Peptide Catalyst for Hydrolysis of Phosphodiesters
A R T I C L E S
promises insights into fundamental aspects of enzyme catalysis.
Phosphodiester linkages are extremely stable and resistant to
hydrolysis5 so that enzymes that have evolved to catalyze their
hydrolysis are among the most efficient ones known, with rate
enhancements of 18 orders of magnitude or more, stemming
from a combination of general acid and general base catalysis,
transition stabilization, and proximity effects. While nature has
evolved a catalytic machinery based on the reactivity of histidine
residues, this is not necessarily the only arrangement capable
of large rate enhancements, though the design of new man-
made artificial nucleases to match the high efficiency of natural
ones constitutes a formidable but very interesting challenge.6
Many artificial nucleases have already been described.7 So
far, most of the effort has been focused on metallonucleases,
since many natural enzymes that cleave phosphodiester bonds
incorporate two or three metal ions in their active site.8 These
enzymes take advantage of the Lewis acid properties of metals
for biochemical reactions, their affinity for basic nitrogen and
oxygen donor ligands, the capacity to support large aromatic
architectures capable of π interactions with the nucleic acid
building blocks, the ability to directly hydrolyze phosphodiester
linkages, and the possibility of promoting redox chemistry or
generate reactive oxygen-derived species.9 But the time-de-
pendent exchange reactions of metal ions7a and the slow
penetration to cells of metal ion chelates tethered to oligonucle-
otide based drugs7a together with the need of a more efficient
binding to the target nucleic acid,7b,c often too dependent on
direct coordination to the metal center, are significant problems
still to be solved.
Figure 1. Modeled structure of the helix-loop-helix motif and amino
acid sequence of HNI. The one letter code for the amino acids is used (A
is alanine, D is aspartic acid, E is glutamic acid, F is phenylalanine, G is
glycine, H is histidine, I is isoleucine, K is lysine, L is leucine, N is
asparagine, P is proline, Q is glutamine, R is arginine, V is valine, and Nle
is norleucine). The C-terminal is amidated, and the N-terminal is acetylated.
Only the side chains of the residues designed for the catalytic site are shown,
and although the active peptide is in the dimer form, only the monomer is
shown for clarity of presentation.
Recently, a very promising result16 from Go¨bel’s group has
demonstrated that tris(2-aminobenzimidazoles) attached to DNA
oligonucleotides act as very efficient nucleases, showing
substrate and site selectivity as well as saturation kinetics, thus
proving that they could compete with the metal-dependent
artificial nucleases.
We have found (unpublished results) that a catalytic site
containing four Arg and two His residues on the surface of a
helix-loop-helix motif, with two Arg and one His in each helix
(Figure 1), is optimal in the reaction of the activated substrate
2-hydroxypropyl p-nitrophenylphosphate, HPNP (1) (Chart 1).
While the study of activated substrates serve as good “early
stage” model systems, the catalysis of, e.g., p-nitrophenyl esters
Among metal-free catalysts, remarkable results showing both
cleavage activity and sequence recognition have been obtained
using diethylenetriamine-ODN (oligodeoxyribonucleotides),10
imidazole containing ODNs,11 peptides conjugated to ODNs,12
methanephosphonate ODNs with diimidazole or an imidazole/
amino cleaving agent,13 a PNA (peptide nucleic acid) conjugate
of neamine,14 and a PNA linked diethylenetriamine moiety.15
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