead to the development of chemical agents suitable for the recognition and hydrolysis of the desired substrates.