Catalysis of the cleavage of uridine 3'-2,2,2-trichloroethylphosphate by a designed helix-loop-helix motif peptide.
A 42-residue peptide that folds into a helix-loop-helix motif and dimerizes to form a four-helix bundle has been designed to catalyze the hydrolysis of phosphodiesters. The active site on the surface of the folded catalyst is composed of two histidine and four arginine residues, with the capacity to provide general acid, general base, and/or nucleophilic catalysis as well as transition state stabilization. Uridine 3'-2,2,2 trichloroethylphosphate (2) is a mimic of RNA with a leaving group pKa of 12.3. Its hydrolysis is energetically less favorable than that of commonly used model substrates with p-nitrophenyl leaving groups and therefore a more realistic model for the design of catalysts capable of cleaving RNA. The second-order rate constant for the hydrolysis of 2 at pH 7.0 by the polypeptide catalyst was 418 x 10(-6) M-1 s-1, and that of the imidazole catalyzed reaction was 1.66 x 10(-6) M-1 s-1. The pH dependence suggested that catalysis is due to the unprotonated form of a residue with a pKa of around 5.3, and the observed kinetic solvent isotope effect of 1.9 showed that there is significant hydrogen bonding in the transition state, consistent with general acid-base catalysis. The rate constant ratio k2(Pep)/k2(Im) of 252 is probably due to a combination of nucleophilic and general acid-base catalysis, as well as transition state stabilization. Substrate binding was weak since no sign of saturation kinetics was observed for substrate concentrations in the range from 5 to 40 mM. The results provide a platform for the further development of catalysts for RNA cleavage with a potential role in the development of drugs.
Razkin
Jesus
J
Department of Applied Chemistry, Public University of Navarra, 31006 Pamplona, Navarra, Spain. jrazk@unavarra.es
Nilsson
Helena
H
Baltzer
Lars
L
D020816:Amino Acid Motifs; D002384:Catalysis; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry*; D012313:RNA / Q000737:chemistry; D014542:Uridine Monophosphate / Q000031:analogs & derivatives* / Q000737:chemistry
D010455:Peptides; C525436:uridine 3'-2,2,2-trichloroethylphosphate; D012313:RNA; D014542:Uridine Monophosphate