Ribonucleotide reductase allosteric regulation

For example, the rate of reaction of ribonucleotide reductase is regulated by deoxyadenosine triphosphate (dATP) which is a product of the pathway for which ribonucleotide reductase is the committing step (Fig. 8.9). Note that dATP is neither a substrate nor a product of ribonucleotide reductase itself rather, it is an allosteric inhibitor. If the pathway (NDP dNTP) is running at a rate too high for the rate at which dNTPs are being used (for DNA synthesis), the concentrations of the dNTPs will rise, including [dATP], The increase will "feed back" to ribonucleotide reductase by the... 

Regulation of the balance of the concentrations of the four deoxyribonucleotides depends on the properties of only two enzymes, the ribonucleotide reductase complex and deoxy-CMP deaminase. The balance between pyrimidine deoxynucleotides is brought about by the properties of the deoxy-CMP deaminase, which is inhibited by deoxy-TTP and stimulated by deoxy-CTP. The ribonucleotide reductase also possesses allosteric sites which bind all four deoxynucleotide triphosphates, the effect of which is to maintain approximately similar concentrations of all the triphosphates. 

The regulation of ribonucleotide reductase is complex. The substrate-specificity and activity of the enzyme are controlled by two allosteric binding sites (a and b) in the R1 subunits. ATP and dATP increase or reduce the activity of the reductase by binding at site a. Other nucleotides interact with site b, and thereby alter the enzyme s specificity. 

The manner in which the reduction of ribonucleotides to deoxyribonucleotides is regulated has been studied with reductases from relatively few species. The enzymes from E. coli and from Novikoff s rat liver tumor have a complex pattern of inhibition and activation (fig. 23.25). ATP activates the reduction of both CDP and UDP. As dTTP is formed by metabolism of both dCDP and dUDP, it activates GDP reduction, and as dGTP accumulates, it activates ADP reduction. Finally, accumulation of dATP causes inhibition of the reduction of all substrates. This regulation is reinforced by dGTP inhibition of the reduction of GDP, UDP, and CDP and by dTTP inhibition of the reduction of the pyrimidine substrates. Because evidence suggests that ribonucleotide reductase may be the rate-limiting step in deoxyribonucleotide synthesis in at least some animal cells, these allosteric effects may be important in controlling deoxyribonucleotide synthesis. 

The reduction of ribonucleotides to deoxyribonucleotides is precisely controlled by allosteric interactions. Each polypeptide ot the R1 subunit of the aerobic E. coli ribonucleotide reductase contains two allosteric sites one of them controls the overall activity of the enzyme, whereas the other regulates substrate specificity (Figure 25.16). The overall catalytic activity of ribonucleotide reductase is diminished by the binding of dATP, which signals an abundance of deoxyribonucleotides. The binding of ATP reverses this teed back inhibition. The binding of dATP or ATP to the substrate-specificity... 

Figure 25.16 Regulation of ribonucleotide reductase. (A) Each subunit in the R1 dimer contains two allosteric sites In addition to the active site. One site regulates ihe overall activity and the other site regulates substrate specificity,...

SiNTCHAK, M. D., ArJARA, G., Kellogg, B. A., Stubbe, J., Drennan, C. L. (2002) The crystal structure of class II ribonucleotide reductase reveals how an allosterically regulated monomer mimics a dimer, Nat. Struct. Biol. 9, 293-300. 

This reaction is the initial step in production of deoxynucleoside triphosphates (dNTPs) for DNA synthesis. Four dNTPs are needed for DNA synthesisdATP, dCTP, dGTP, and TTP (TTP comes from dUDP). The proportions of these dNTPs need to be balanced for efficient synthesis. The feedback molecules, or effectors, are the final products of the pathway, the dNTPs, and they act on ribonucleotide reductase to modify its substrate specificity in order to balance the production of dNTPs. In addition to the specificity control site, an additional allosteric control site determines the overall rate of the reaction. At this site, ATP acts as a positive regulator and dATP as a negative regulator. 

Reichard, P. (2002). Ribonucleotide reductases The evolution of allosteric regulation. Archives of Biochemistry and Biophysics, 397, 149-155. 

DNA synthesis depends on a balanced supply of the four deoxyribonucleotides [1]. In all living organisms, with no exception so far, this is achieved by reduction of the corresponding ribonucleotides (substrates can be either ribonucleoside diphosphates NDP or ribonucleoside triphosphates NTP) by NADPH (Scheme 10-1), through a complex free radical chemistry. The substrate specificity is modulated by a sophisticated allosteric mechanism which makes it possible for a single protein to regulate the reduction of all four conunon ribonucleotides. This aspect will not be discussed here. Three well-characterized classes of ribonucleotide reductases (RNRs) have been described so far, which all are radical metalloenzymes [2-5]. 

The formation of deoxyribonucleotides requires ribonucleotide reductase activity, which catalyzes the reduction of ribose on nucleotide diphosphate substrates to 2 -deoxyribose. Substrates for the enzyme include adenosine diphosphate (ADP), guanosine diphosphate (GDP), cytidine diphosphate (CDP), and uridine diphosphate (UDP). Regulation of the enzyme is complex. There are two major allosteric sites. One controls the overall activity of the enzyme, whereas the other determines the substrate specificity of the enzyme. All deoxyribonucleotides are synthesized using this one enzyme. 

The regulation of ribonucleotide reductase is quite complex. The enzyme contains two allosteric sites, one controlling the activity of the enzyme and the other controlling the substrate specificity of the enzyme. ATP bound to the activity site activates the enzyme dATP bound to this site inhibits the enzyme. Substrate specificity is more complex. ATP bound to the substrate site activates the reduction of pyrimidines (CDP and UDP), to form dCDP and dUDP. The dUDP is not used for DNA synthesis rather, it is used to produce dTMP (see below). Once dTMP is produced, it is phosphorylated to dTTP, which then binds to the substrate site and induces the reduction of GDP. As dGTP accumulates, it replaces dTTP in the substrate site and allows ADP to be reduced to dADP. This leads to the accumulation of dATP, which will inhibit the overall activity of the enzyme. These allosteric changes are summarized in Table 41.3. 

An increase in dCTP levels signals that the cell has ample deoxynucleotides for DNA synthesis and that there is a need for thymidylate synthesis. An increase in dTTP levels signals that the activity of thymidylate synthase can be decreased, and the inhibition of dCMP deaminase by dTTP reduces the input of dUMP into the pathway. While ribonucleotide reductase is subject to regulation by other deoxynucleotides, it is not subject to allosteric regulation by dCTP. Instead it appears that regulation of dCMP deaminase provides a second control point for the generation of deoxynucleotides in the cell. 

A huge amount of data, often still fragmentary, on allosteric regulation of all different types of ribonucleotide reductases awaits unambiguous analyses as the ones described. It is certainly not overstating to say that, next to the chemistry of ribonucleotide reduction, the enzymes interaction with nucleotides and the physiologic consequences is one of the most peculiar pieces of biochemistry. 

Reichard for allosteric regulation of ribonucleotide reduction. One has to conclude that replitase-associated ribonucleotide reductase remains freely accessible for effectors from outside ... 

The ribonucleotide reductases form deoxycytidine phosphates which are destined for incorporation into DNA these nucleotides are diverted to some extent into the deoxyuridylate pool, and thence into the thymine nucleotides, through the action of deoxycytidylate deaminase. This diversionary flow into the thymine pathway is regulated by the demand for the terminal products, dTTP and dCTP the valve controlling this flow is deoxycytidylate deaminase, the activity of which is subject to allosteric regulation by dTTP and dCTP. 

As with the bacterial reductases, a complex pattern of activation and inhibition by nucleoside triphosphates has been demonstrated for the tumor reductase dATP inhibits reduction of all four substrates (S2). A similar pattern of nucleotide regulatory effects was found with partly purified reductase from rat embryo extracts (S5). Thus, presuming that analogy with the bacterial ribonucleotide reductases is valid, it would appear that the animal reductases are allosteric enzymes subject to a complicated regulation by nucleotides again, the function of such regulation would seem to be that of ensuring a balanced supply of deoxyribonucleo-tides for DNA synthesis. 

Reichard P. 2002. Ribonucleotide reductases the evolution of allosteric regulation. Arch Biochem Biophys 397 149-155. 

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