©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Directed Mutagenesis of Deoxyguanosine Site at Arginine 79 Up-regulates Turnover on Deoxyadenosine Kinase Subunit of Heterodimeric Enzyme from Lactobacillus acidophilus R26 (*)

(Received for publication, November 9, 1994; and in revised form, January 23, 1995)

Young Soo Hong (§) Grace T. Ma (¶) David H. Ives (**)

From the Department of Biochemistry and the Ohio State Biochemistry Program, The Ohio State University, Columbus, Ohio 43210-1292

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

Examination of conserved motifs on the cloned subunits of the deoxyguanosine kinase/deoxyadenosine kinase (dGK/dAK) of Lactobacillus acidophilus R-26 has begun with the Asp-Arg-Ser (DRS) motif. Replacement of Asp-78 of both subunits with Glu, Ala, or Asn reduced dGK and dAK activities to less than 0.2%, whereas replacement of Arg-79 with Lys, either on both subunits in tandem (R79K), or on the dGK subunit only (R79K:dGK), yielded active but kinetically modified enzymes. These were partially purified, and their kinetic and regulatory properties were analyzed. For dAK activity, the V(max) of the R79K:dGK enzyme was increased 28-fold, with no change in the limiting K for dAdo, but with a slightly reduced K for MgATP. The V/K efficiency ratio of dAK was also increased 29-fold, but that of dGK was decreased to 5-10% due to a 10-fold increase in K for dGuo and a reduced V(max). Therefore, the R79K substitution seems to have a greater effect on dGuo binding than on that of dAdo, but dGK modification appears to produce a stimulatory conformational effect on the opposite subunit, resembling the known unidirectional activation of dAK by either dGuo or dGTP.


INTRODUCTION

The four deoxynucleoside kinases of Lactobacillus acidophilus R26 exhibit a level of specificity for their respective deoxynucleoside substrates previously unknown among this class of enzymes. Although the lactobacilli contain a thymidine kinase (TK), (^1)an activity common to nearly all prokaryotes and eukaryotes, and which resembles the Escherichia coli enzyme in its regulation, (^2)the other three activities are apparently unique in terms of their nucleoside specificities, structural organization, and regulation. In contrast with human and other mammalian deoxycytidine kinases(1, 2, 3) , which actually phosphorylate dAdo and dGuo, as well as dCyd, at a common site, these three activities are found in lactobacilli as two heterodimers, dGK/dAK (deoxyguanosine kinase/deoxyadenosine kinase) or dCK/dAK (deoxycytidine kinase/deoxyadenosine kinase), and separate catalytic functions have recently been assigned to each subunit(4) .

Each subunit's activity is selectively controlled by feed-back inhibition from its respective dNTP end product, which (mounting evidence strongly suggests) binds to both the nucleoside and ATP sites as a bisubstrate inhibitor(4, 5) . The other remarkable regulatory aspect of these enzyme is the unidirectional activation of dAK, which in its unactivated state has only about one-fifth the activity of its associated dCK or dGK. However, when dCyd or dGuo, or their triphosphate homologs, occupy the respective dCK or dGK subunits, the turnover on dAK is increased up to 6-fold. (Curiously, dAdo or dATP exert very little stimulatory effect on dCK or dGK, in the opposite direction.) This effect is purely a turnover phenomenon; the K values for dAdo are unchanged by dCyd or dGuo(6, 7) . Recent evidence suggests that this heterotropic activation of dAK is due to conformational changes, analogous to the classical taut-to-relaxed inter-conversion, induced upon the respective binding of dCyd or dGuo (or their triphosphates) to the dCK or dGK active site (4, 8) . Under these circumstances, the dAK of either dimer becomes more susceptible to limited protolysis, presumably by toggling into an open or relaxed state, whereas selective proteolytic inactivation of dGK abolished this effect(4) . In a related experiment, photoaffinity labeling of the dGK active site with 8-azidoadenine permanently locks dAK into an activated configuration, again revealing conformational communication between the two subunits of the heterodimer(8) .

It is of great interest to identify the structural basis for these unusual regulatory phenomena as well as the sequences contributing to the enzymes' active sites. Scanning for homology with structural elements shown to be related to function in other kinases reveals that dGK/dAK contains the glycine- and arginine-rich motifs associated with the ATP- or GTP-binding sites of many nucleotide-binding proteins (9, 10) and which are also highly conserved in the adenylate kinases (11) and herpesviral thymidine kinases (HSV sites 1 and 5)(12, 13) . Another small conserved motif, -DRH- (HSV-TK site 3), found between the G-loops and the arginine-rich regions of herpesviral thymidine kinases(12) , may be analogous to the similarly located DRS motif of dGK/dAK. Mutations 4-12 residues further on the HSV-1 TK map (around HSV-TK site 4) produced increased K values for thymidine, leading Darby et al.(14) to suggest the region may be part of a thymidine-binding site. Random oligonucleotide mutagenesis leading to the replacement of a number of residues in the intervening region between sites 3 and 4 has also produced mutants with both increased and decreased K and k values for thymidine(15) , whereas a double amino acid substitution within this region of varicella-zoster TK (which is partially conserved relative to HSV TKs) resulted in reduced thymidine and thymidylate kinase activities and larger dissociation constants for these and analogous substrates(16) . Significantly, random mutagenesis of HSV-1 TK produced no active enzymes with modifications within the DRH motif, an indication of its possible importance, and site-directed mutagenesis of the corresponding Asp-162 completely inactivated the enzyme(17) , making it difficult to say whether the mutation affected substrate binding or turnover, or if it produced global disruption of structure. But, the essential character of Asp-162 and its proximity to site 4 would make it seem likely that sites 3 and 4 are both part of a larger nucleoside-binding or recognition domain for HSV-TKs.

The homology between the DRS motif of dGK/dAK and the DRH of HSV-TKs, although very limited in size, suggested the possible importance of these residues in some aspect of nucleoside binding and/or catalysis in Lactobacillus dGK/dAK. Furthermore, since the regulatory events described appear to be mediated through deoxynucleoside sites, it seemed more desirable to probe potential deoxynucleoside sites initially, rather than the recognizable ATP-binding elements.


EXPERIMENTAL PROCEDURES

Materials

2`-Deoxyadenosine and 2`-deoxyguanosine were purchased from Sigma; 2`-[2,8-^3H]deoxyadenosine and 2`-[8-^3H]deoxyguanosine were from Moravek Biochemicals, Inc. Deoxyadenosine 5`-[alpha-S]thiotriphosphate was provided by Amersham Corp. Bradford reagent and reagents for polyacrylamide gel electrophoresis were obtained from Bio-Rad. Escherichia coli XL1-Blue was from Stratagene. The Sequenase version 2 kit was purchased from U. S. Biochemical Corp., and all other materials, including protocols, cell lines, and enzymes for the site-directed mutagenesis, were from a Muta-Gene phagemid in vitro mutagenesis version 2 kit, which was purchased from Bio-Rad. Electroporation was done in a Gene Pulser from Bio-Rad, using 0.4-cm cuvettes.

Enzyme Assays

Deoxynucleoside kinase activities were assayed using DE-81 ion exchange paper by the method of Ives (18) with minor modifications as follows. In the standard assay, the final concentration of each reagent in the reaction mixture (in a 1.5-ml Eppendorf tube) was: ATP, 10 mM; MgCl(2), 12 mM; Tris-HCl, pH 8.0, 0.1 M; [^3H]dAdo (for dAK), [^3H]dGuo (for dGK), 0.2 mM (0.5 µCi/assay), respectively. The reactions were started by adding 20 µl of enzyme diluted in 15 mM potassium phosphate buffer, pH 8.0, containing 20% (v/v) glycerol to the warmed reaction mixture tube (final volume, 80 µl). After 30 min of incubation at 20 °C, 0.2 ml of 0.1 M formic acid was added to stop the reaction. Aliquots (20 µl) of the mixture were spotted on Whatman DE-81 anion exchange papers for the measurement of radioactivity(18) . The protein concentration of enzyme solutions was determined by the method of Bradford(19) . Specific enzyme activity is defined as the nanomoles of product per min per mg of protein. SDS-polyacrylamide gel electrophoresis was as described by Laemmli (20) .

Expression of Mutant Enzymes

E. coli XL1-Blue cells containing mutants of the recombinant pBlueScript KS(+) clone GTM-K48 were cultured in LB liquid medium, containing 100 µg/ml ampicillin, for expression of enzyme. Since the cloned genes are activated by an endogenous promotor, it was not necessary to add isopropyl-1-thio-beta-D-galactopyranoside for the stimulation of protein expression. The cell pellets were washed in ice-cold cell suspension buffer (0.1 M Tris-HCl, pH 8.0, 25 mM EDTA, 20% glycerol) and used immediately or stored at -20 °C for later use. The washed cell pellets were suspended in cell suspension buffer, broken by ultrasonication, and centrifuged at 13,500 times g for 1 h at 4 °C. Fraction IV enzyme(21) , prepared with only minor modifications yielded reproducible linear kinetics.

Site-directed Mutagenesis

The materials and protocols of the Muta-Gene phagemid in vitro site-directed mutagenesis kit (version 2) from Bio-Rad was used. Mutagenic oligodeoxynucleotides synthesized in the Biochemical Instrument Center of The Ohio State University were unblocked at 58 °C for 24 h, dried completely under N(2) gas, and purified with a Qiagen column (tip-20) by the manufacturer's protocol. Pure oligomers were dried in air, dissolved in 1 ml of 10 mM Tris buffer, pH 8.0, and stored at -20 °C until use. The quality of each oligomer was checked by 6% polyacrylamide gel electrophoresis. The 5` end of each mutagenic oligomer was phosphorylated with polynucleotide kinase to improve the frequencies of mutagenesis.

After the polymerization/ligation reaction was continued for 90 min at 37 °C, the reaction was stopped by adding Tris-EDTA buffer. Closed-circular heteroduplex recombinant pBlueScript KS(+) was extracted from agarose electrophoresis gel and used in the transformation of E. coli XL1-Blue by electroporation in a Gene Pulser (Bio-Rad), according to the manufacturer's protocol. Transformants were grown on LB plates containing ampicillin, and each instance of mutagenesis was confirmed by DNA sequencing, using the dideoxynucleotide termination method(22) .

Steady State Kinetics

Limiting K(m) values for deoxynucleoside substrates and MgATP were determined by steady state kinetics, varying both nucleoside and ATP concentrations. Slope and intercept replots yielded the limiting K(m) and V(max) values(23) . When ATP concentrations were varied, the concentrations of Mg in the reaction mixtures were kept at 120% of the corresponding ATP concentrations. Dissociation constants for the dNTP end product inhibitors were estimated by means of Dixon plots that were constructed by replotting reciprocal velocities, measured at several substrate concentrations in the presence of several concentrations of inhibitor, versus inhibitor concentration.


RESULTS

Site-directed Mutagenesis and Preliminary Assays

The -DRS- motif or its conservatively substituted analog has been implicated as part of the deoxynucleoside site in the HSV TKs. Its presence in Lactobacillus dGK/dAK and its location at the N-terminal boundary of a consensus sequence (D/E-R-S-I/V-X-D) shared with mammalian deoxycytidine kinases (24, 25, 26) makes it an attractive initial target in the search for the deoxynucleoside active site. We began by examining the functional significance of the first two charged residues, Asp-78 and Arg-79, on both the dAK and dGK peptides, by means of site-directed mutagenesis using the Kunkel (27) method. Eight different mutagenic oligomers were synthesized and are shown in Table 1. Aspartate 78 of both subunits was replaced with glutamate, a supposedly conservative replacement except for the longer side chain, with asparagine to eliminate the negative charge and with alanine to minimize the volume occupied by the side chain. Since the target amino acids may be located in the inner pocket, bulky substituents more likely to cause global disruption of the tertiary structure were avoided. At the Arg-79 residue substitution with lysine only was performed, providing a single positively charged center with a smaller side chain. However, two types of mutants were constructed; one bears the R79K mutation on both the dAK and dGK subunits, while the other has the R79K mutation on the dGK subunit only (R79K:dGK). As it is not yet clear whether the dAK subunit is processed in E. coli host like the wild-type Lactobacillus enzyme(24) , the product of the unmutated parental gene will be referred to as UMCE (unmodified cloned enzyme) in this report.



Both UMCE and mutant Lactobacillus enzymes were expressed in E. coli at levels amounting to at least 3% of the total soluble protein in crude cell extracts, judging from the intensities of the 27-kDa bands resolved by 12% SDS-polyacrylamide gel electrophoresis (Fig. 1). The quantities of dAK/dGK peptides expressed by UMCE and mutants apparently are identical, and they remain unchanged after 16 h of incubation with good aeration at 37 °C, which suggests that there is no specific or selective degradation of the expressed foreign proteins by E. coli proteases. The sizes of all of the expressed dAK and dGK peptides appear to be identical at about 27 kDa, in close agreement with the wild-type L. acidophilus R26 dAK/dGK peptides(4) . Crude extracts of all mutants were screened for activity by the standard assay. After partial purification of the expressed cloned enzyme activities, the relative quantities and molecular sizes were unchanged.


Figure 1: SDS-polyacrylamide gel electrophoresis profiles of expressed UMCE and mutant enzymes in crude extracts. Lanes 1 and 9, molecular mass markers (Bio-Rad low range); lane 2, pBlueScript transformed cell control; lane 3, UMCE; lane 4, D78A; lane 5, D78E; lane 6, D68N; lane 7, R79K; lane 8, R79K:dGK. Expressed protein is indicated by the arrow.



All three types of Asp-78 mutations (D78A, D78E, and D78N, involving both subunits) virtually eliminated both dAK and dGK activities, being less than 0.2% of the activities found in comparable UMCE fractions. As the activities of these mutants were deemed to be too low for reliable kinetics, further analysis was not attempted. While it is quite likely that Asp-78 is directly essential for the activities of each subunits, indirect effects such as disruption of folding cannot yet be ruled out as an explanation for the loss of activities.

Charge-conservative mutations at Arg-79, on the other hand, had a much more remarkable effect. While preliminary assays (not shown) revealed a reduction in the dGK activities of both the R79K and R79K:dGK mutants, the dAK activities were increased dramatically, whether or not the dAK subunit was altered. However, there was no apparent increase in the amount of peptide expressed. Preliminary enzyme activation and inhibition studies were carried out to determine whether these regulatory mechanisms might have been altered by R79K mutations. While both mutant enzymes remain susceptible to inhibition by end products dGTP or dATP, activation of dAK by dGuo is nearly eliminated in the case of the R79K:dGK mutant, while for the tandemly mutated R79K, there was even a 30% inhibition of the dAK activity upon addition of dGuo. In the opposite direction, dAdo produces, at most, a 10% stimulation of UMCE dGK, and this small stimulation is replaced by a comparable inhibition of the R79K:dGK mutant.

Effects of Arg-79 Mutations on Steady State Kinetics-Because mutations at or near the active site of an enzyme may alter its K(m) values, or even its kinetic mechanism, it is necessary to compare limiting K(m) and V(max) values (i.e. extrapolating to saturation by both substrates rather than the apparent values obtained by varying only one substrate) lest variable second-substrate saturation produces apparent differences in reaction velocities. Therefore, these parameters were determined for UMCE and each of the Arg-79 mutants by steady-state kinetics in which both deoxynucleoside and MgATP concentrations were varied (Table 2). The limiting K(m) for dAdo was increased severalfold in the tandem mutant (7.5 µM in R79K) but was unchanged by the dGK-only mutation. A greater effect is seen upon the binding of dGuo, with the K(m) for dGuo increasing about an order of magnitude on both the single and tandem mutants.



An interesting contrast in the effect of mutation on the K(m) values for MgATP occurs between the two subunits. Whereas both the single and tandem mutations resulted in decreased K(m) values for MgATP on the dAK subunit, i.e. whether or not the dAK subunit was altered, the K(m) for MgATP on dGK was doubled for both mutants. No change was produced in the K(m) for MgATP on dAK upon the binding of dGuo to dGK of UMCE (data not shown).

Effects of Arg-79 Mutations on Substrate Turnover

The other striking effect of mutations at Arg-79 is seen upon comparison of true maximum velocities which are proportional to the turnover number (Table 2). The turnover of dAK was increased 8-fold by the tandem R79K mutation and nearly 30-fold above that of UMCE by the R79K:dGK mutation, respectively. Under V(max) conditions these stimulatory affects cannot be explained in terms of any alteration of substrate binding at the dAK active site, whereas the relatively large changes in the K(m) values of both substrates for the dGK signal significant conformational changes at the dGK active site which may be capable of altering the ``tautness'' of the dAK subunit, like the positive heterotropic effector dGuo, but greater in magnitude. This is believed to be analogous to the somewhat smaller stimulatory effects on dAK activity which were detected following chemical modifications of the dGK active site(8) . The accompanying loss of stimulation by dGuo is certainly consistent with the notion that the dAK subunit was already locked into a much more active conformation by the mutation on the dGK subunit.

The other conclusion which can be inferred from these data is that, in terms of the V(max)/K(m) ``efficiency ratios,'' the R29K mutations can be said to have had opposite effects on the activities of the two subunits. Whereas the efficiency of dAdo binding and turnover combined increased from 3- to 29-fold, that of dGuo was decreased 90% or more. Since limiting values, i.e. real V(max) values and K(m) values, make up these ratios, it is even more apparent that asymmetry exists between the two subunits, despite their identical sequences within the motif which was mutagenized.

Confirmation of Kinetic Mechanisms

One other possibility remains to be examined: whether or not these mutations alter the kinetic mechanism of either subunit, which are known to differ in the wild-type dGK/dAK(5) . For example, might the ordered kinetic path followed by wild-type dAK be shifted to a random mechanism, e.g. possibly allowing for a faster off-rate? This does not appear to be the case, judging from the following observations. The first, possibly trivial, observation is that the mutations produced no qualitative changes in the steady-state kinetics patterns. For example, with the dAK of UMCE, plots of 1/V versus 1/MgATP at various fixed concentrations of dAdo, or of 1/V versus 1/[dAdo] at various fixed concentrations of MgATP, gave a family of straight-line plots converging at one point on the horizontal axis (results not shown). However, for the dGK component of UMCE, the plots of 1/V versus the reciprocal concentration of MgATP or dGuo, at various fixed concentrations of the other substrate, gave a family of straight lines intersecting in the upper left-hand quadrant. The Arg-79 replacement mutants yielded patterns qualitatively identical to those just described. Neither pattern, by itself, defines whether the sequential kinetics is ordered or random, of course, but the absence of any mutationally induced shift in the point of intersection of either pattern of lines, at least suggests that the mechanism is unchanged.

Far more compelling evidence for unchanged kinetic mechanisms is provided by end product inhibition studies. Earlier work from this laboratory has established that with these enzymes, as well as with the dCyd kinase/dAdo kinase pair from L. acidophilus R26, the respective dNTP end products mimic the effects of multisubstrate analogs(5) . Thus, in the case of random substrate binding, the multisubstrate analog or the homologous dNTP should compete with either substrate for the free enzyme, while for an ordered kinetic path these compounds will compete only with the leading substrate. For the wild-type dGK, dGTP competes with both dGuo and MgATP, consistent with a random mechanism, while for dAK dATP competes only with MgATP, the leading substrate in an ordered path, but is noncompetitive toward dAdo (5) . Identical conclusions as to differing kinetic mechanisms for the two activities were drawn earlier from classical product-inhibition analyses. However, the end product inhibition experiments used in the present work are practically simpler than the classical product-inhibition experiments used to establish the kinetic mechanisms of the wild-type enzymes, but provide clear-cut results. Dixon plots (1/V versus [dNTP] at several substrate concentrations) were used in obtaining numerical values for the inhibitor dissociation constants(23) . The K(i) values for end product inhibition against each substrate were decreased up to 75% in dAK, but were increased about 2-fold in dGK by both the R79K and R79K:dGK mutations (Table 3). However, neither the dNTP inhibition pattern nor, therefore, the kinetic mechanism of either enzyme was affected by mutations at Arg-79; the kinetic path for dAK remains ordered, while that for dGK remains random. Therefore, mutation at Arg-79 affects only the substrate binding and turnover of the paired enzymes, not their kinetic mechanisms.




DISCUSSION

The heterodimeric deoxynucleoside kinases of Lactobacillus present an array of important fundamental questions relating to the control of DNA precursor biosynthesis. These include the mechanism of end product inhibition of each activity, the structural basis of the exquisite deoxynucleoside specificity exhibited by each catalytic subunit and the positive heterotropic stimulation of dAdo turnover by dCyd or dGuo, respectively.

We elected to probe first the dGK/dAK DRS motifs, the conserved elements which by analogy with HSV TKs seemed likely to be associated with the deoxynucleoside sites. While we have no details yet of the dGK/dAK heterodimer's tertiary structure, a very preliminary prediction of the protein's secondary structure by the method of Chu and Fasman (28) or Garnier, et al.(29) suggests that the -DRS- motif may be at the turn of a loop. Therefore, if this local structure is involved in the binding of substrates or the stabilization of intermediates in the enzyme reaction, most substitutions should cause dramatic effects on enzyme activities. Accordingly, site-specific replacement of Asp-78 of both subunits with Glu, Asn, or Ala virtually abolished both the dGK and dAK activities of the mutants.

From the standpoint of uncovering the subtleties of enzyme regulation by subunit interaction, a much more interesting site of mutagenesis is found at Arg-79 of the Lactobacillus enzyme upon charge-conservative replacements with Lys. Whereas the dGK activities of R79K and R79K:dGK were reduced substantially, the dAK activities of those mutant enzymes were elevated dramatically above that of UMCE. This phenomenon is thought to be analogous to the opposite-substrate activation effect reported previously with wild-type dCK/dAK and dGK/dAK (6, 7) in which up to 6-fold stimulation of dAK activity occurs upon adding the opposite substrate, dCyd or dGuo, respectively, to the dAK reaction mixture. Arg-79-Lys mutations of the dGK subunit, either singly or in tandem with modification of the dAK subunit, essentially abolish this stimulation by dGuo, but, at the same time, produce an 8-28-fold increase in the V(max) of dAK. Affinity-labeling of the dGK active site with 8-azidoadenine produced a comparable, if smaller, effect(8) . We may presume that these modifications at or near what appears to be part of the dGK active site induce a conformational change in the dGK subunit similar to that produced by dGuo binding, and which, in turn, toggles the dAK subunit into its activated conformation. The smaller 8-fold stimulation in the case of the tandem mutant is probably due to some offsetting reduction of efficiency of the dAK subunit upon its mutation. It is important to note, however, that for R79K:dGK the K(m) for dAdo is unchanged, whereas it increases severalfold in the R79K mutant where both subunits are altered. Stimulation by dGuo has been shown to be purely a V(max) effect, producing no change in the K(m) for dAdo(7) , and the R79K:dGK mutation seems to have had an identical effect. Recent work on the wild-type dGK/dAK in this laboratory suggests that selective modification of the dGK subunit by photoaffinity labeling may also lock dAK into its activated configuration(8) , an effect which is mediated therefore through the dGK subunit.

The R79K mutation has opposite effects on the K(m) values for MgATP at the two active sites, but lower K(m) values on dAK would not account for the stimulation as the assay is carried out under near-saturating conditions with respect to MgATP. Since these mutations are not within either of the putative ATP-binding domains of the protein, we tentatively assume that global folding or interactions between domains are affected. On dGK the K(m) for MgATP is already much larger than on dAK, and is doubled again by the Arg-79 mutation. It should be noted that, even for the wild-type enzyme, the K(m) for MgATP on the dGK subunit (1.7 mM) is an order larger than on dAK (0.11 mM). Although the conserved ATP-binding sites at the N termini are genetically identical, they do not appear to be processed alike after translation in either Lactobacillus or the E. coli host(24) .

The evidence pointing to asymmetry between the tertiary and/or quaternary structures of the two subunits can be summarized as follows. (i) We note that they follow different kinetic pathways, one ordered and one random, and the activation of dAK by dGuo or by the R79K:dGK mutation does not alter this. However, the K(i) for dATP is decreased by about the same extent as the K(m) for MgATP, the leading substrate in the ordered mechanism. This is consistent with the understanding that dATP behaves as a multisubstrate analog bridging the deoxynucleoside and ATP-phosphate sites. (ii) The one-way stimulation of the dAK activity by dGuo or dGTP, by affinity labeling of the dGK subunit (8) and by R79K mutagenesis also supports this notion. (iii) The opposite effects of R79K replacement on the binding of MgATP to the two subunits, despite similar sequences, also suggests differences in folding or contact points of the heterodimer. For example, the subunits could be arranged in a head-to-tail fashion, effectively burying one of the N termini.

The detailed mechanism of the enhancement of dAK activity by R79K replacement is still unknown, but it seems most likely that Arg-79 is located near the contact area, possibly in a loop, between the dAK and dGK subunit, and the amino acid residues involved in the substrate binding site of one subunit might effect on the overall rate-limiting reaction steps of the paired subunit. Since the K(m) for dAdo is not changed by the mutation on dGK, turnover of dAK rather than substrate-binding is affected, and we may regard dGuo as a positive heterotropic effector of a rate-limiting step in dAK catalysis.


FOOTNOTES

*
This work was supported in part by National Institutes of Health Grants CA-47828 and GM49635. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Present address: Dept. of Biochemistry, State University of New York at Buffalo, Buffalo, NY 14214.

Present address: Dept. of Biochemistry, Molecular and Cellular Biology, Northwestern University, Evanston, IL 60208-3520.

**
To whom correspondence should be addressed: Dept. of Biochemistry, The Ohio State University, 484 W. 12th Ave., Columbus, OH 43210-1292. Tel.: 614-292-0485; Fax: 614-292-6773.

(^1)
The abbreviations used are: TK, thymidine kinase; dGK, deoxyguanosine kinase; dAK, deoxyadenosine kinase; dCK, deoxycytidine kinase; UMCE, unmodified cloned enzyme; HSV, herpes simplex virus.

(^2)
S. Ikeda, personal communication.


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