Regulation of Ribonucleotide Reductase

CURRENT TOPICS IN CELLULAR REGULATION, VOLUME 19

Regulation of Ribonucleotide Reductase ARNE HOLMGREN Department Karolinska Stockholm,

of Chemistry Institute Sweden

I. Introduction A. General Outline B. History C. Assay of Ribonucleotide Reductase II. Structure of Enzymes A. Ribonucleoside Diphosphate Reductase of E. coli Β . Ribonucleoside Triphosphate Reductase of L . leichmannii C. M a m m a l i a n Reductases D. Viral Reductases III. Hydrogen Transport Mechanism A. Thioredoxin System B. Glutaredoxin System C. Phage T 4 Thioredoxin D. Thiol Redox Control of Activity IV. Allosteric Control A. E. coli Enzyme B. Lactobacillus Enzyme C. M a m m a h a n Enzymes D. P h a g e T 4 Enzyme V. Regulation of Enzyme Synthesis VI. Ribonucleotide Reductase and Regulation of DNA Synthesis A. Deoxyribonucleotide Pools B. Multienzyme Complexes and Metabolic Channeling VII. Drugs Affecting Ribonucleotide Reductase VIII. Ribonucleotide Reductase and Immune Dysfunction References

47 47 49 51 52 53 55 56 57 57 57 60 62 63 64 64 66 66 68 68 69 69 70 71 72 73

I. Introduction A. General Outline Ribonucleotide reductase catalyzes the first unique step of DNA syn­ thesis by converting the four ribonucleotides to the corresponding deoxyribonucleotides. Deoxyribonucleotides are highly specialized metabolites playing only limited roles apart from their function as DNA precursors. They occur in surprisingly low overall concentrations in cells. A rate-limiting function of ribonucleotide reduction in DNA replication is observed in many systems. The regulation of ribonu47 Copyright © 1981 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-152819-7

48

ARNE HOLMGREN

cleotide reductase is thus of major interest for the understanding of growth regulation under normal and pathological conditions. All ribonucleotide reductases catalyze the replacement by hydrogen of the OH group at the 2' position of the ribose moiety of a ribonu­ cleotide (Fig. 1). The enzymes have been purified from many sources, including Escherichia coli, Lactobacillus leichmannii, and various mammalian tissues, and can be divided into two classes {119), One class is represented by the enzyme from Lactobacillus leichmannii; this is a monomer (MW 76,000) using adenosylcobalamin (coenzyme B12) as a dissociable cofactor and ribonucleoside triphosphates as substrates. The second class is represented by the E, coli enzyme (MW 240,000). This uses ribonucleoside diphosphatase as substrates; it is composed of two nonidentical subunits, proteins B l and B2, both required for ac­ tivity. The E. coli enzyme has no requirement for adenosylcobalamin; instead protein B2 contains two bound iron atoms and a tyrosyl free radical as part of the polypeptide chain. The iron and tyrosyl radical are involved in the enzyme mechanism. The mammalian reductases show properties similar to the E, coli enzyme. During purification, ribonucleotide reductase is separated from its natural hydrogen donor substrate; the purified enzymes require certain dithiols such as dihydrolipoate or dithiothreitol. Efforts to find the physiological hydrogen donor have resulted in the discovery of two novel types of hydrogen carrier systems both involving small proteins (thioredoxin and glutaredoxin) with oxidation-reduction active di­ sulfides. The first to be identified was the thioredoxin system, where the dithiol form of thioredoxin is the immediate substrate for the reductase and is kept reduced by NADPH and the flavoprotein, thoredoxin reduc­ tase. The second is the glutaredoxin system where glutathione (GSH) is the hydrogen donor substrate in the presence of glutaredoxin. The true in vivo hydrogen donor is presently unknown; genetic results show that the thioredoxin system is nonessential for ribonucleotide reductase.

OH

OH

F I G . 1. Reduction of a ribonucleotide to a deoxyribonucleotide. P „ denotes a diphosphoryl or a triphosphoryl residue.

REGULATION OF RIBONUCLEOTIDE REDUCTASE

49

Both systems may possibly substitute for each other under different growth conditions; together they may regulate ribonucleotide reduc­ tase activity. The purified ribonucleotide reductases can reduce all four ribonu­ cleotides. The enzyme from Escherichia coli and mammalian cells is subject to an unprecedented type of allosteric regulation by positive and negative effectors. The regulatory properties of the enzjone may mark an evolutionary adaptation to the unique situation of a single enzyme catalyzing the first reaction in each of four parallel pathways. The outstanding feature of this regulation is that end products of these pathways (nucleoside triphosphates) modify the substrate specificity of the enzyme as well as the overall activity. The allosteric behavior of the mammalian enzyme has provided an interesting explanation for hered­ itary immune-deficiency diseases caused by a lack of either adenosine deaminase or purine nucleoside Phosphorylase. The level of ribonu­ cleotide reductase synthesis is regulated at the genetic level; higher enzyme activity is observed in growing cells. Enzyme synthesis is in­ duced by genetic derepression during inhibition of DNA synthesis and "SOS repair" of DNA. The larger DNA viruses carry genes for ribonu­ cleotide reductases that are expressed upon infection of the host cells. The activity of the reductase is highly influenced by its environment; in vivo it may be part of a multienzyme complex of precursorsynthesizing enzymes that channels deoxyribonucleotides to the repli­ cation fork to give a local high effective deoxyribonucleotide concen­ tration. The presumed rate-limiting function of ribonucleotide reductase in DNA synthesis has been utilized for designing inhibitors of the enzyme with chemotherapeutic potential. Drugs affecting the reaction mecha­ nism of the enzymes as well as inhibitors operating via the allosteric mechanism have been described. This article will mainly focus on general principles of wellcharacterized systems and recent data. No attempt is made to cover all published results. For further information the reader will be referred to the excellent reviews by Thelander and Reichard (119), Hogenkamp and Sando (58), and Follmann (44). B. History The first experiments suggesting that deoxyribonucleotides are formed by direct reduction of ribonucleotides were in vivo experiments in the rat (55). In 1950, Hammarsten al. (55) found that pyrimidines in the form of ribonucleosides were efficiently incorporated into DNA.

50

ARNE HOLMGREN

Subsequently, Rose and Schweigert (104) administered pyrimidine ribonucleotides labeled in both the sugar and the base and showed that conversion of ribonucleosides to the corresponding deoxy derivatives took place without the cleavage of the N-glycosidic bond. Larsson and Neilands (74) performed a similar experiment in which [^diphosphate and uniformly labeled [^^CJcytidine were administered to rats with regenerating liver. Analysis of cytidylate from RNA and deoxycytidylate from DNA showed the same ^^P: ^^C ratio, demonstrating that both compounds were derived from a common precursor, evidently a ribonucleotide. Many investigators have since contributed to the present knowledge of the enzymology of ribonucleotide reductase in various systems. (119). Isolation of the proteins required for ribonucleotide reduction was started in the early 1960s by the pioneering work of Reichard and collaborators using J5. coli (101). Important steps were the development of assay methods and the identification of cofactor requirements, in­ cluding the use of a dithiol such as dihydrolipoic acid as hydrogen donor substrate (100). Fractionation of the extract resulted in the isola­ tion of the proteins required for in vitro activity, including thioredoxin (77) and thioredoxin reductase as a NADPH-dependent hydrogen donor system (94). The requirement for ATP as a positive allosteric eflFector (67) and the allosteric properties (19, 20, 75, 76) of the pure E. coli enzyme (17) were described. Thelander (115, 116) characterized theJS*. coli ribonucleotide reductase by physicochemical methods. Doubts about the essential nature of ribonucleotide reductase for cell growth due to its low in vitro activity were eflPectively dispelled when Fuchs et al. (48) found that a temperature-sensitive mutant in DNA synthesis (DNA F) affected the gene for the enzyme. The active center disulfide bridge of E. coli thioredoxin-Sa (59, 68) was found to be located in a unique protrusion of the molecule, as determined by X-ray crystallography. Doubts about the in vivo role of the thioredoxin system in ribonucleotide reduction came from analysis of anS. coli mutant lacking thioredoxin (60, 66). This led to the discov­ ery of the glutaredoxin system by Holmgren (60) and the analysis of GSH-dependent deoxyribonucleotide synthesis (62, 63). The Bi2-dependent ribonucleotide reductase present in Lactobacillus leichmannii and its reaction mechanism was extensively characterized by Blakely and co-workers (16, 97, 107) and Hogenkamp and Sando (58). Berglund et al. (7, 11) isolated a phage T4-induced ribonucleotide reductase and a T4 thioredoxin (12). The knowledge of the properties of mammalian ribonucleotide reductases has been lagging. Moore (92) has characterized the rat Novikoff tumor reductase system and The-

REGULATION OF RIBONUCLEOTIDE REDUCTASE

51

lander and co-workers (35) have obtained a highly purified calf thymus reductase and described its allosteric regulation (36). C. Assay of Ribonucleotide Reductase The basic in vitro assay methods for ribonucleotide reductases have been summarized (15, 70,120). Spectrophotometric assay based on the oxidation of NADPH is possible only with purified enzyme fractions. It also requires the use of pure thioredoxin or glutaredoxin systems. In crude extracts the conversion of a ^H- or ^Φ-labeled ribonucleotide to the deoxy derivative is used. For the E. coli enzyme (120), the assay mixture contains: 200 nmol ATP, 1.6 Mmol MgCl2, 80 nmol NADPH, 5 Mmol Ar-2-hydroxyethylpiperazine-Ar'-2-ethanesulfonic acid (HEPES) buffer (pH 7.6), 300 pmol thioredoxin, 40 pmol thioredoxin reductase, 10 nmol EDTA, 65 nmol dithiothreitol, and 75 nmol [^H]CDP in a final volume of 0.13 ml. After incubation for 10 minutes at 25°, the reaction is broken by addition of 0.5 ml of 1 Μ perchloric acid. The nucleotides are then hydrolyzed to monophosphates, and [^HJCMP and [^HJdCMP are separated on Dowex-50 columns eluted with 0.2 Μ acetic acid (120). The activity of the reductase in a crude cell-free extract is always very low and often not detectable at all. This is due to real variations in the absolute level of the enzyme but also frequently due to complica­ tions in the assay. The following points are of special importance. 1. SUBSTRATE

The enzyme requires either a ribonucleoside di- or triphosphate; however, extracts also contain highly active kinases and phosphatases that change the phosphorylation state of the substrate. In fact, a potent nucleoside diphosphate kinase is present in nearly homogeneous ribonucleotide reductase from E. coli (30). The presence of catabolic enzymes may lead to degradation of both substrates and products. Thus, CDP and dCDP may be degraded by deamination to uridine compounds or by breakage of the glycosidic bonds. 2. ALLOSTERIC EFFECTORS

Generally, enzyme activity is absolutely dependent on the presence of ATP or another positive allosteric effector. The products of the reac­ tion after kinase action (except dCTP) are positive or negative allo­ steric effectors that, when accumulated, will influence the activity of the enzyme. Crude extracts may also contain or generate nucleotides that will act by the allosteric mechanisms and severely limit detection of reductase activity.

52

ARNE HOLMGREN

3. PROTEIN INTERACTIONS

The enzymes from E. coli or animal cells (119) are composed of weakly bound subunits. Assays of dilute crude extract thus give very pronounced sigmoidal enzyme concentration curves, indicating associa­ tion of subunits. Reliable measurements will require the addition of excess of one subunit or very high protein concentrations. Eriksson (37) described a high activity form of the reductase fromiJ. coli obtained by "gentle" lysis of cells. However, this high activity form is not stable to storage; its activity has a half-life of about 8 hours at +4°. Some prog­ ress in the isolation of a membrane-bound form of the E. coli reductase has been reported by Lunn and Pigiet (81). These findings point to yet-to-be-discovered protein interactions in the cellular organization of the reductases, including in particular the hydrogen donor system. 4. HYDROGEN DONORS

GSH or NADPH are the apparent ultimate hydrogen donors via either the glutaredoxin or thioredoxin systems. In crude extracts, NADPH, GSH, and thioredoxin-(SH)2 are rapidly oxidized by unspecific processes; they are also diluted below their eflFective concentra­ tion. NADP^ can be inhibitory through competitive inhibition of the thioredoxin and glutathione reductase enzymes. To overcome these problems, the general reductant used for in vitro assays is a dithiol such as dihydrolipoate or dithiothreitol. 5. INHIBITION

It is clear from mixing experiments that strong unknown inhibitors exist in crude extracts (37). The general lability of purified ribonu­ cleotide reductase is a complication for storage of enzyme fractions. Methods to overcome this problem have been described (15, 70, 120). II. Structure of Enzymes Organisms contain either the Β12 dependent or the iron-containing class of ribonucleotide reductase (119). Gleason and Hogencamp (53) observed the Bi2-dependent enzyme to be common among prokaryotes and rare among eukaryotes, but no general rules were found; even closely related species could contain either type of enzyme. The ironcontaining reductase occurs in green algae, yeast, higher plants, and mammalian species (40, 53, 54). The molecular characteristics of the two enzyme classes are very distinct, as will be described later. An interesting evolutionary link may be the enzyme from Corynebacterium nephridii described by Tsai and Hogenkamp (121). This

REGULATION OF RIBONUCLEOTIDE REDUCTASE

53

adenosylcobalamin-dependent enzyme consists of two subunits and uses ribonucleoside diphosphate as substrates. A. Ribonucleoside Diphosphate Reductase of £.

coll

The enzyme is composed of two nonidentical subunits called protein B l and B2 (20). These are coded for by two closely linked structural genes (called nrdA and nrdB), located at 48 minutes on the E. coli linkage map (4). Mutants in both genes have been described by Fuchs et al. (48-50). Eriksson et al. (39) constructed a strain of E. coli lysogenic for a defective λ phage carrying both the nrdA and nrdB genes. On induction such cells yield up to 10% of the soluble protein as Bl + B2, in a roughly 1:1 ratio. Affinity chromatography on dATPSepharose, described by Berglund and Eckstein (9), allows purification of large amounts of highly active enzyme from such cells (39). A schematic model ofE. coli ribonucleotide reductase is given in Fig. 2. The active enzyme consists of the two dissimilar proteins, B l and B2, in a 1:1 stoichiometry bound together by Mg^^ (19,115). The binding is weak (19), and during purification the two subunits dissociate easily and were originally purified as separate entities (17), each inactive in the overall reaction. Thelander (115) found that protein B l has a MW of 160,000 and is a dimer of the general structure aa'. The two polypeptides are of similar or identical size and show the same COOH-termini but have different NH2-terminal sequences (115). As yet this difference has not been ex­ plained; it may represent a preparation artifact or indicate that B l is coded for by separate structural genes, with possible functional conse­ quences. The isolated B l subunit contains three classes of binding sites for nucleotides; one for the substrates (31) and two different sites (20) for the allosteric effectors (see Fig. 2). Equilibrium dialysis experSubstrate specificity (ATP, dATP, / dTTP, dGTP)

/

V

w ·* ^-site

Activity (ATP. dATP) ^1

B2 -subunit

F I G . 2. Model of E. coli ribonucleotide reductase. Taken from Reichard (102) with permission.

54

A R N E HOLMGREN

iments demonstrated the presence of two substrate binding sites per B l (31) and that all four NDPs* are bound by the same site. Two classes of allosteric effector-binding sites (h- and /-sites, Fig. 2) have been found on Bl, each class consisting of two subsites (19, 20). Λ-Sites are defined by their high affinity (Ka = 0,03 μΜ) for dATP, /-sites by their low affinity (K^ = 0.1-0.5 μΜ). Competition experiments showed that Λ-sites also bind the allosteric effectors ATP, dTTP, and dGTP, whereas /-sites only bind ATP in addition to dATP. The regulation of enzyme activity by allosteric effectors is further discussed in Section IV. Protein B2 (MW 78,000) consists of two apparently identical polypep­ tide chains (115) and has two atoms of iron, presumably one per pep­ tide chain (18). The iron is required for enzyme activity; its removal by dialysis against 8-hydroxyquinoline gives inactive apoprotein B2. This can be reactivated to more than 100% by reconstitution with Fe(II)ascorbate. Atkin et al. (3) studied the state of the iron in protein B2 by Mössbauer spectroscopy on ^Te-enriched protein. The results (Fig. 2) suggest the presence of two nonidentical high spin Fe(III) ions in a antiferromagnetically coupled binuclear complex (3). Protein B2 also contains a unique free radical that is characterized by a sharp absorbance peak at 410 nm and a doublet EPR signal centered around^ = 2.0047 (32). By isotope substitution experiments, Sjöberg et al. (109) assigned the radical to a tyrosine residue in the B2 protein; the radical spin density is localized over the aromatic ring (110). The free radical of protein B2 is closely linked to the presence of iron. Removal of iron leads to loss of the radical and it is reformed on reconstitution of the apoenzyme (3, 18, 32). It appears that the function of the iron, as it binds to the protein, is both to generate the tyrosyl radical, probably by an iron-catalyzed one-electron aerobic oxidation, and to stabilize the radical in the enzyme by some continued interaction. The maximal content offi:'eeradical in B2 preparations is estimated as at least 1 mol per 2 iron atoms and 78,000 gm of protein (39). This may be due to loss during purification or it may suggest that the radical partic­ ipates in one of the active sites at a time (Fig. 2). This would be consis­ tent with a mechanism of half-site reactivity (109). The involvement of thefi:-eeradical in enzyme activity is demonstrated by its irreversible destruction by hydroxyurea (18). The active center of ribonucleotide reductase involves elements from both protein Bl and B2 (Fig. 2). Thelander (116) found that protein B l contains oxidation-reduction active sulfhydryls that are able to reduce stoichiometric amounts of substrate in the absence of an external hyAbbreviations: rNDP, ribonucleoside diphosphates; dNDP, diophosphates; dNTP(s) deoxyribonucleoside triphosphates.

deoxyribonucleoside

REGULATION OF RIBONUCLEOTIDE REDUCTASE

55

drogen donor. They are then oxidized to a disulfide. The function of thioredoxin-(SH)2 is to act as a "ping-pong" substrate to reversibly reduce these disulfides in oxidized protein Bl. The reaction mechanism of E. coli ribonucleotide reductase involves direct replacement of an OH group by a hydrogenfiroma dithiol. The new hydrogen enters at the 2' position in ribose, with the configuration at this carbon atom retained. A free radical and the dithiol are impli­ cated in the mechanism. Possible mechanisms for ribonucleotide re­ ductase are discussed in previous reviews (44, 58), B. Ribonucleoside Triphosphate Reductase from L. leichmannii The enzyme is composed of one large polypeptide chain with a MW of 76,000 and a s§o,w of 5.13 S (24, 97); it does not aggregate in the pres­ ence of absence of substrate or allosteric effectors, and thus lacks subunit interactions. The enzyme has been purified to homogeneity both by conventional methods (24, 97) and by affinity chromatography on dGTP-Sepharose (56,107), Vitrols et al, (122) showed that the activity of the enzyme is absolutely dependent on the presence of the B12 coen­ zyme, 5'-deoxy-5'-adenosylcobalamin. The coenzyme is rather weakly bound to the polypeptide chain and its affinity for the enzyme is one factor influencing activity, which in turn is regulated by the presence of allosteric effectors (107), The substrates for the enzyme are the four ribonucleoside triphos­ phates; kinetic experiments showed that the apparent üTm for GTP, the best substrate, was quite high (240 μΜ), It is assumed that a single catalytically active site binds all four different substrates. Equilibrium dialysis experiments demonstrated a second common binding site for regulatory deoxyribonucleoside triphosphates with ranging from 9-80 μΜ, This regulatory site can also bind substrates, but with a 100to 1000-fold lower affinity (24, 107), The adenosylcobalamin-dependent reaction mechanism of the L, leichmannii enzyme is unique for a B12 coenzyme because an intermolecular hydrogen transfer is involved. The coenzyme is implicated as an intermediate hydrogen carrier between a dithiol substrate such as thioredoxin-(SH)2 and the ribonucleoside triphosphate. An intermedi­ ate, observed by EPR spectroscopy and similar to cob(II)alamin (Bi2r) fbrmed by a hemolytic cleavage of the carbon-cobalt bond, is sug­ gested, together with a stabilized deoxyadenosyl radical (105), The Bi2-dependent ribonucleotide reductases catalyze an isotope exchange reaction between tritium at the 5'-methylene group (5'-^H2) of the 5'deoxyadenosylcobalamin coenzyme and water in the presence of a sub­ strate dithiol (1, 57), This reaction may be used as a simple alternative way to determine activity.

56

ARNE HOLMGREN

The pure L. leichmannii reductase enzyme contains a disulfide that is reduced by thioredoxin-(SH)2 or dithiothreitol. Kim et al. {72) found that the isolated dithiol form of the enzyme cannot transfer the reduc­ ing equivalents to the nucleotide substrate. Their results (72) suggest that the dithiol form of the enzyme is required for overall activity rather than as an intermediate in the reaction mechanism. C. Mammalian Reductases Enzyme activity has been demonstrated in actively growing normal and malignant cells of different origin, including rats {92), calf (35, 36, 117), rabbit {69, 70), and man (23). The low levels of the enzyme and its lability has prohibited the isolation of a homogeneous enzyme. So far, the purest preparations have been obtained from Novikoff hepatoma of rats {92) and from calf thymus (35, 36, 117). The mammalian reduc­ tases contain easily separable subunits {69), do not require cobalamin coenzymes for activity, are inhibited by hydroxyurea, and most proba­ bly contain iron. Thus, they resemble the E. coli rather than the Lac­ tobacillus enzyme. Thelander et al. {117) separated the calf thymus ribonucleotide re­ ductase into two nonidentical subunits, called protein Ml and M2. Protein Ml was purified to homogeneity and was shown to behave as a monomer (5.7 S) with a MW of 84,000. Addition of dTTP leads to dimer formation (8.8 S; MW 170,000) whereas addition of dATP induces tetramer formation (15.2 S). The oligomerization probably refiects con­ formational changes induced by the nucleotides. Binding of 1.8 mol of dATP or 0.7 mol of dTTP per 170,000 gm of protein was demonstrated by equilibrium dialysis {117). Protein M2 has an apparent MW of 110,000, with subunits of 55,000. It has not yet been obtained pure. The calf th5nnus enzyme was also purified 3400-fold without separa­ tion of subunits (35). The enzyme preparation contained mainly Ml and only low nonstoichiometric amounts of the iron-containing M2 subunit. It is not clear if this is due to the lability of M2, or if Ml occurs in excess also in cells. The calf thymus enzyme is inactivated by EDTA (35); activity can be fully restored by iron or manganese. Although generally similar to the E. coli enzyme, a different structure or environment for the putative free radical in the mammalian enzyme is suggested (35). These two inhibitors show reversible inhibition of the calf thymus reductase (35) as their removal by gel filtration resulted in a fully active enzyme. With theE. coli reductase, the two drugs act by irreversibly scavenging the t5n:Osyl radical of the B2 subunit {3, 118).

REGULATION OF RIBONUCLEOTIDE REDUCTASE

57

D. Viral Reductases Infection ofE. coli with T2, T4, T5, and T6 phages results in the induction of virus-coded ribonucleotide reductases {11,13,28). Neither T7 nor λ seems to carry a gene for ribonucleotide reductase {38). The phage T4 ribonucleotide reductase has been purified to homogeneity by Berglund (8), using dATP Sepharose affinity chromatography. The genes {nrdA and nrdB) coding for the subunits have been identified {126). The T4-coded reductase has many similarities to the E. coli en­ zyme (8), with a MW of 225,000 and «2/^2 structure. The enzyme uses diphosphates as substrates. «2 has a MW of 160,000, contains binding sites for substrates and effectors, and thus resembles B l fromS. coli. ß2 contains 2 mol of iron as well as the paramagnetic species indicating a free radical and corresponds to B2 fromJS. coli {8). It differs from theE. coli reductase in that the binding of the subunits is tighter and does not require Mg^"^ {8). The T6-induced proteins resemble those of T4 closely and cross-react immunologically {38). T5 ribonucleotide reductase, although not purified, appears to be different and uses triphosphates as substrates (38). The regulation of ribonucleotide reduction in the T4-infected cell is also discussed in Section III,C. III. Hydrogen Transport M e c h a n i s m In vitro either thioredoxin-(SH)2 or GSH in the presence of glutaredoxin may function as hydrogen donor substrate for ribonu­ cleotide reductase {60,62,63). The in vivo hydrogen donor is not known, although it is evident from genetic results that the thioredoxin system is not essential for ribonucleotide reductase activity. The two systems may possibly have specific roles in reduction of ribonucleotides under different growth conditions; together they may regulate the activity of the enzyme by "thiol redox control." A. Thioredoxin System The thioredoxin system consists of NADPH, thioredoxin reductase, and thioredoxin and functions by a combination of reactions 1 and 2: thioredoxin reductase

Thioredoxin-S2 + N A D P H + H^

> thioredoxin-(SH)2 + NADP^

(1)

ribonucleotide reductase

Thioredoxin-(SH)2 + r N D P

> thioredoxin-Sa + dNDP

(2)

It was originally discovered in E. coli {77, 94). Similar systems were later found inL. leichmannii {96) and eukaryotes {90, 98,123). The key

58

ARNE HOLMGREN

component, thioredoxin, is a small heat-stable protein (MW 11,700) that has been extensively studied, particularly in E. coli, [for a review, see Ref (64)]. Holmgren (59) determined the complete amino acid sequence of the 108 amino acid residues of E, coli thioredoxin and found the active center to be an oxidation-reduction active disulfide with the sequence: Ί

Γ

-Trp-Ala-Glu-Trp-Cys-Gly-Pro-Cys-Lys-Met32

35

The three-dimensional structure of thioredoxin-S2 from E. coli at 2.8-Ä resolution, by X-ray crystallography, was established by Holmgren al. (68). The active disulfide (Fig. 3) is located on a protru­ sion formed by residues 27-39, making thioredoxin-Sa a (so far) unique example of a "male" protein. The function of E. coli thioredoxin-(SH)2 in NADPH-dependent ribonucleotide reduction has been proposed (116) to involve a shuttle of disulfide-dithiol interchanges as summarized in Fig. 4. Thus, thioredoxin reductase (MW 70,000) (64) contains both FAD and

F I G . 3. Schematic drawing of the three-dimensional structure oiE. coli thioredoxin-S2 designed by Dr. Bo. Furugren. ßi to represents five strands of jS-pleated sheet; «ι to «4 represents four α-helices.

REGULATION OF RIBONUCLEOTIDE REDUCTASE

59 Bl

NADPH

.)( NADP

Thioredoxin reductase-(SH)2

FAD

) (

)( FADH2

Thioredoxin reductase-$2

· . {SH)2 B2 ^

Thioredoxin-$2

Μ

)(

Thioredoxin-(SH)2

rNDP

81 52" ^2

dNDP

F I G . 4. Involvement of oxidation-reduction active disulfides in E. coli ribonucleotide reduction via the thioredoxin system.

I

1

oxidation-reduction active disulfides (-Cys-Ala-Thr-Cys- in E, coli); protein B l contains disulfides that accept the hydrogens fi:-om thioredoxin-(SH)2 in a ping-pong type of reaction. No stable ternary complex has been detected between thioredoxin and either thioredoxin reductase or ribonucleotide reductase (64, 116), The sulfhydryl group of Cys-32 inE, coli thioredoxin-(SH)2 shows an abnormally low apparent value of 6.7, as determined by the pHdependent rate of alkylation with iodoacetic acid (71), Cys-35 has a corresponding p/f value close to 9.0 (71), These results have led to the formulation of a mechanism with thioredoxin-(SH)2 as a protein di­ sulfide reductase (71), This is based on the thiol-disulfide interchange reaction and involves the initial nucleophilic attack by the thiolate of Cys-32 on the disulfide, with formation of a transient mixed disulfide involving Cys-32 and one of the sulfurs of the disulfide substrate. After a conformational change and a nucleophilic attack of Cys-35, the di­ sulfide bridge in thioredoxin-S2 and the dithiol of the substrate are formed. In spite of the clear demonstration that the thioredoxin system in vitro is a hydrogen donor for ribonucleotide reductase, its true physio­ logical functions in E, coli are presently unclear. This became evident as a result of studies of prototrophic coli mutants isolated by Chamberlin (22); these mutants had lost the ability to support the growth of bacteriophage T7. One class of such mutants (tsnC) had mutations in the gene for thioredoxin (66, 83), Mark and Richardson (83) found that extractsfi:OmT7-infected tsnC cells lacked T7 DNA polymerase activ­ ity, as thioredoxin combines with the phage-coded gene 5 protein (MW 84,000) to form the active phage T7 DNA polymerase. One mutant (tsnC 7004) appeared to be a nonsense or deletion mutant (66) that contained no thioredoxin activity as measured by sensitive enzymatic or immunological assays. Yet, the tsnC 7004 mutant showed no de­ creased capacity to reduce ribonucleotides (60). The same is true for an E, coli mutant lacking thioredoxin reductase activity (46), Thus, the only genetically proved essential function ofE, coli thioredoxin is as a

60

ARNE HOLMGREN

subunit of phage Τ7 DNA polymerase (83); its role in the polymerase remains an exciting mystery. E. coli contains about 10,000 molecules of thioredoxin per cell (66), equivalent to an intracellular concentration of about 15 μΜ. Mammalian cells also have relatively high thioredoxin concentra­ tions irrespective of the active growth and ribonucleotide reductase activity (65). The mammalian thioredoxins are homologous to the E. coli protein (64); the active center sequence: -Cys-Gly-Pro-Cys- is iden­ tical (A. Holmgren, unpublished results). There is a general crossreactivity between bacterial and mammalian thioredoxin-(SH)2 and ribonucleotide reductase (82). The subcellular distribution of calf thymus thioredoxin shows mul­ tiple locations, including membrane associations (65). These results suggest that the bulk of thioredoxin in mammalian cells functions in protein thiol-disulfide interchange reactions (64), unrelated to ribo­ nucleotide reductase. Among such reactions are reversible reduction of disulfides of an enzyme, resulting in changes in the catalytic activ­ ity ("thiol redox control," see Section III,D). B. Glutaredoxin System Glutaredoxin enables the monothiol GSH to be hydrogen donor for ribonucleotide reductase and functions by a combination of reactions 3 and 4: ribonucleotide reductase 2 GSH + r N D P

^ GSSG + d N D P + H 2 O glutaredoxin glutathione reductase GSSG + N A D P H + H^ > 2 G S H + NADP^

(3)

(4)

Glutaredoxin was discovered (60) in the thioredoxin-negative E. coli mutant tsnC 7004, and purified to homogeneity fromjB. coli Β wild-type cells (62). Glutaredoxin is a small, acidic protein consisting of about 89 amino acid residues including a single catalytically active disulfide bridge (62). This is reduced to a dithiol by GSH and NADPHglutathione reductase, collectively called the glutaredoxin system. It is not reduced by NADPH and thioredoxin reductase. Tryptic peptide maps of reduced and carboxymethylated glutaredoxin and thioredoxin demonstrated that the two proteins are structurally unrelated and are the products of two separate genes (62). Furthermore, the two proteins are antigenically different (60). The properties of thioredoxin and glutaredoxin are compared in Table I. Glutaredoxin has inherent

61

REGULATION OF RIBONUCLEOTIDE REDUCTASE TABLE

I

P R O P E R T I E S OF T H I O R E D O X I N AND G L U T A R E D O X I N FROM E.

Property" Molecular weight Amino acid residues Active center* GSH-disulfide transhydrogenase Substrate for thioredoxin reductase Molecules/cell for ribonucleotide reductase ( μ Μ ) Turnover number ( m i n ' )

coli

Thioredoxin

Glutaredoxin

11,700 108 P-S-S , -Cys-Gly-Pro-Cys-

11,000 89 -Cys-Pro-Tyr-Cys-

No

Yes

Yes 10,000

No 100-200

1.3 13-15

0.13 110-150

i-s-s—I

" Taken from Holmgrem {62, 63). ^ Results for glutaredoxin of A. Holmgren and M.-L. Persson, unpub­ lished findings.

GSH-disulfide transhydrogenase (or oxidoreductase) activity in a coupled system with 2-hydroxyethyl disulfide as substrate and GSH, NADPH, and glutathione reductase as reductant {62), Holmgren al, (unpublished results) have determined the sequence of the disulfide in I

-1

E. coli glutaredoxin to be -Cys-Pro-Tyr-Cys-. Thus, as in E, coli thioredoxin-(SH)2 the active center consists of a 14-membered disulfide ring. Glutaredoxin shows an apparent of 0.13 μΜ with ribonucleotide reductase in the presence of excess CDP and 4 mM GSH (63). The apparent ÜLm for GSH in 0.4 mM in the presence of excess NADPH and glutathione reductase {63), The molecular activity of glutaredoxin is about 10-fold higher than that of thioredoxin and similar to the corresponding values for the B l and B2 subunits of ribonucleotide reductase {63), The molecular mechanism of glutaredoxin in ribonucleotide reduction is relatively unknown; it is not known if the reduction occurs via the oxidation-reduction active disulfides of protein B l (Figs. 2 and 4). The binding of glutaredoxin to ribonucleotide reductase has not been investigated so far. The level of glutaredoxin in wild-type £J. coli Β corresponds to about 200 molecules/cell as determined by a radioimmunoassay (A. Holmgren et al., unpublished). Variations in this value are seen in

62

ARNE HOLMGREN

diflFerent Ε. coli strains. Low apparent levels are found in derepressed, thymine-starved cells whereas high levels are found in a thioredoxin reductase mutant (46), Luthmanei aL have identified a glutaredoxin from calf thymus (82). When the cross-reactivity of E. coli and calf thymus glutaredoxin was studied using the corresponding ribonucleotide reductases, a high species specificity was observed (82). C. Phage T4 Thioredoxin Phage T4 induces a thioredoxin upon infection ofE. coli cells (12). This is a small protein that in its dithiol form is the specific hydrogen donor for T4-induced ribonucleotide reductase and in its oxidized form is a substrate for the bacterial thioredoxin reductase (12). T4 thiore­ doxin contains only 87 amino acid residues of known sequence and show no primary structure homology with Ε. coli thioredoxin (108). Even the active center of T4 thioredoxin

I

1

-Cys-Val-Tyr-Cys14

17

is diflferent (108). The overall three-dimensional structure of T4 thioredoxin-S2 obtained by X-ray crystallography (113) shows, how­ ever, large overall structural similarities to that ofE. coli thioredoxin. Clear structural diflFerences are present around the disulfide bridge (113). E. coli thioredoxin-(SH)2 is not active as a substrate for the T4induced ribonucleotide reductase (12). The redox potential of T4 thioredoxin is - 0 . 2 3 V at pH 7.0 (12) as compared to a value of - 0 . 2 6 V for the E. coli protein (64). Thioredoxin reductase also catalyzes the reduction of T4 thioredoxin-S2 by bacterial thioredoxin-(SH)2 (10), making possible the preferential use of the T4 ribonucleotide reductase system in the infected cells. The discovery of glutaredoxin helped to explain some of the proper­ ties of T4 thioredoxin (61). As reported by the author (61) T4 thiore­ doxin has GSH-disulfide transhydrogenase activity. T4 thioredoxin also catalyzes GSH-dependent ribonucleotide reduction by the T4 ribonu­ cleotide reductase and behaves as a glutaredoxin. Indeed, T4 thiore­ doxin has properties as a functional hybrid between thioredoxin and glutaredoxin. Furthermore, E. coli glutaredoxin is an excellent hydro­ gen donor for T4 ribonucleotide reductase. However, its lower concen­ tration would be insufficient to serve the phage-induced reductase. In summary, the hydrogen transport after T4 infection ofE. coli leads to a preferential use of the T4-induced ribonucleotide reductase (61).

REGULATION OF RIBONUCLEOTIDE REDUCTASE

63

D. Thiol Redox Control of Activity The control of ribonucleotide reductase activity by the hydrogen transport system had, until the discovery of the glutaredoxin system, received little attention. Because the reductase requires a thiol as hy­ drogen donor, the general thiol-disulfide state in vivo will be of impor­ tance for activity. The glutathione-dependent reduction of CDP to dCDP in the pres­ ence of glutaredoxin and ribonucleotide reductase is highly influenced by the ratio of GSH to GSSG (63). Even small amounts of GSSG inhibit strongly. This gives NADPH-glutathione reductase a pivotal role in ribonucleotide reduction by controlling the GSH: GSSG ratio (63). Also the rate of synthesis of GSH by glutathione synthetase and its utiliza­ tion in other reactions will influence the function of glutaredoxin. Thus, the inhibition by GSSG is a possible control mechanism for deoxyribonucleotide synthesis. Protein B l ofE. coli ribonucleotide reductase is inactivated by in vitro storage in the absence of dithiothreitol, through oxidation of structural SH groups to disulfides (225). The glutaredoxin system is not active with such oxidized enzyme preparations (63), whereas the thioredoxin system shows activity. This suggests that thioredoxin(SH)2 can reduce the disulfides of B l and also act as hydrogen donor at the active center (63). "Thiol redox control" has been used {74) to de­ scribe thoredoxin-dependent regulation of enzyme activity through the covalent modification involving reversible reduction of disulfides to dithiols. Thioredoxin may thus regulate the activity of ribonucleotide reductase: thioredoxin-(SH)2 by activating and thioredoxin-S2 by inac­ tivating the enzyme. Do glutaredoxin and thioredoxin both serve as hydrogen donors but in difierent forms of ribonucleotide reduction? One hypothesis is that the glutaredoxin system is involved in DNA replication as part of an organized multienzyme complex with high turnover. The thioredoxin system may function together with a "free" form of ribonucleotide re­ ductase producing deoxyribonucleotides for DNA repair and errorprone DNA replication. As yet, no mutants in the gene for glutaredoxin are available to prove or disprove this hypothesis. Other results of interest for the understanding of the function of glutaredoxin and thioredoxin are the following: 1. Mutants lacking the thioredoxin system show no decreased capacity to make deoxyribonucleotides. They have the high-activity form of ribonucleotide reductase of "gentle" lysates (60). 2. Mutants in the biosynthesis of GSH inE. coli (2, 52), containing

64

ARNE HOLMGREN

very low levels of GSH, have derepressed and increased levels of ribonucleotide reductase; possibly a consequence of the lack of GSH as a hydrogen donor substrate for the enzyme. Such cells may also use the thioredoxin system as hydrogen donor for ribonucleotide reductase. 3. GSH-deficientÄ. coli mutants are more sensitive to X-ray irradia­ tion than wild-type cells (95). This suggests a relation between GSH metabolism and DNA synthesis perhaps originating from a changed activity of ribonucleotide reductase and its functional organization. 4. GSH is related to cell growth by its continuous direct synthesis (2). The formation of glutathionylspermidine at the end of logarithmic growth mE. coli {114) may serve to regulate DNA synthesis by effects on both DNA polymerase and deoxyribonucleotide production {63). IV. Allosteric Control A. E. coli Enzyme The E. coli reductase has been extensively studied; data from ki­ netic experiments (75, 76), effector binding studies {20, 31), and phys­ icochemical characterization of the enzyme {19, 115, 116) are avail­ able. The overall enzyme activity, and perhaps more significantly, the substrate specificity are regulated by nucleoside triphosphate effec­ tors. Binding studies {20) showed that the B l subunit of the enzyme (see Fig. 2, Section II,A) contains two classes of sites {h and I). The I sites (i^d = 0.1-0.5 μΜ) regulate the general level of activity with dATP acting as the negative and ATP as the positive effector. The Λ-sites {K^ = 0.03 μΜ) regulate the specificity of the enzyme (20). The presence of ATP, dATP, dTTP, or dGTP at the A-sites results in con­ formational changes at the active site that will induce preferential binding of one substrate. The presence of dATP > 10"^ Μ leads to ag­ gregation of the enzyme {19). The active 1:1 B1-B2 complex has a sedi­ mentation coefficient of 9.7 S; in the presence of high dATP this is con­ verted to an inactive 15.5 S complex proposed to be a dimer {19, 20). The multiplicity of effectors and the two classes of binding sites per­ mits the enzyme to assume a large number of conformation states with different activities. Some of these are summarized in Table II. In the absence of effectors the activity of the enzyme is very low. Addition of ATP or low concentrations of dATP (ca. 10"^ M) selectively stimulates the reduction of CDP and UDP, whereas the presence of dGTP enhances the conversion of ADP and to a smaller extent that of GDP. Also dCTP stimulates the reduction of ADP and GDP but not to the same extent as dGTP. Reduction of all four substrates is stimulated by dTTP. The positive effectors simultaneously increase the Vmax of ^he reac-

REGULATION OF RIBONUCLEOTIDE REDUCTASE

65

T A B L E II ALLOSTERIC REGULATION OF RIBONUCLEOTIDE REDUCTASE FROM E. coli" Effector binding to /-sites 0 0 0 ATP ATP ATP dATP

Λ-sites ATP dTTP dGTP ATP or dATP dTTP dGTP Any effector

Reduction of CDP

UDP

GDP 0

+ 0

+

-

ND

-

ADP 0

0

+ 0

0

-

-1-

(+) +

ND

-

(+)

-

-

Taken from Thelander and Reichard (119) with permission. 0, No effect; - f - , stimulation; - , inhibition; ND, not determined.

tion and decrease the for the respective substrates {75, 76). The binding of a positive allosteric eiSFector may give up to a 50- or 100-fold increase in the rate of the enzyme reaction at low substrate concentra­ tion. coli ribonucleotide reductase shows substrate saturation curves that are hyperbolic both in the absence and presence of allosteric nu­ cleotides {20). The enzyme does not show homotropic cooperative effects (20), and its regulation thus appears more complex than the simple two-state model of Monod al. {89). The complexity also derives from the many diflFerent conformational states of the enzyme obtained by combinations of eflFectors in vitro. The enzyme in vivo probably has its Z-sites occupied by either ATP or dATP and the ratio of the dATP to ATP concentrations will determine the activity. As seen from Table II, three active and one inactive state exist: the active states contain ATP bound to Z-sites and will reduce CDP or UDP with ATP (or dATP) at the Λ-sites, reduce GDP (and ADP) with dTTP at the A-sites, and reduce ADP (and GDP) with dGTP at the A-sites. All combinations containing dATP at Z-sites are inactive. A scheme of how the allosteric regulation of ribonucleotide reductase might turn the enzyme on and oflF in vivo can be obtained by regarding the reduction of the four substrates as a sequential process {102). The sequence would then start with the reduction of CDP and UDP, stimu­ lated by ATP. Through some intermediate steps these reduced pyrimidines are converted to dTTP, which triggers the reduction of GDP and ADP. Finally, accumulation of high concentrations of dATP gives rise to inhibition of the enzjone.

66

ARNE HOLMGREN

Β. Lactobacillus Enzyme This Bi2-dependent, monomeric enzyme shows allosteric regulation of activity and substrate specificity by nucleoside triphosphates. The allosteric properties of the reductase has been reviewed in detail previ­ ously (44, 58). The most efficient positive effectors are dATP for the reduction of CTP, dCTP for UTP reduction, dTTP for GTP reduction, and dGTP for ATP reduction (122). Other combinations may give weak inhibition (44, 58). In contrast to£^. coli or mammalian enzymes, no strong negative effector is known for the Lactobacillus enzyme (44, 58). A unique feature of the Lactobacillus reductase is that the enzyme's affinity for the cobalamin coenzyme is increased by the binding of posi­ tive effectors to the regulatory site (107). Binding studies demon­ strated that very small amounts of coenzyme are bound to the enzyme when the regulatory site is empty (107). The regulatory site may also bind ribonucleoside triphosphates and these can thus act both as sub­ strates and effectors. This explains the kinetic substrate activation observed for GTP reduction at low adenosylcobalamin and in the ab­ sence of deoxynucleoside triphosphate effectors. Addition of higher coenzyme concentration or effectors abolished the effect. The stimula­ tion of cobalamin binding shows little specificity for the base of the effector. Evidence for conformational changes of the protein due to effectorbinding was obtained by the observation of significant changes in the S value of the enzyme on binding of dGTP (107). This effector also caused significant changes in the aromatic region of the CD spectrum. Other indications of conformational changes in the enzyme were a slower rate for inactivation of an essential SH-group on the enzyme by N-ethylmaleimide after the binding of dATP (72). C. Mammalian Enzymes Reichard et al. (103) were the first to describe regulatory effects of nucleotides on the reductase in an extract from chick embryos. They found that formation of dCDP and dGDP was inhibited by purine deoxyribonucleotide triphosphates and that dGTP formation was stimulated about 75% by dTTP. Later, Moore and Hurlbert (93) exam­ ined the behavior of a somewhat more purified rat Novikoff ascites tumor reductase and found a complicated pattern of activations and inhibitions of the reduction of all four ribonucleoside diphosphates by nucleoside triphosphates. The complexity of the effects suggested that more than one enzyme was present, possibly a separate reductase for each of the four substrates.

REGULATION OF RIBONUCLEOTIDE REDUCTASE

67

The highly purified calf thymus ribonucleotide reductase obtained by Thelanders group (35, 36) catalyzed the reduction of all four ribonucleoside diphosphates at almost identical rates. Furthermore, the activities toward CDP and GDP were purified in parallel and the two nucleotides competed for the same catalj^ic site. A simimary of the allosteric eflFects is given in Table III. In the absence of positive eflFec­ tors, the enzyme was inactive with any ribonucleoside diphosphate. Reduction of CDP and UDP was stimulated by ATP, reduction of GDP by dTTP, and reduction of ADP by dGTP. Reduction of the purine ribonucleotides was further stimulated by ATP combined with dTTP and dGTP. dATP served as a general inhibitor whose negative eflfects could be reversed by ATP. Inhibition of the ATP-stimulated enzyme was also caused by dTTP or dGTP (see Table III) (35, 36). The kinetic studies have recently (117) been complemented with binding studies using the separated Ml subunits. Protein Ml behaves as a monomer in solution (5.7 S), which upon addition of dTTP forms a dimer (8.8 S), whereas addition of dATP induces tetramer formation (15.2 S) (117). In the presence of ATP, the protein exists as a mixture of dimers and tetramers. The binding of nucleotides to protein Ml was measured in equilibrium dialysis experiments (117). A maximum of 1.8 mol of dATP or 0.7 mol of dTTP was bound by 170,000 gm of protein. In both cases, cooperativity of binding was observed, with final dissociation constants of 0.3 and 2 μΜ for dATP and dTTP, respecT A B L E III SUMMARY OF THE STIMULATORY AND INHIBITORY EFFECTS OF DIFFERENT NUCLEOSIDE TRIPHOSPHATES ON THE ACTIVITY OF CALF THYMUS RIBONUCLEOTIDE REDUCTASE"

Substrates

Positive effector* or efifector combination

CDP UDP

ATP (1 X 10-3 M)

GDP

dTTP (5 X 10-^ M) dTTP + A T P (1 X 10-3 M) dGTP (5 X 10-^ M) dGTP H- ATP (1 X 10-3 M)

ADP

Inhibition*" dATP (5 dTTP (1 dGTP (1 dATP (5 dGTP (5 dATP (5 dTTP (5

x X X x x x x

10"« M) 10-« M) 10-^ M) 10"« M) lO'^ M) 10"« M) 10"^ M)

° Taken from Eriksson et al. (36) with permission. *The concentrations given represent the values for halfm a x i m a l stimulation. ^ The concentrations of effectors t h a t give half-maximal inhibi­ tion of the reaction.

68

A R N E HOLMGREN

tively. These data indicate the presence of two classes of effector sites, one specific for ATP and dATP, whereas the second class in addition bound dTTP and dGTP {117). D. Phage 14 Enzyme Berglund has shown that the T4-induced ribonucleotide reductase has an allosteric regulation similar to that of the bacterial enzyme (7). A major difference is the absence of inhibition by dATP; this nucleotide acts only as a positive effector for reduction of CDP and UDP (7). Apparently, the T4 enzyme lacks the activity or /-sites of the E. coli enzyme (see Fig. 2), and its activity is only modulated by substrate specificity sites. The situation might mark the adaptation of the phage enzyme to the requirements of the phage-infected cells where only a fine control of the dNTP pools is required and no cell cycle-dependent regulation of activity is needed following the accumulation of dNTPs. V. Regulation of Enzyme Synthesis The level of ribonucleotide reductase is correlated to the extent of DNA synthesis {119). In E. coli, the amount of enzyme activity in­ creases with shorter generation time {41,42). Also in cells with blocked DNA synthesis, and hence unbalanced growth, large increases of ribonucleotide reductase occur. Filpula and Fuchs {41, 42) observed that any condition in E. coli that specifically retards replication fork velocity causes an increase in reductase activity. Thus, when DNA synthesis is blocked by treatment with the drugs nalidixic acid or bleomycin, by thymine starvation of thymine auxotrophs, or by shift­ ing cultures of temperature-sensitive dna mutants to nonpermissive conditions, 5- to 10-fold increases in reductase activity are observed {41,42). It is not known if the mechanism of increase in enzyme synthe­ sis is the same for rapid balanced growth and for cells with blocked DNA synthesis. In E. coli it has been suggested that cells starved for thymine accumulate an unknown compound that both stimulates in­ itiation of DNA synthesis and induces ribonucleotide reductase {41). In animal cells, the reductase is difficult to detect in tissues with low DNA synthesis; the highest activities were reported in rapidly growing tumor cells {33). Larsson (73) found that during liver regeneration, the activity of ribonucleotide reductase rises 10- to 20-fold. Similarly, cells induced to synthesize DNA by virus infection {80) rapidly increase reductase activity. As in E. coli, the mechanism of induction of reduc­ tase synthesis in animal cells is unknown. The regulation of the level of the hydrogen donor system(s) may differfi:Omthe reductase. Thioredoxin and thioredoxin reductase occur

REGULATION OF RIBONUCLEOTIDE REDUCTASE

69

in both E. coli and mammahan cells in excess over ribonucleotide re­ ductase (65, 66) and the level is not changed by induction of ribonu­ cleotide reductase {33, 41, 42). The regulation of glutaredoxin levels has not yet been investigated. VI. Ribonucleotide Reductase and Regulation of D N A Synthesis A. Deoxyribonucleotide Pools The intracellular concentration of deoxyribonucleotides limits capac­ ity of the cells to produce new DNA and undergo cell division {119). Total pools of deoxyribonucleotides have to be synthesized with each round of DNA synthesis, except in rare cases where preformed deoxyribonucleotides appear to be stored as DNA precursors [e.g., in sea urchin eggs (85)]. Measurement of dNTP pools in cultured cells by Skoog et al. {Ill) demonstrated that resting cell populations contain small but definite amounts of all four triphosphates. When cells in culture are stimulated to synthesize DNA, the pools increase signifi­ cantly before the onset of DNA synthesis and reach a maximum during S phase {112). Even though DNA synthesis requires an equal supply of all four bases, very large differences in pool size exist during S phase. The dGTP pool is always the smallest; the size of this pool may only suffice for 15 seconds of DNA synthesis. The dCTP pool is usually the largest; the difference from dGTP in some cases being up to 100-fold {111). Among the four pools, the variations in dCTP concentration most closely reflect the rate of DNA synthesis {102). Bjursell and Reichard {14, 102) suggested that the dCTP pool could serve a regulatory func­ tion in DNA synthesis. Why the different dNTP pools differ so much and what they actually represent in terms of DNA replication and repair synthesis is not un­ derstood. The pool sizes in animal cells have been related to the allo­ steric behavior of the mammalian reductase, Reichard {102) has pro­ posed a scheme (Fig. 5) that links ribonucleotide reduction to DNA synthesis. The ATP-activated enzyme starts to reduce CDP and UDP, proceeds to GDP reduction via a dTTP-activated enzyme, and finally reaches ADP reduction by a dGTP-activated enzyme. Accumulation of dATP completely turns off the reductase {102). Accumulation of dTTP shuts off the reduction of pyrimidine substrates; accumulation of dGTP turns off GDP reduction {102). Mutant cell lines with increased resistance toward deoxyadenosine contain an altered reductase with decreased sensitivity to dATP inhibition {88). These cells also con­ tained larger dNTP pools {87), consistent with the idea that the dATP

70

A R N E HOLMGREN

CDP

UDP

-^DNA GDP

ADP

FIG. 5. Scheine of the proposed physiological regulation of deoxyribonucleotide synthe­ sis in m a m m a l i a n cells. The broken arrows stand for positive eflfects, the open bars for negative eflfects. Taken from Reichard (102) with permission.

pool normally regulates the size of the other dNTP pools. Another re­ sult that supports the validity of the scheme in Fig. 5 is that addition of thymidine to cells inhibits DNA synthesis by a specific depletion of the dCTP pool, with increased dTTP, dGTP, and dATP pools (14), B. Multienzyme Complexes and Metabolic Channeling The in vitro activity of the purified ribonucleotide reductases (39) is not able to support all cellular DNA synthesis. This suggests that a degenerate enzyme is being studied (39) and that the enzyme in the cell performs its function more efficiently. Evidence for this is the finding that permeabilized cells ofi^. coli (124) and cell lysates on cellophane discs (37) show up to 50-fold higher activities than crude extracts pre­ pared by alumina grinding. Similar results have been obtained for mammalian cells (78, 99), Organization of enzymes in the cell, permitting channeling of meta­ bolic intermediates or maintenance of concentration gradients (with the highest levels existing at the sites of utilization) has been discov­ ered in recent years (125). Several features of DNA replication (86) make it attractive a priori to consider the existence of enzyme interac­ tions that compartmentalize DNA precursors and maintain high local concentrations at sites of replication. First, dNTPs are highly special­ ized metabolites, thus the dNTPs need not be distributed throughout the cell. Second, DNA chains grow quite rapidly and at a limited num­ ber of intracellular sites. In prokaryotes, the DNA chain growth is

REGULATION OF RIBONUCLEOTIDE REDUCTASE

71

about 1000 nucleotides per second at 37°. Third, most studies of DNA synthesis in vitro indicate that much higher dNTP concentrations are needed to sustain maximal incorporation rates than the average in­ tracellular dNTP levels as measured in vivo. Association of ribonu­ cleotide reductase with other DNA-synthesizing enzymes is thus strongly indicated. Mathews and co-workers (86) and Greenberg and co-workers (25,43) have demonstrated that in bacteriophage T4-infected cells, multien­ zyme complexes exist with protein-protein interactions favoring eflFec­ tive channeling of nucleotides for DNA replication. Such rapidly sedimenting complexes in T4-infected E, coli cells has also been shown to involve T4 ribonucleotide reductase and six other T4-induced early enzymes (86). In rat liver, Baril et al. (6) have reported that DNA replication en­ zymes during active synthesis are attached to nonnuclear membranes. Ribonucleotide reductase and several other enzymes were also found in aggregates (8.5-12 nm) associated with postmicrosomal membrane fragments of unknown origin in NovikoflF tumor cells (5). Results of Reddy and Pardee (99) using permeabilized fibroblasts, demonstrate a multienzyme complex, probably including ribonu­ cleotide reductase, for metabolic channeling in DNA replication. The complex is present in the nucleus of S-phase cells in contrast to its absence from the nucleus in quiescent and Gi-phase cells. Cells, made permeable by treatment with lysophosphatidylcholine, selectively channeled incorporation of ribonucleoside diphosphates into DNA in preference to deoxyribonucleoside triphosphates if ribonucleotide re­ ductase was active. This was accomplished by addition of dithio­ threitol. More efficient incorporation of dNTPs was obtained when ribonucleotide reductase was inactivated by hydroxyurea. The name replitase was proposed by Reddy and Pardee for this multienzyme complex; its assembly may possibly signal the initiation of the S phase of the cell cycle. Evidence for channeling of deoxyribonucleotides has also been ob­ tained by Forsdyke and Scott (45). They suggested a nonconvergence of de novo and salvage pathways of purine deoxyribonucleotide synthesis by showing that exogenous purine deoxyribonucleosides did not pre­ vent inhibition of DNA synthesis by hydroxyurea in rat thymus cells (45). VII. Drugs Affecting Ribonucleotide Reductase Hydroxyxirea destroys the tyrosylfi:*eeradical of the B2 subunit ofE. coli ribonucleotide reductase (109). The mammalian reductases are

72

A R N E HOLMGREN

also inhibited by hydroxyurea (91). Only 3 minutes after addition of the drug to cells in S phase (112), the enzyme is strongly inhibited and the dGTP pool and DNA synthesis have both decreased to 30%. Later, the dATP pool also decreases. Thus, hydroxyurea inhibits DNA synthesis by depletion of dGTP and dATP pools. After removal of the drug (112), both dGTP and dATP pools expand rapidly, followed by a resumption of DNA synthesis. This may be explained by the finding of Engström al. (35) that the inhibition of the highly purified thymus ribonucleotide reductase was reversible; removal of the drug by gel filtration resulted in a fully active enzyme. In contrast, the E. coli enzyme is irreversibly inactivated by hydroxyurea. Hydroxyurea has been used to isolate resistant CHO-mutant cells with altered levels of ribonucleotide reductase (79). Some of the cell lines isolated contained a modified drug-resistant form of the enzyme; others contained elevated levels of drug-sensitive ribonucleotide reduc­ tase activity. Elford et al. (34) have synthesized a series of hydroxy- and aminosubstituted benzohydroxamic acids and analyzed these for inhibition of ribonucleotide reductase and antitumor activity in leukemia-bearing mice. Thus, 2,3,4-trihydroxybenzohydroxamic acid was 160 times more potent as an inhibitor of ribonucleotide reductase than hy­ droxyurea (34). One class of DNA synthesis inhibitors acting on mammalian reduc­ tase is substituted a-(Ar)-heterocyclic carboxaldehyde thiosemicarbazones (106). The Novikoff hepatoma reductase was inhibited 50% by inhibitor concentrations of about 0.1 μΜ (106); in contrast no inhi­ bition was observed with theE. coli enzyme. The mechanism of action is not known; the drugs are strong iron chelators and it has been sug­ gested (106) that a preformed metal chelate interacts with the enzyme at or near the site occupied by the dithiol substrate. VIII. Ribonucleotide Reductase and Immune Dysfunction Hereditary immunodeficiency diseases in man (52, 84) may ulti­ mately be caused by an imbalance of the allosteric control of ribonu­ cleotide reductase. In patients (52) having a deficiency in the purine salvage enzyme, adenosine deaminase (which in normal individuals rapidly catabolizes both adenosine and deoxyadenosine), deoxy­ adenosine can instead be phosphorylated to dATP. An accumula­ tion of dATP up to 50-fold has been shown in erythrocytes of these patients (27, 29). These high levels of dATP can strongly inhibit ribonucleotide reductase and DNA synthesis. The specific effects in the patients are expressed in the development of the lymphoid tissues. How

REGULATION OF RIBONUCLEOTIDE REDUCTASE

73

remains unclear. It has been suggested that the particularly high phosphorylation activity of deoxyadenosine in that tissue (21) makes it more sensitive. A second form of the immunodeficiency disease is caused by the lack of purine nucleoside Phosphorylase (26). Cells deficient in that enzyme cannot catabolize deoxyguanosine and will accumulate dGTP. The high dGTP pool (26) inhibits ribonucleotide reductase (Fig. 5), resulting in a lowered dCTP pool. Administration of deoxycytidine has been sug­ gested (26) as a potential therapy to correct for the adenosine deaminase or purine nucleoside Phosphorylase defect in immunodeficient patients. ACKNOWLEDGMENTS The excellent secretarial work of Mrs. Delphi Post is gratefully acknowledged. The work of the author is supported by the Swedish Medical Research Council (Projects 1 3 X - 3 5 2 9 and 1 3 P - 4 2 9 2 ) and the Swedish Cancer Society (961). REFERENCES 1. Abeles, R. H., and Beck, W. S.,t7. Biol. Chem. 2 4 2 , 3 5 8 9 - 3 5 9 3 ( 1 9 6 7 ) . 2. Apontoweil, P., and Berends, W.,Biochim. Biophys. Acta 3 9 9 , 1 0 - 2 2 ( 1 9 7 5 ) . 3. Atkin, C. L . , Thelander, L . , Reichard, P., and L a n g , G., J . Biol. Chem. 2 4 8 , 7 4 6 4 7 4 7 2 (1973), 4. B a c h m a n n , Β . J . , Low, K. B . , and Taylor, A. L., Bacteriol. Rev. 4 0 , 1 1 6 - 1 6 7 ( 1 9 7 6 ) . 5 . Baril, Ε . , Baril, B . , Elford, H., and Luftig, R. B . , in "Mechanism and Regulation of DNA Replication" (A. R. Kober and M. Kohiyama, eds.), pp. 2 7 5 - 2 9 1 . Plenum, New York, 1 9 7 3 . 6. Baril, Ε . , Baril, B . , and Elford, K.^Proc. Am. Assoc. Cancer Res. 13, 8 4 - 8 6 ( 1 9 7 2 ) . 7. Berglund, O., J . Biol. Chem. 2 4 7 , 7 2 7 6 - 7 2 8 1 (1972). 8. Berglund, O., J. Biol. Chem. 2 5 0 , 7 4 5 0 - 7 4 5 5 ( 1 9 7 5 ) . 9. Berglund, O., and Eckstein, F., Methods Enzymol. 3 4 B , 2 5 3 - 2 6 1 (1974). 10. Berglund, O., and Hohngren, Α., J. Biol. Chem. 2 5 0 , 2 7 7 8 - 2 7 8 2 ( 1 9 7 5 ) . 11. Berglund, 0 . , Karlström, Ο., and Reichard, P., Proc. Natl. Acad. Sei. U.S.A. 6 2 , 8 2 9 - 8 3 5 (1969). 12. Berglund, 0 . , and Sjöberg, B.-M., J . Biol. Chem. 2 4 5 , 6 0 3 0 - 6 0 3 5 ( 1 9 7 0 ) . 13. Biswas, C , Hardy, J . , and Beck, W. S., J. Biol. Chem. 2 4 0 , 3 6 3 1 - 3 6 4 0 (1965). 14. Bjursell, G., and Reichard, Ρ , J. Biol. Chem. 2 4 8 , 3 9 0 4 - 3 9 0 9 (1973). 15. Blakely, R. L . , Methods Enzymol. 5 1 , 2 4 6 - 2 5 9 ( 1 9 7 8 ) . 16. Blakely, R. L . , Ghambeer, R. K., Nixon, Ρ R , and Vitols, E.,Biochem. Biophys. Res. Commun. 2 0 , 4 3 9 - 4 4 5 (1965). 17. Brown, N. C , Canellakis, Z. N., Lundin, B . , Reichard, P., and Thelander, L.,Eur. J. Biochem. 9, 5 6 1 - 5 7 3 (1969). 18. Brown, N. C , Eliasson, R., Reichard, P., and Thelander, L . , Eur. J. Biochem. 9 , 5 1 2 - 5 1 8 (1969). 19. Brown, N. C , and Reichard, R , J. Mol. Biol. 4 6 , 2 5 - 3 8 ( 1 9 6 9 ) . 20. Brown, N. C , and Reichard, Ρ , J. Mol. Biol. 4 6 , 3 9 - 5 5 ( 1 9 6 9 ) . 21. Carson, D. Α., K a y e , J . , and Seegmiller, J . E . , Proc. Natl. Acad. Sei. U.S.A. 7 4 , 5 6 7 7 - 5 6 8 1 (1977).

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