- 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 × 10-6 M-1 s-1, and that of the imidazole catalyzed reaction was 1.66 × 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,Nilsson, Helena,Baltzer, Lars
-
-
Read Online
- Evaluation of Natural and Synthetic Phosphate Donors for the Improved Enzymatic Synthesis of Phosphate Monoesters
-
Undesired product hydrolysis along with large amounts of waste in form of inorganic monophosphate by-product are the main obstacles associated with the use of pyrophosphate in the phosphatase-catalyzed synthesis of phosphate monoesters on large scale. In order to overcome both limitations, we screened a broad range of natural and synthetic organic phosphate donors with several enzymes on a broad variety of hydroxyl-compounds. Among them, acetyl phosphate delivered stable product levels and high phospho-transfer efficiency at the lower functional pH-limit, which translated into excellent productivity. The protocol is generally applicable to acid phosphatases and compatible with a range of diverse substrates. Preparative-scale transformations using acetyl phosphate synthesized from cheap starting materials yielded multiple grams of various sugar phosphates with up to 433 g L?1 h?1 space-time yield and 75% reduction of barium phosphate waste. (Figure presented.).
- Tasnádi, Gábor,Jud, Wolfgang,Hall, Mélanie,Baldenius, Kai,Ditrich, Klaus,Faber, Kurt
-
supporting information
p. 2394 - 2401
(2018/05/14)
-