deoxyguanosine-triphosphate has been researched along with cobamamide* in 2 studies
2 other study(ies) available for deoxyguanosine-triphosphate and cobamamide
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Thermodynamic and kinetic studies on carbon-cobalt bond homolysis by ribonucleoside triphosphate reductase: the importance of entropy in catalysis.
In the catalytic mechanism of nucleotide reduction, ribonucleoside triphosphate reductase (RTPR) from Lactobacillus leichmannii catalyzes the homolytic cleavage of the carbon-cobalt bond of adenosylcobalamin (AdoCbl) at a rate approximately 10(11)-fold faster than the uncatalyzed reaction. Model systems have suggested hypotheses for the thermodynamic basis of this reaction, but relevant measurements of the enzymatic reaction have been lacking. To address this question in a system for which the microscopic rate constants can be measured as a function of temperature, we examined the RTPR-catalyzed exchange reaction. RTPR, in the presence of allosteric effector dGTP and in the absence of substrate, catalyzes carbon-cobalt bond homolysis and formation of a thiyl radical from an active-site cysteine in a concerted fashion [Licht, S., Booker, S. , Stubbe, J. (1999) Biochemistry 38, 1221-1233]. Both the kinetics of cob(II)alamin formation and the amounts of cob(II)alamin formed have been studied as a function of AdoCbl concentration and temperature. Analysis of these data has allowed calculation of a DeltaH of 20 kcal/mol, a DeltaS of 70 cal mol-1 K-1, a DeltaH of 46 kcal/mol, and a DeltaS of 96 cal mol-1 K-1 for carbon-cobalt bond homolysis/thiyl radical formation. The results further show that the enzyme perturbs the equilibrium between the reactant (AdoCbl-bound) state and the product (cob(II)alamin/5'-deoxyadenosine (5'-dA)/thiyl radical state, making them approximately equal in energy. The thermodynamic perturbation, in addition to transition-state stabilization, is required for the large rate acceleration observed. Entropic, rather than enthalpic, factors make the largest contribution in both cases. Topics: Allosteric Regulation; Allosteric Site; Calorimetry; Catalysis; Cobalt; Cobamides; Deoxyguanine Nucleotides; Entropy; Kinetics; Lactobacillus; Ribonucleotide Reductases; Thermodynamics | 1999 |
Adenosylcobalamin-dependent ribonucleoside triphosphate reductase from Lactobacillus leichmannii. Rapid, improved purification involving dGTP-based affinity chromatography plus biophysical characterization studies demonstrating enhanced, "crystallographic
Ribonucleoside triphosphate reductase (RTPR, EC 1.17.4.2) from Lactobacillus leichmannii is a 5'-deoxyadenosylcobalamin-dependent (AdoCbl; Coenzyme B12) enzyme. RTPR is also a prototypical adenosylcobalamin-dependent ribonucleotide reductase, one that, as its name indicates, converts ribonucleoside triphosphates (NTP) to deoxyribonucleoside triphosphates (dNTP). Upon substrate binding to RTPR, AdoCbl's cobalt-carbon bond is cleaved to generate cob(II)alamin, 5'-deoxyadenosine, and the cysteine (C408) derived thiyl radical. Five key cysteines (Cys 119, 408, 419, 731, and 736), from among the ten total cysteines, are involved in RTPR's catalytic mechanism. A critical examination of the RTPR isolation and purification literature suggested that the purification protocol currently used results in RTPR which contains 2040% microheterogeneity, along with minor contamination by other proteins. In addition, no report of crystalline RTPR has ever appeared. The literature indicates that irreversible cysteine oxidation (e.g., to -SO2H or -SO3H) is one highly plausible reason for the microheterogeneity of RTPR. The literature also indicates that improvement in the level of enzyme purity is the most effective next step in coaxing enzymes to crystallize that have previously failed to do so. A shortened, improved purification of RTPR has been developed, one involving a shorter purification time, a lower pH, a higher concentration of the more effective reductant DTT (all designed to help protect the cysteines from oxidation), and a final step utilizing our recently reported, improved dGTP-based affinity chromatography resin. The resultant RTPR is approximately 20-30% higher in both specific activity and in its ability to undergo single turnovers, and is homogeneous by mass spectrometry and dynamic light scattering. Additionally, the revised purification procedure eliminates > 30 proteins present in 2-3% amounts along with damaged RTPR that does not bind properly (i.e. tightly) to the dGTP-affinity resin. Finally, dGTP-based affinity chromatography purified RTPR has yielded the first reported, albeit small, single crystals of RTPR. Topics: Allosteric Regulation; Allosteric Site; Chromatography, Affinity; Chromatography, Agarose; Cobamides; Crystallization; Crystallography; Deoxyguanine Nucleotides; Electrophoresis, Polyacrylamide Gel; Escherichia coli; Hydrogen-Ion Concentration; Lactobacillus; Mass Spectrometry; Models, Chemical; Resins, Synthetic; Ribonucleotide Reductases; Scattering, Radiation; Sodium Acetate; Substrate Specificity | 1999 |