Aerobic Ribonucleotide Reductase Research Articles

Regulation of T4 Phage Aerobic Ribonucleotide Reductase: SIMULTANEOUS ASSAY OF THE FOUR ACTIVITIES

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.

Allosteric effectors are required for subunit association in T4 phage ribonucleotide reductase.

Bacteriophage T4 encodes its own aerobic ribonucleotide reductase (RNR), which reduces ribonucleoside diphosphates to the corresponding deoxyribonucleoside diphosphates. T4 RNR is composed of homodimeric large (R1) and small (R2) subunits. Intricate regulation of enzymatic activity is accomplished by the binding of nucleotide effectors to R1. Berglund (Berglund, O. (1972) J. Biol. Chem. 247, 7270-7275) described similarities between T4 RNR and the corresponding enzyme from aerobic Escherichia coli. An important difference, however, is that T4 RNR forms a tight R1.R2 complex, while the E. coli R1 and R2 more readily dissociate. In this study we purified the phage R2 subunit from an overexpression vector constructed by Tseng et al. (Tseng, M., Hilfinger, J., He, P., and Greenberg, R. (1992) J. Bacteriol. 174, 5740-5744) and used this as an immunogen to generate polyclonal antiserum. Using co-immunoprecipitation techniques, we probed in vitro for interactions between the phage-induced R1 and R2 subunits. Our studies indicate that tight binding of the phage RNR subunits is completely dependent upon the known allosteric effectors of the enzyme. Once the R1.R2 holoenzyme has been formed it appears to be remarkably stable when in the presence of dATP. However, if dATP is removed, the R1.R2 complex readily dissociates.