Proc. Natl. Acad. Sci. USAVol. 90, pp. 4012-4016, May 1993Biochemistry

Thymidine kinase mutants obtained by random sequence selection(random nucleotide sequences/evolution/mutations)

KHAN M. MUNIR, DAVID C. FRENCH, AND LAWRENCE A. LOEB*Joseph Gottstein Memorial Cancer Research Laboratory, Department of Pathology, SM-30, University of Washington, Seattle, WA 98195

Communicated by Earl Benditt, December 30, 1992

ABSTRACT Knowledge of the catalytic properties andstructural information regarding the amino acid residues thatcomprise the active site of an enzyme allows one, in principle,to use site-specific mutagenesis to construct genes that encodeenzymes with altered functions. However, such informationabout most enzymes is not known and the effects of specificamino acid substitutions are not generally predictable. Analternative approach is to substitute random nudeotides forkey codons in a gene and to use genetic selection to identify newand interesting enzyme variants. We describe here the con-struction, selection, and characterization of herpes simplexvirus type 1 thymidine kinase mutants either with differentcatalytic properties or with enhanced thermostablity. From alibrary containing 2 x 106 plasmid-encoded herpes thymidinekinase genes, each with a different nucleotide sequence at theputative nucleoside binding site, we obtained 1540 activemutants. Using this library and one previously constructed, weidentified by secondary selection Escherichia coli harboringthymidine kinase mutant clones that were unable to grow in thepresence of concentrations of 3'-azido-3'-deoxythymidine(AZT) that permits colony formation by E. coi harboring thewild-type plasmid. Two of the mutant enzymes exhibited areduced Km for AZT, one of which displayed a higher catalyticefficiency for AZT over thymidine relative to that of the wildtype. We also identified one mutant with enhanced thermosta-bility. These mutants may have clinical potential as the promiseof gene therapy is increasingly becoming a reality.

Genetic diversity can be achieved in vitro by insertingrandom nucleotide sequences into cloned genes. By geneticcomplementation, new mutants that encode active proteinscan be identified in these random nucleotide sequence librar-ies. This approach offers the promise of obtaining enzymeswith different substrate specificities or unique physical prop-erties. The underlying premise is that multiple amino acidsequences can carry out the same or similar reactions andthat during the course of evolution many of these sequenceswere discarded on the basis of fitness criteria that are nolonger relevant or ones that are different from those imposedby the experimenter. These techniques of applied molecularevolution (1) could be used to generate entirely new enzy-matic activities and could provide insights into pathways thatgoverned natural selection.We and others have used positive genetic selection to

demonstrate that new biologically active molecules can beobtained from random nucleotide sequences. Random se-quence substitutions within the -35 region ofthe promoter ofthe Escherichia coli tetracycline-resistance gene yielded acollection of 190 new active promoters (2). New mutants withdifferent specificities toward a series of penicillin analogueswere generated by substituting random sequences within aportion of the active site of 8-lactamase gene (3, 4). Parallelstrategies based on screening of random sequence libraries

have been used to define the sequence specificity of DNAbinding proteins (5, 6), to generate active ribozymes (7, 8),and to pan for new ribo- and deoxyribooligonucleotides thatbind to specific ligands or cellular receptors that mimicpolypeptide drugs (9-12). A further advance has been theinsertion of random nucleotide sequences into phage displaylibraries for the identification of new binding proteins (13,14).We demonstrate here the use ofrandom sequence selection

to change the catalytic properties of an enzyme withoutknowledge of its three-dimensional structure or even knowl-edge of the specific amino acid residues involved in catalysis.Herpes simplex virus type 1 (HSV-1) thymidine kinase (TK)catalyzes the phosphorylation ofdT to thymidine monophos-phate in the presence of ATP (15). HSV-1 TK, unlike themammalian or E. coli TK, can also phosphorylate dTMP,deoxycytidine, and a variety of nucleoside analogues such as3'-azido-3'-deoxythymidine (AZT) and acyclovir (15, 16).This phosphorylation of nucleoside analogues that inhibit orterminate DNA replication is the basis of drug therapyagainst herpetic infections. The gene for HSV-1 TK has beencloned, sequenced (17, 18), and expressed in E. coli (19); acrystal structure is not yet available (20). The ATP bindingsite has been mapped and the nucleoside binding site has beenputatively identified as encompassing amino acid residues165-176 (21-23). The informational content of each aminoacid residue within this sequence has been assessed bysubstituting an oligonucleotide that was 20% degenerate forcodons 165-175 (24). Here we present the results of substi-tuting a 100% random nucleotide sequence for 33 nucleotidesthat span positions 165-175. From -2 x 106 transformantswe obtained 1540 new active TK mutants. By screeningmutants from this and previous studies (24) we identified TKsthat selectively phosphorylate AZT as well as one that resiststhermal denaturation.

MATERIALS AND METHODSBacterial Strain, Plasmids, and Transformations. The E.

coli K-12 TK-deficient strain KY895 (K12, tdk-, F-, ilv 276)and anti-TK antibody were gifts of William Summers (YaleUniversity, New Haven). The TK expression vector, pMCC,and a "dummy vector," pMDC (which expresses an inactiveTK), were constructed as described (24). This vector alsocontains a 3-lactamase gene to render transformed bacteriacarbenicillin resistant. Bacterial transformations by plasmidDNAs were carried out by electroporation (24).

Oligonucleotides. A 52-mer with wild-type tk sequence,5'-d(TG GGA GCT CAC ATG CCC CGC CCC CGG CCCTCA CCC TCA TCT TCG ATC GCC AT)-3', and a 56-mercontaining random nucleotides, 5'-d(ATG AGG TAC CGNNNNNNN NNNNNN NNNNNN NNN NNNNNNNNNNNA TGG CGA TCG AA)-3', where N = equimolar concen-

Abbreviations: AZT, 3'-azido-3'-deoxythymidine; HSV-1, herpessimplex virus type 1; TK, thymidine kinase; DTT, dithiothreitol.*To whom reprint requests should be addressed.

4012

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Proc. Natl. Acad. Sci. USA 90 (1993) 4013

trations of G, A, T, or C, were synthesized by OperonTechnologies (Alameda, CA). The oligonucleotides wereseparated by electrophoresis through a 20% denaturing poly-acrylamide gel followed by purification on a reverse-phasemini column (Glen Research, Sterling, VA).Random Sequence-Containing Libraries. The random se-

quence library was constructed by inserting an oligonucleo-tide containing a stretch of 33 random nucleotides betweentwo unique Kpn I and Sac I restriction sites within theputative nucleoside binding site in the HSV-1 tk gene. Asynthetic 52-mer corresponding to the wild-type HSV-1 tksequence containing a Kpn I site at the 5' end was hybridizedwith a 56-mer containing random nucleotides correspondingto HSV-1 tk codons 165-175 (N = A, C, G, or T) andcontaining a Sac I site at the 3' end. The methods forextension, PCR amplification, and ligation of this fragmentinto the E. coli expression vector pMDC to replace thenonfunctional insert have been described (24). The ligatedproduct was introduced into tk- E. coli strain KY895. Thetotal number of transformants was determined by plating onLB agar containing 50 ,ug of carbenicillin per ml and thenumber of transformants that produced catalytically activethymidine kinase was determined by plating on TK-selectionmedium [2% BBL peptone, 0.5% NaCl, 0.2% glucose, 0.8%Gel-Rite (Scott Laboratories, Carson, CA)], 50 ,g of car-benicillin per ml, 10 ,ug of fluorodeoxyuridine per ml, 2 ,ug ofdT per ml, and 20 ug of uridine per ml (24).

Selection of AZT-Sensitive TK Mutants. A subset of 690mutants from the 100% random library and 190 previouslyisolated mutants from the 20% degenerate library (24) weresubjected to secondary screening to identify AZT-sensitiveclones. The mutants were first grown as individual colonieson TK-selection medium (1.0 ,g of dT per ml) and thenreplica plated onto AZT-selection medium (0.05 ,mg of AZTper ml, 1.0 ug of dT per ml). All other components in theAZT-selection medium are the same as the TK-selectionmedium. Those TK mutants that failed to grow on theAZT-selection medium were picked and retested for growthon TK- and AZT-selection media separately.

Affinity Purification. Purification of wild-type and mutantTKs was performed by affinity chromatography on CH-Sepharose 4B (Pharmacia) coupled to p-aminophenylthymi-dine 3'-phosphate (25, 26). Crude bacterial extract (24) waspassed three times through a 7-ml bed-volume affinity col-umn. The column was then washed sequentially using 30 mleach of buffer A [0.1 M Tris HCl, pH 7.5/5 mM dithiothreitol(DTT)/10% glycerol], buffer B (0.1 M Tris-HCl, pH 7.5/0.5M KCl/5 mM DTT/10% glycerol), and buffer A. TK waseluted using a 60-ml linear gradient of 0-600 ,uM dT in bufferC (0.3 M Tris HCl, pH 7.4/50mM KCl/10% glycerol). Activefractions were pooled and dialyzed against three changeseach of 2 liters of 50 mM Tris HCl, pH 7.4/5 mM DTT/10%glycerol. Except in the final dialysis, all the above bufferscontained 50 ,ug of aprotinin per ml and 2 jig each of pepstatinand leupeptin per ml.

RESULTSConstruction and Characterization of Random Sequence-

Containing Library. The scheme for the insertion of a 33-nucleotide 100% random sequence within the herpes tk issimilar to that previously used in the construction of a librarywith 20% degeneracy (24). Functional tk mutants were iden-tified by colony formation on TK-selection medium based ontheir ability to phosphorylate dT. We screened 2 x 106transformants from the 100% random library, of which 1540formed colonies on the TK-selection medium.

Selection Based on the Enhanced Phosphorylation of AZT. Asubset of 690 mutants from the 100% random library (TKI)and 190 mutants previously identified from the 20% degen-

erate library (TKF) (24) were subjected to secondary negativeselection on medium containing AZT to identify mutants thatexhibit enhanced phosphorylation of AZT. This screen isbased on the premise that mutants with increased ability tophosphorylate AZT relative to dT would be unable to formcolonies on the AZT-selection medium, since the product,AZT monophosphate, would be further phosphorylated bythe host cell's nonspecific nucleotide kinases or possibly themutant TK and then incorporated into bacterialDNA by hostDNA polymerases, terminate DNA synthesis, and preventreplication of the host chromosome. We first determined that1 ,ug ofdT per ml was the lowest concentration that supportedcolony formation by E. coli harboring wild-type TK as wellas most of the TK mutants obtained in the primary TK-selection protocol. We also determined that wild-type tkharboring E. coli could form colonies on medium containing1.0 ,ug of dT per ml plus 0.05 ug of AZT per ml. Of 880primary selectants that we screened, only two, TKF 105(from the 20% library) and TKI 208 (from the 100% library),formed colonies on the TK-selection medium at an efficiencysimilar to that of E. coli harboring the wild-type plasmid andyet failed to form colonies on the AZT-selection medium(Fig. 1A). The nucleotide and deduced amino acid sequencesof TKF 105 and TKI 208 are presented in Fig. 1B. Bothmutants contain a single amino acid substitution at the sameposition: Leu-170 was changed to Ile in TKF 105 and to Valin TKI 208. No other substitutions were observed in thesurrounding 220 nucleotides. To make sure that this differ-ence was not due to differential expression of TK in E. coliharboring mutant and wild-type plasmids, we comparedWestern blots ofextracts from cells containing either TKI 208or wild type. No significant difference was observed in theamount or electrophoretic mobility of immunoreactive stain-ing protein (data not shown). Also, the rate of dT phosphor-ylation per mg of protein was similar in extracts of E. coliharboring TKI 208, TKF 105, and wild-type plasmids (datanot shown).That the lack of growth of these mutants on AZT-selection

medium is due to enhanced phosphorylation ofAZT is furtherindicated by two criteria. (i) We determined the rate of[3H]AZT uptake relative to [3H]dT into E. coli harboringwild-type and mutant plasmids. Studies have indicated thatdT uptake in E. coli is correlated with the amount of TKactivity (27, 28). We found that E. coli harboring the AZT-sensitive mutants, TKF 105 and TKI 208, exhibited a 4-foldincrease in the ratio ofAZT to dT uptake compared to E. coliwith the wild-type plasmid (results not shown). (ii) Wedetermined the kinetics of AZT phosphorylation (Table 1).The AZT-sensitive variant TKI 208 (Table 1) exhibits a lowerKm (4.4 uM) compared to that of the wild type, 8.5 u.M. Bycomparing the kcat/Km between the two substrates (AZT vs.dT), we find that TKI 208 selectively phosphorylates AZT2.3-fold more efficiently than dT. Our preliminary observa-tion with purified TKF 105 TK also showed lower Km (3.7,M) for AZT but similar values for kcat/Km compared to thewild type (data not shown).Enhanced Thermostability. We analyzed the thermostabil-

ity of 50 TKF mutants. One of the mutants, TKF 2, was morethermostable at 42°C than any ofthe other mutants or than thewild type. Except for TKF 2, all of the mutants tested,including the wild type, had ratios of residual activity afterpreincubation at 42°C compared to 34°C of 0.05-0.30; TKF2 had a ratio of 0.7. TKF 2 contains three amino acidsubstitutions: Pro-165 -* His, Ala-167 -- Ser, and Ala-174 -*Val (Fig. 1B). We also examined our collection ofmutants forthe corresponding single amino acid substitutions. TKF 75contained an Ala-167 - S ersubstitution and TKF 56 con-tained Ala-174 -* Val, whereas TKI 440 with Pro-165 -+ Alawas the closest to the Pro-165 -- His substitution. Analysesof the thermostability of the unfractionated extracts from

Biochemistry: Munir et al.

Proc. Natl. Acad. Sci. USA 90 (1993)

A. TK-selection AZT-selection B. Sequence

165 166 167 168 169 170CCC ATC GCC GCC CTC CTGPro lie Ala Ala Leu Leu

171 172 173 174 175TGC TAC CCG GCC GCGCys Tyr Pro Ala Ala

CCC ATC GCC GCC CTC TGC TAC CCG GCC GCGPro lie Ala Ala Leu lie Cys. Tyr Pro Ala Ala

CCC ATC GCC GCC CTC [ TGC TAC CCG GCC GCGPro lie Ala Ala Leu | Cys Tyr Pro Ala Ala

)I ATC E GCC....

jm piIle Ala

CTC CTG TGC TAC CCG GCGLeu Leu Cys Tyr Pro [aI| Ala

FIG. 1. Selection of AZT-specific TK mutants. (A) Two AZT-sensitive clones, TKF 105 and TKI 208, along with an AZT-insensitive mutant,TKF 2, and the wild type were grown on TK- and AZT-selection media. The wild type and TKF 2 each formed a similar number of colonieson TK- and AZT-selection media. In contrast, TKF 105 and TKI 208 showed almost no visible colonies in the presence of AZT. In otherexperiments, TKF 105 and TKI 208 formed colonies on AZT-selection medium but they were 40-60%o fewer in number and smaller in size thanthose formed on the TK-selection medium. (B) The nucleotide and amino acid sequences within the targeted region are indicated; codon andamino acid substitutions are boxed and the codon numbers are presented above the wild-type nucleotide sequence.

each of these mutants at 42°C are presented in Fig. 2. Thethermolability of mutants TKF 56 and TKF 75 with Ala-174-* Val and Ala-167 -* Ser substitutions, respectively, was

similar to that of the wild type. Both lost >80% of theiractivity after incubation for 5 min at 42°C. TKF 440 with aPro-165 -* Ala is more stable but not as stable as TKF 2, the

triple mutant.Two types of experiments were carried out to verify the

thermostability of TKF 2. (i) We purified to near homoge-neity TKs from TKF 2 and the wild-type plasmid harboringE. coli and determined that the loss of activity is less in TKF2 than in the wild type after preincubation at 42°C (Fig. 2E).(ii) We transferred TKF 2 and the wild-type tk genes into a

vector with a promoter for T3 RNA polymerase. The RNAproduced by transcription in vitro was translated using arabbit reticulocyte lysate with [35S]methionine. An autoradi-ograph ofthe labeled proteins after SDS/PAGE (Fig. 3 Inset)indicates that the translation products migrate as doublebands corresponding to a protein of 43 kDa, which is inaccord with the reported size of HSV-1 TK expressed in E.coli (19, 20). The two bands could be due to the proteolyticdegradation of a 32-residue fragment at the amino-terminalend, which does not detectably alter TK activity of theHSV-1 TK (19, 20). The loss of TK activity of the in vitrosynthesized proteins from the wild-type and TKF 2 tk genesas a function of preincubation at 42°C is shown in Fig. 3. The

Table 1. Ability of wild-type and mutant HSV-1 TKs to phosphorylate AZT and dT

kcat/Km, kcat/Km (AZT)Phosphorylation Km, ,uM kcat, s-1 s-LM-1 kcat/Km (dT)AZT

Wild-type 8.46 ± 1.3 3.6 x 10-2 4.2 X 103 1.7 x 10-3TKI 208 4.40 ± 0.43* 3.0 x 10-2 6.5 x 103 4.0 x 10-3

dTWild-type 0.475 ± 0.10 1.21 2.5 x 106TKI 208 0.35 ± 0.008 0.56 1.57 x 106

Phosphorylation ofAZT: Kinetic analyses ofTK purified from wild-type and AZT-sensitive mutants.Reactions were carried out in a final volume of 100 ,ul containing 50mM Tris HCl (pH 7.5), 5 mM ATP,4 mM MgCl2, 2.5 mM DTT, 12 mM KCI, 0.18 mg of bovine serum albumin per ml, 5% glycerol, 0.08,Ci of [3H]AZT (Sigma), various concentrations of unlabeled AZT (0-4.0 ,uM), and purified enzymes(4 and 1.2 units, respectively, for wild-type and TKI 208). One unit ofenzyme is defined as that amountthat can phosphorylate 1.0 pmol of dT to TMP in 1 min under the conditions described above.Incubation was at 34°C ± 1C for 10 min and reactions were stopped by adding 1.0 mM unlabeled dTand cooling on ice. Half of the reaction mixtures were pipetted onto a DEAE-cellulose disc (25 mm),dipped in distilled water (1 min), followed by four washes in absolute ethanol. The amount ofradioactivity adsorbed to the disc was determined by scintillation spectroscopy. Km and Vmax valueswere determined by using the Cleland SUBIN program (29). The values for kcat were calculated usingthe equation Vm = kcat4[Elo, where [Elo = total enzyme concentration. Phosphorylation of dT. TKassays were carried out in a final volume of 50 ,ul using 0.3 ,Ci (3H-methyl]dT; 87 Ci/mmol;Amersham) and various concentrations of unlabeled dT (0-4.0 uM) and 1.1 and 0.5 unit ofTK for thewild type and TKI 208, respectively. All other components in the reaction mixtures and the incubationconditions were as described for phosphorylation of AZT.*Statistically significant values compared with wild type, P < 0.02.

Wild-Type

:. I

- i

.7.3

TKF 105

TKI 208

TKF 2

4014 Biochemistry: Munir et al.

.z\

Proc. Natl. Acad. Sci. USA 90 (1993) 4015

TFA B C DETKF56 TKF75 TKF 440

a) AlX>-tyAa174--a174 Ala167-*-Ser167 Pro -.Ala165cr 10- TKF 2

Wild-typeWidTp0-0

0 10 20 30 0 10 20 30 0 10 20 30 0 10 20 30 0 10 20 30 40

Preincubation (min)

FIG. 2. (A-D) Thermostability of wild-type and TK mutants using crude extracts. Twenty-five micrograms of each extract in 0.3 ml of 28mM Tris*HCl, pH 7.5/0.28 mg of bovine serum albumin per ml/28 ,ug of aprotinin per ml/2 ug (each) of pepstatin and leupeptin per ml waspreincubated at 42°C for 0, 5, 10, 20, 30, or 40 min. At each time point 30-,ul (2.5 ,ug) aliquots were assayed for residual TK activity in a totalreaction volume of 50 ,ul containing 50 mM Tris HCl (pH 7.5), 5 mM ATP, 4 mM MgCl2, 2.5 mM DTT, 12 mM KCI, 0.18 mg of bovine serumalbumin per ml, 5% glycerol, and 1 ,uM [3H-methyl]dT (60 x 103 dpm/pmol). Incubation was at 34°C for 10 min. The reaction was stopped bycooling on ice, and 25 ,ul was pipetted onto a DEAE-cellulose disc. Washing and assaying radioactivities ofthe discs were performed as describedfor the AZT assay in Table 1. (E) Thermostabilities of purified wild-type and TKF 2 enzymes. Affinity-purified enzymes were concentrated witha Centricon 30 filtration unit prior to use in the assays. The amount ofenzyme used was adjusted so that a similar amount of activity was presentin each sample prior to preincubation.

protein encoded by TKF 2 lost <10% of its activity afterpreincubation for 45 min. In contrast, the protein encoded bythe wild-type gene lost >80% ofits initial activity. The degreeof thermostability exhibited by the in vitro synthesized TKF2 was similar to or greater than that of crude extractsharboring the original TKF 2 plasmid.

DISCUSSIONWe have obtained >1700 active TK mutants from twolibraries by selection from -2.5 x 106 recombinant plasmidclones containing a segment of 33 random nucleotides span-

14 , _

0)

c

Ea)

.:

0en

I-TKF 2

\ V..:4":

Wild-Type TKF 2

Wild-1

3 kDa

rype

0 10 20 30 40 50Preincubation (min)

FIG. 3. Heat-inactivation profiles of in vitro translated wild-typeand TKF 2 TK. The full-length Bgl II-Pvu II fragments of tk genesfrom wild-type and TKF 2 plasmids were isolated and subcloned intothe pBluescript SK+ (Stratagene) vector between the Spe I andEcoRI sites with the use of synthetic linkers. In vitro transcriptionusing the T3 promoter was carried out using the Promega transcrip-tion system. In vitro translation was carried out using a reticulocytelysate system (Promega) following the supplier's protocol. ForSDS/PAGE analysis, the translated products were labeled with[35S]methionine. (Inset) Autoradiograph of the SDS/PAGE-fractionated in vitro translated products. The arrow indicates theexpected size of translated TKs as judged by molecular massstandards (Bio-Rad). For thermostability studies, TKs were synthe-sized in the presence of unlabeled methionine. Three experimentswere carried out on different days using different amounts of crudepreparations and, without exception, TKF 2 was more stable than thewild type.

ning codons 165-175 of the tk gene. Two of the mutantsstudied contain amino acid substitutions for Leu-170 and aresensitive to AZT. Another mutant, containing three differentamino acid substitutions, encodes a TK that is highly ther-mostable relative to the wild-type enzyme. This method forrandom sequence selection can be scaled up and yield evenlarger collections of mutants with different properties. Basedon the efficiency ofDNA transformation by current methods('1%) and on procedures for identifying mutants by geneticselection, we estimate that it is feasible to select mutants thatcode for active enzymes from as many as 1011 E. colitransformed with plasmids containing random sequences.Once information is obtained about permissible substitutions(24), the inserts can be redesigned to randomize only thosesubstitutions that are most likely to yield enzymes withdesired properties.Many current methods for protein engineering utilize site-

specific nucleotide substitutions to change the amino acidsequence of proteins. Specified amino acid substitutionswithin enzymes have been used to alter substrate specificity,to determine functional residues, or to redesign active sites(30-34). However, tailoring enzymes by site-specific muta-genesis is currently limited: (i) it is conveniently guided bystructural knowledge (only a few are known), (ii) the effectsof most single amino acid substitutions are not easily pre-dictable, and (iii) the rules to analyze the effects of multipleamino acid substitutions are only beginning to be developed(35), even though multiple substitutions are more likely toyield mutants with unusual properties (1, 36). We have shownhere that random sequence selection offers an attractivealternative to site-specific mutagenesis for the generation ofenzymes with improved properties and that random sequenceselection does not require a detailed knowledge of the three-dimensional structure of the enzyme.The secondary screening procedure for AZT-specific mu-

tants that we used relies on negative genetic selection. Theassumption is that the enhanced phosphorylation of AZTrelative to dT results in the incorporation of this analogue intoDNA and the termination of DNA replication. Presumably,an analogous protocol can be designed to search for mutantsthat preferentially phosphorylate other nucleoside ana-logues. Only 2 of the 880 active tk mutants that we screened,TKF 105 and TKI 208, did not form colonies on AZT-selection medium containing 0.05 ,g ofAZT per ml and 1 ,ugof dT per ml. E. coli harboring either of these mutants exhibita >3.5-fold increase in the uptake of AZT relative to that ofdT when compared to either the wild type or to two other

vvI

Biochemistry: Munir et al.

Proc. Natl. Acad. Sci. USA 90 (1993)

mutants that were tested (not shown). Studies on purified TKfrom TKI 208 and TKF 105 indicate that the enhancedcatalytic efficiency with AZT as a substrate is predominantlydue to a reduction in the Km. The substituted amino acids,valine and isoleucine, in the two AZT-sensitive mutants arenot structurally very different from that of the wild-typeLeu-170. These results indicate that an isosteric change withno addition or removal of polar groups and no major confor-mational alteration is likely to have taken place in producingthe mutant enzymes. The finding that the two mutantsidentified as AZT-sensitive contain a single amino acid sub-stitution at Leu-170 highlights the importance of this residuein substrate binding.Random sequence selection of P-lactamase mutants in our

laboratory (3) and Struhl's laboratory (4) as well as studies onHSV-1 TK (37) indicate that the many mutant enzymesobtained by random sequence selection are thermolabile.Only 1 of the 50 mutants that we have analyzed was signif-icantly more thermostable than the wild type. The loss ofactivity at 42°C for up to 40 min in extracts from either TKF2 or the wild type was not the result of proteolysis since therewas no degradation in immunoreactive protein upon electro-phoresis after incubation at 42°C. The thermostability ofTKF2 TK was verified by analyzing the purified enzyme and theenzyme synthesized in vitro.We analyzed the contribution of each of the three amino

acid substitutions in TKF 2 to its enhanced thermostability bystudying extracts of mutants that contained only single aminoacid substitutions at the same positions. Only one mutantwith Pro-165 -- Ala is more thermostable than the wild typebut is not as thermostable as TKF 2 (Fig. 2). These resultssuggest that none of the single substitutions is adequate toaccount for the large enhancement in thermostability exhib-ited by TKF 2. The identification of a thermostable mutantsuggests that a systematic approach to selecting thermostableenzymes from random sequence-containing libraries mightbe fruitful. Mutants could be selected directly based on thethermostability of the transformed gene products in thermo-philic bacteria (38, 39). The production of enzymes that arestable at elevated temperatures and presumably resistant toother denaturing agents could have useful industrial andclinical applications.The broad substrate specificity of HSV-1 TK renders it a

target for nucleoside analogues that abate herpetic infections.The success of treating herpetic infections with nucleosideanalogues has brought into the forefront the possibility ofinserting the HSV-1 tk gene into target cells and then selec-tively killing them by growth on specific nucleoside ana-logues. Our present study demonstrates the efficacy of ran-dom sequence selection to obtain mutant TKs that preferen-tially phosphorylate specific nucleoside analogues such asAZT. This methodology can also be extended to produceTKs that preferentially phosphorylate gancyclovir and othernucleoside analogues. The introduction of these tk mutantgenes into cells may render the cells particularly susceptibleto analogues that are preferentially phosphorylated by themutant enzymes. Culver et al. (40) have recently shown theefficacy of the in vivo transfer of the HSV-1 tk gene for thetreatment of experimental brain tumors. The use of specificTKs should facilitate specific killing of tumor cells in thepresence of less toxic concentrations of the therapeuticnucleoside analogues.

We thank Drs. M. S. Z. Horwitz, A; Blank, and A. Mildvan forcritical comments. This work was supported by Grants OIG R35-CA-39903 (L.A.L.) and AG-00057 (K.M.M.) from the NationalInstitutes of Health.

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