Abstract
We have devised an assay procedure that permits simultaneous monitoring of the four activities of ribonucleotide reductase. Using this assay, we have compared the reduction of all four substrates by the T4 bacteriophage aerobic ribonucleotide reductase within different allosteric environments. Specifically, we compared the relative turnover rates by the enzyme when activated with "in vivo" concentrations of the known allosteric effectors versus activation by ATP alone. Consistent with the known allosteric properties of this enzyme, our results show that ATP does act as a general activator, although the rate of purine nucleotide reduction was approximately 5% of the rate for the pyrimidine nucleotides. However, addition of the allosteric effectors at their estimated physiological concentrations dramatically changed the relative rates of substrate reduction, creating a more "balanced" pool of products. Addition of the substrates at their respective in vivo concentrations further pushed rates of product formation toward a ratio similar to the base composition of the T4 genome. The similarity of the product profile produced under in vivo conditions to the genomic composition of T4 phage is discussed.
Highlights
The first committed step in DNA biosynthesis occurs by direct reduction of the 2Ј-hydroxyl of ribonucleotides and is catalyzed by the enzyme ribonucleotide reductase
In other words, when the known allosteric effectors and substrates are supplied together in a single reaction mixture at their estimated physiological or in vivo concentrations, what are the relative rates of formation of each of the products? This question is of particular interest because, in vivo, T4 ribonucleotide reductase functions as part of an enzyme complex [4] and the possibilitiy that intracellular reaction fluxes are controlled by protein-protein interactions must be considered
The HPLC method gives good resolution of all possible ribonucleotide reductase dNDP products as well as resolution of the dNTPs that may be added as allosteric effectors
Summary
In other words, when the known allosteric effectors and substrates are supplied together in a single reaction mixture at their estimated physiological or in vivo concentrations, what are the relative rates of formation of each of the products? The ability to follow the reduction of all substrates simultaneously and in a quantitative way, under varied conditions, was the goal of this method development We describe such an assay and its application to the T4 bacteriophage aerobic ribonucleotide reductase, in which the product profile was determined under different allosteric states. When a mixture of ribo- and deoxyribonucleotides is applied to a boronate column the deoxyribonucleotides pass through while the ribonucleotides are retained This type of affinity chromatography is frequently used to separate dNTPs from rNTPs derived from cell lysates when doing dNTP pool assays [5]. The HPLC step uses a strong anion exchange column and an ammonium phosphate gradient to resolve the individual deoxyribonucleotides into quantifiable peaks
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