De novo metallonucleases based on helix-loop-helix motifs
2004 (English)In: Chemistry - A European Journal, ISSN 0947-6539, Vol. 10, no 17, 4163-4170 p.Article in journal (Refereed) Published
Three new 42-mer peptides (PRI-III) designed to fold into a hairpin helix-loop-helix motif have been prepared. In the peptide sequence two (PRII-III) or four (PRI) copies of an unnatural amino acid bearing a triazacyclononane metal-ion binding site (ATANP) have been inserted in appropriate positions to allow the ligand subunits to face each other either within the same helix or between the two helices of the hairpin motif. Circular dichroism (C(d) studies in solution have shown that the apopeptides adopt a well-defined helix-loop-helix tertiary structure that dimerizes in solution at concentrations above 2001 µM to form a four-helix bundle. However, the helical content is strongly dependent on pH and metal-ion binding. Both protonation of the amines of the triazacyclononane units present in the ATANP lateral arm and complexation with ZnII ions cause a significant decrease of the helical content of the sequences. The ZnII complexes of the three peptides catalyze the transesterification of the RNA model substrate 2-hydroxypropyl-p-nitrophenyl phosphate (HPNP) with different efficiency. The best catalyst appears to be PRI-4ZnII. that is, the peptide incorporating four ATANP units. Michaelis-Menten saturation kinetics allowed us to estimate that substrate fully bound to the catalyst reacts 380 times faster than in its absence. The kinetic evidence suggests cooperativity between (at least two) metal ions: one activating the nucleophilic species (directly or indirectly) and the other facilitating nucleophilic attack by coordination of the phosphate.
Place, publisher, year, edition, pages
2004. Vol. 10, no 17, 4163-4170 p.
Enzyme models, Kinetics, Peptides, Phosphate cleavage, Zinc
Engineering and Technology
IdentifiersURN: urn:nbn:se:liu:diva-45637DOI: 10.1002/chem.200400160OAI: oai:DiVA.org:liu-45637DiVA: diva2:266533