84-52-6Relevant articles and documents
Isothermal titration calorimetric study of RNase-A kinetics (cCMP → 3′-CMP) involving end-product inhibition
Spencer, Shawn D.,Raffa, Robert B.
, p. 1642 - 1647 (2004)
Purpose. Isothermal titration calorimetry (ITC) and progress curve analysis was used to measure the enzyme kinetic parameters (K M and k cat) of the hydrolysis of cCMP by RNase-A, a reaction that includes end-product competitive inhibition by 3'-CMP. Methods. The heat generated from injection of 9-15 μl cCMP (20 mM) into bovine pancreatic RNase-A (600 nM) in 50 mM Na+ acetate buffer (pH 5.5; 37°C) was monitored for 1500-2000 s. Thermal power (dQ/dt), equal to (1) /ΔH app × d(cCMP)/dt was recorded every 1 s. The end-product inhibition constant (K p) and enthalpy of the inhibitor binding interaction was obtained from the saturation data of 60 sequential injections of 3′-CMP (1.2 mM) into 0.05 mM RNase-A. The data of the plot of -d[cCMP]/dt against [cCMP] were fitted to kinetic equations incorporating K p to yield K M and k cat. Results. ΔH app for each run was obtained by integration of the progress curve. The plot of -d[cCMP]/dt against [cCMP] yielded the kinetic parameters K M = 105.3 μM, 121.6 μM, and 131.3 μM; k cat = 1.63 s-1, 1.56 s-1, and 1.71 s-1. The end-product bound with 1:1 stoichiometry and K p = 53.2 μM. Conclusions. The combination of progress curve analysis and ITC allowed rapid and facile measurement of the kinetic parameters for catalytic conversion of cCMP to 3′-CMP by RNase-A, a reaction complicated by end-product inhibition.
Guanidine-based polymer brushes grafted onto silica nanoparticles as efficient artificial phosphodiesterases
Savelli, Claudia,Salvio, Riccardo
, p. 5856 - 5863 (2015/03/31)
Polymer brushes grafted to the surface of silica nanoparticles were fabricated by atom-transfer radical polymerization (ATRP) and investigated as catalysts in the cleavage of phosphodiesters. The surfaces of silica nanoparticles were functionalized with an ATRP initiator. Surface-initiated ATRP reactions, in varying proportions, of a methacrylate moiety functionalized with a phenylguanidine moiety and an inert hydrophilic methacrylate species afforded hybrid nanoparticles that were characterized with potentiometric titrations, thermogravimetric analysis, and SEM. The activity of the hybrid nanoparticles was tested in the transesterification of the RNA model compound 2-hydroxypropyl para-nitrophenylphosphate (HPNP) and diribonucleoside monophosphates. A high catalytic efficiency and a remarkable effective molarity, thus overcoming the effective molarities previously observed for comparable systems, indicate the existence of an effective cooperation of the guanidine/guanidinium units and a high level of preorganization in the nanostructure. The investigated system also exhibits a marked and unprecedented selectivity for the diribonucleoside sequence CpA. The results presented open up the way for a novel and straightforward strategy for the preparation of supramolecular catalysts.
Dinuclear Zn2+ complexes in the hydrolysis of the phosphodiester linkage in a diribonucleoside monophosphate diester.
Yashiro, Morio,Kaneiwa, Hideki,Onaka, Kenichi,Komiyama, Makoto
, p. 605 - 610 (2007/10/03)
Dizinc complexes that were formed from 2:1 mixtures of Zn(NO3)2 and dinucleating ligands TPHP (1), TPmX (2) or TPpX (3) in aqueous solutions efficiently hydrolyzed diribonucleoside monophosphate diesters (NpN) under mild conditions. The dinucleating ligand affected the structure of the aquo-hydroxo-dizinc core, resulting in different characteristics in the catalytic activities towards NpN cleavage. The pH-rate profile of ApA cleavage in the presence of (Zn2+)(2)-1 was sigmoidal, whereas those of (Zn2+)(2)-2 and (Zn2+)(2)-3 were bell-shaped. The pH titration study indicated that (Zn2+)(2)-1 dissociates only one aquo proton (up to pH 12), whereas (Zn2+)(2)-2 dissociates three aquo protons (up to pH 10.7). The observed differences in the pH-rate profile are attributable to the various distributions of the monohydroxo-dizinc species, which are responsible for NpN cleavage. As compared to that using (Zn2+)(2)-1, the NpN cleavage using (Zn2+)(2)-2 showed a greater rate constant, with a higher product ratio of 3'-NMP/2'-NMP. The saturation behaviors of the rate, with regard to the concentration of NpN, were analyzed by Michaelis-Menten type kinetics. Although the binding of (Zn2+)(2)-2 to ApA was weaker than that of (Zn2+)(2)-1, (Zn2+)(2)-2 showed a greater kcat value than (Zn2+)(2)-1, resulting in higher ApA cleavage activity of the former.
