Probing the infra-structure of thymidylate synthase and deoxycytidylate deaminase

PROBING THE INFRA-STRUCTURE OF THYMIDYLATE SYNTHASE AND DEOXYCYTIDYLATE DEAMINASE FRANK MALEY, MARLENE BELFORT and GLADYS MALEY Center for Laboratories and Research,New York State Department of Health, Empire State Plaza, Albany, New York 12201

INTRODUCTION At the Eighth Symposium on Enzyme Regulation we presented data on regulatory processes affecting both deoxycytidylate (dCMP) deaminase (EC 3.5.4.12) and thymidylate (dTMP) synthase (EC 2.1.1.45) (1). The sequential relationship of these enzymes within the pyrimidine deoxynucleotide pathway, coincident with the marked responsiveness of dCMP deaminase to allosteric end-product regulation (2), provides most animal cells with a particularly sensitive means of regulating dTMP for DNA synthesis. The fact that nature has committed the responsibility for the synthesis of dUMP to 3 allosteric enzymes, dCMP deaminase (2), ribonucleotide reductase (3), and deoxyuridine (thymidine) kinase (4), cannot help but suggest that the formation of this compound plays an important if not rate controlling step in cell division. Our findings at the previous symposium dealt primarily with kinetic responses of the deaminase, from which information regarding the functional groups involved in substrate and effector binding was derived. Responses of the synthase to methotrexate were demonstrated in vivo, and the elevated levels of this enzyme were shown subsequently to be due to an altered steady state resulting from an impairment in enzyme degradation (5). At the time of the earlier meeting an in depth analysis of the structures of the synthase and deaminase was not possible due to the absence of methods which would provide the necessary amounts of both enzymes required for this type of study. These problems have been resolved since by the development of procedures for, 1) isolating methotrexate resistant high dTMP synthase (6, 7) producing strains ofLactobacillus caseL 2) incorporating the dTMP synthase gene from Escherichia coli and phage T4 into amplification plasmids, and 3) the synthesis of a folate analog which has provided an extremely efficient affinity column for the purification of dTMP synthase from various sources (8). In addition a large scale fermentation procedure was developed to isolate dCMP deaminase from T2-phage infectedE, coll. Although they are not from a eucaryotic source, the similarity in properties of these enzymes from 413

414

FRANK MALEY, et al.

bacterial and animal sources suggests that meaningful comparisons can be made, particularly with respect to their phylogenetic evolution. The studies to be presented are but a start in this direction, from which it is hoped that a more complete picture will be available in the near future.

MATERIALS

AND METHODS

Chemicals. Labeled deoxynucleoside mono- and triphosphates were purchased from New England Nuclear Corp., while [214C]5-fluorodeoxyuridylate (FdUMP) and [63H]FdUMP were from Moravek Biochemicals. The unlabeled ribo- and deoxyribonucleotides were obtained from P-L Biochemicals or from Sigma. Hn-dUMP,4-N-hydroxy-dCMP, 5-hydroxymethyl(HM)-dCMP and HM-dCTP were prepared as described previously (911); 5-nitro-dUMP was a generous gift of Dr. Daniel V. Santi. The folylpolyglutamates employed were kindly provided by Dr. Charles M. Baugh. Oligonucleotide linkers were purchased from New England Biolabs and dideoxynucleoside triphosphates and heptadecanucleotide universal primer were from P-L Biochemicals. Enzyme preparation and assay. L. casei dTMP synthase was purified and crystallized as previously described (12). The enzyme was stored routinely at 4°C in 20 mM potassium phosphate, pH 7.0, containing 20 mM 2mercaptoethanol. The E. coli and T-even phage dTMP synthases were purified from crude extracts using primarily the quinazoline affinity column procedure of Rode et al. (8). The affinity column was generously provided by Drs. Wojciech Rode and Joseph R. Bertino. To assay these enzymes, the spectrophotometric procedure of Wahba and Friedkin (13) was employed. Homogeneous T2-phage dCMP deaminase was prepared as described earlier (14), and assayed by a continuous recording method (15). T4-DNA ligase and BAL-31 nuclease were purchased from New England Biolabs. The Klenow fragment ofE. coli DNA polymerase was obtained from Bethesda Research Labs. Restriction enzymes, such as BamHI, ClaI, EcoRv, EcoRI, HindlII, PvulI, PstI, and others were from either of the above suppliers and used according to their instructions. Bacterial strains, phage strains and plasmids. The various recombinant plasmids containing the T4- and E. coli synthase genes were constructed from the plasmid vector pBR322 and then transferred to the high amplifying expression plasmid pKC30 (16). The methodology employed in both cases has been described in detail (17, 18). DNA andprotein sequence analysis. Both strands of the thyA gene from E. coli were sequenced by the dideoxy chain termination method of Sanger (19).

d T M P SYNTHASE A N D d C M P D E A M I N A S E

415

Single-stranded DNA templates of either BAL-31 generated deletions or specific rest riction fragments were cloned into sequencing vectors M 13 mp8 or M 13mp9 (20). Both DNA and protein sequence data were stored and analyzed by programs 1 and 2 of Larson and Messing (21). Protein sequencing. The methods employed for determining the complete amino acid sequences of both L. casei dTMP synthase (22-24) and T2-phage dCMP deaminase (25) have been described, as has the procedure for isolating and analyzing the FdUMP-ternary complex peptide (23, 26). Most of the isolated peptides were sequenced using a Beckman 890B sequencer.

RESULTS

AND

DISCUSSION

Thymidylate Synthase Structural analysis of substrate and inhibitor binding sites. The development of MTX resistant strains ofL. casei (6, 7) that contained a 100-fold enrichment in dTMP synthase, coupled with a modification in the original purification procedure (12), were essential steps in providing adequate amounts of enzyme for the desired structural studies. As a consequence the enzyme was verified to be a 73,000 dalton dimer composed of identical subunits, each consisting of 316 amino acids (22-24). An added dividend was the clarification of the 5fluorodeoxyuridylate (FdUMP) binding region of this protein, first isolated as a nonapeptide (26) and then located more precisely at cysteine residue 198 (Fig. 1) (23). The involvement of a nucleophile at the catalytic site was originally suggested by Santi and Brewer (27) from model studies and subsequently verified by the elegant mechanistic studies of Santi and his group with the synthase from L. casei (28). However, since FdUMP was employed in these studies it was not completely certain that dUMP and FdUMP are bound to the same amino acid, although equilibrium dialysis studies reveal these 2 nucleotides to compete for the same region (29), if not the same amino acid. These studies have, in addition, verified our original kinetic studies with the chick embryo synthase (30), showing that dUMP binds to the enzyme prior to the second substrate, 5,10-methylenetetrahydrofolate (5,10-CH2H4PteGlu). However, there still remains the unexplained anomaly of dUMP binding to a single site, possibly by a half-the-sites mechanism, while FdUMP binds to two sites (one per subunit) (29, 31). To locate the dUMP binding region precisely requires the fixation of dUMP in a manner tight enough to facilitate its isolation as a peptide, similar to that described for the FdUMP ternary complex (26, 32). We have been able to affect this fixation recently by exposing the enzyme to UV light in the presence of dUMP alone (Table 1). The fact that compounds which are mechanism based inhibitors of dUMP, such as FdUMP (28) and 5-nitro-dUMP (33, 34), impair the fixation, with the latter obviously a more tightly bound compound than FdUMP (about 1000× better), suggests

416

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THR-LE~-GLN-LEu-A~N-PR~-A~P-LY~HIs-AsP~ILE-PHE-Asp-PHE-AsP-MET-LYs-A~P~ILE-LYsLEU-LEu-AsN-TYR-AsP-PRo-TYR-PRO-ALA-I~-LYS-ALA-PRo-VAL-ALA-VAL-OH FIG. 1. The primary sequence ofL. casei thymidylate synthase designating the active site region for FdUMP (area encompassing cysteine-198) and the region labeled by PteGlu7 (area encompassing lysine 50, 51 and lysine 58). Reproduced from (36).

that d U M P is binding to cysteine-198 or to an amino acid in its proximity. If this bond is not labile, we should be able to locate the precise binding site of dUMP. The fact that U M P and 4-N-hydroxy-dCMP inhibit is not surprising in view of the fact that the former can act as a substrate and the latter as an inhibitor of the synthase (30). Since folate exists in nature primarily as a polyglutamate, which can bind to the synthase in the absence of d U M P in contrast to the monoglutamate derivatives of folate (29, 35), the location of this binding site in the synthase was undertaken. For this purpose labeled pteroyl heptaglutamate was used, with the most distal glutamate to the pteroic acid moiety being activated with a water soluble carbodiimide (36). To enhance the specificity of binding, d U M P was included in the reaction, resulting in the fixation of the activated

FIG. 2. Scanning electron micrographs of crystals of L. casei and T2-phage thymidylate synthases.

dTMP SYNTHASE AND dCMP DEAMINASE

417

TABLE 1. EFFECT OF UV254nm ON THE FIXATION OF dUMP BY dTMP SYNTHASE IN THE PRESENCE OF VARIOUS NUCLEOTIDES Added nucleotide (nmol) 0 FdUMP UMP 4-N-HO-dCMP 5-Nitro-dUMP dCMP

(25) (25) (25) (2.5) (25)

dUM32P Incorporated (pmol) 88O 310 550 370 0 640

Each reaction mixture contained the following components (in nmol): N-ethylmorpholine pH 7.0, 200; MgCI2, 2.5; dUM32P (1.11 × l0s cpm/nmol), 11.4; nucleotide as indicated; L. casei thymidylate synthase, 2.0; and H20 to 50 #1. The mixtureswere irradiated in the cold with an overhead UV254nm source at 3 mwatts/cm 2 for 20 rain at which time a 10/~1aliquot was removedand added to a solution of 200 #g of bovine serum albumin in 0.2 ml of cold water. Similar aliquots were removedprior to irradiation as a zero time control. Following addition of 1.5 ml of cold 5% trichloroacetic acid the precipitates were filtered through a 25-mm HAWP Millipore nitrocellulose filter. After washing with two 3 ml vol of trichloroacetic acid, the filters were dried under a heat lamp, placed in a scintillation vial with 6 ml of a toluene based fluor and counted. g l u t a m a t e to lysine 58 of one s u b u n i t a n d lysine 50, 51 o f the other (Fig. 1). It s h o u l d be n o t e d that this region is considerably distant from cystein-198, no d o u b t reflecting the length of the h e p t a g l u t a m a t e residue, a n d the folding of the p r o t e i n itself. F u t u r e studies will establish whether 5,10-CH2H4PteGIu binds to the same lysines or not. The d T M P synthase from L. casei a n d that i n d u c e d in E. coli by T2-phage have been crystallized a n d the u n i q u e n e s s of each is readily a p p a r e n t in the s c a n n i n g electron micrographs of Figure 2.

Construction o f plasmids containing dTMP synthase genes and the amplification o f their gene products. The lengthy a n d a r d u o u s procedures required for sequencing the L. casei enzyme deterred us from u n d e r t a k i n g a similar study with synthases from other sources, that is until we were able to o b t a i n d T M P synthase c o n t a i n i n g gene segments from E. coli (37) a n d T4phage (38). The strategy for s u b c l o n i n g these D N A fragments into a high amplifying plasmid adjacent to the ?,PL phage p r o m o t e r is indicated in Figures 3 a n d 4. W h e n the plasmids were g r o w n in a lysogen c o n t a i n i n g a t e m p e r a t u r e sensitive repressor a n d placed at the restrictive t e m p e r a t u r e an increase of at least 200-fold was o b t a i n e d for both the T4-phage (17) a n d E. coli (18) synthases. This level of activity represents at least 5% of E. coil cellular

418

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FIG. 3. Construction of pBTd and pKTd plasmids. (A) cloning the T4 td fragment into pBR322. The transducing phage hT4tdl, constructed by Mileham et al. (38), comprises the 2.7-kb tdcontefiningEcoRI fragment of phage T4 cloned into theEcoRI replacement interval of the Aimm21 vector NM816. The arrows show the direction of transcription ofpL. which is located beside the immunity region (imm), as well as the transcriptional orientation of the td gene. The solid circle marks the position of the h attachment site. The 2.7-kb T4 td fragment was subcloned into the EcoRI site of pBR322. These constructions are designated pBTd. (B) cloning the T4 td fragment into expression plasmid pKC30. The 2.7-kb EcoRl restriction fragment was excised from a pBTd derivative, purified, and made blunt-ended by treatment with E. coli DNA polymerase I in the presence of the four deoxynucleoside triphosphates. This blunt-ended td fragment was then ligated into the HpaI sites of pKC30, 321 nucleosides downstream from the ApL promoter. Recombinants carrying the T4 td insert in the correct transcriptional orientation relative topL are designated pKTd. Reproduced from (17).

protein. T h e degree to which the e n z y m e is am p l i f i ed is easily seen in the gel p a t t e r n o f the crude E. coli extracts before and after the t e m p e r a t u r e was raised f r o m 32°C to 42°C (Fig. 5). T h a t the gene was derived f r o m E. coli was d e m o n s t r a t e d by a S o u t h e r n blot analysis o f the restriction f r a g m e n t s f r o m the c l o n e d gene a n d E . coli D N A and by the a m p l i f i ed e n z y m e ' s reactivity with a n t i b o d y to pure E. coli d T M P synthase (18). O b t a i n i n g b o t h enzymes in a pure state was facilitated by use o f the 5 - f o r m y l q u i n a z o l i n e affinity c o l u m n p r o c e d u r e o f R o d e et al. (8). T h e c o m p l e t e sequence o f the 1.16 Kb f r a g m e n t e n c o d i n g the t h y A gene o f E. coil was d e t e r m i n e d in b o t h strands using the Sanger d i d eo x y chain t e r m i n a t i o n m e t h o d (19). F r o m this f r a g m e n t a 792 nucleotide o p e n r ead i n g

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420

F R A N K MALEY, et al.

flame encompassing the 264 amino acids of the gene was deduced, and confirmed by amino acid sequencing of selected peptides in regions where ambiguities in the DNA sequence existed (39). The fact that the -10 a n d - 3 5 RNA polymerase interaction regions of the thyA gene fragment do not conform exactly to the normal E. coil consensus sequences may be the reason why this enzyme is only expressed to the extent of 250 molecules/rapidly dividing E. coli KI2 cell (39). This important problem is currently under investigation. Comparison of dTMP synthases from E. coli and L. casei. Although theE. coli dTMP synthase dimer is about 10,000 daltons (51 amino acids) smaller than that from L. casei, there appears to be a 62% conservation of amino acid sequences between the two when the region spanning residues 89-139 is deleted from the L. casei enzyme (Fig. 6). This homology is seen in the matrix comparison of the two enzymes in Figure 6. By lining these proteins up in the manner indicated, it becomes apparent from a hydrophilicity plot (Fig. 7) that most of the homology occurs in the hydrophobic regions, which appear to be more conserved than the hydrophilic regions. In fact, it is readily seen that the 51 amino acid deletion occurs primarily in an area containing mostly polar amino acids (Fig. 7). It would be of interest to determine the effect that this deletion has on the L. casei synthase's activity. The most highly conserved

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E. coh FIG. 6. Matrix comparison of the thymidylate synthase sequences ofE. coli andL. casei. Three consecutive amino acid matches between the 264-residue sequence of the E. coli enzyme and the 316-residue L. casei sequence have been plotted by using the matrix comparison option of version 2 of the Larson and Messing sequence program (21). The 51-residue E. coli 'deletion' is represented by a corresponding break in the diagonal homology plot. The inset is an amino acid comparison of the two sequences in the area immediately surrounding the 51-residue disparity.

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FIG. 5. Identification and purification of the amplified thyA gene product. Crude extracts prepared from cultures of U C 5 8 2 6 / p K H A T (lanes 1 and 3) or U C 5 8 2 6 / p K T A H (lanes 2 and 4) before (lanes 1 and 2) and after (lanes 3 and 4) shifting the cultures to the elevated temperature were separated on a 10% gel. The amplified synthase band, which appears only when the thyA gene is correctly oriented and only after incubation at 42°C (lane 4), corresponds to the enzyme purified from such an extract on an affinity column (8) (lane 5). Reproduced in part from (18).

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FIG. 7. Comparative hydrophilicity plots of E. coli and L. casei dTMP synthases. This comparison based on a running average of seven amino acids using version 2 of the Larson and Messing sequence analysis program (21), revealed a 62% overall homology between the two enzymes. However, this homology increased to 82% for a 40 amino acid stretch surrounding the FdUMP binding region. Note the complete absence ofa hydrophilic 51 amino acid region in theE. coli synthase, which is however present in the L. casei enzyme. The average hydrophilicity for the amino acid sequences was calculated using values from +3 for hydrophilic amino acids (arginine, glutamic acid) to -3.4 for hydrophobic residues such as tryptophan. Reproduced from (39). region, a m o u n t i n g to over 80% homology, is a 40 amino acid segment encompassing the active site, which occurs in a h y d r o p h o b i c pocket in both enzymes. By analogy to the active site cysteine-198 residue in L. casei, this residue is located at cysteine-146 in E. coli (Figs. 6 and 7). The strong conservation of the active site region is even more apparent when the amino acid sequences of several synthases are c o m p a r e d as below:

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422

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Even in those cases where amino acids are not the same, one of comparable hydrophobicity is usually substituted. The F d U M P binding region, putatively the 'active site', was confirmed for all of the above enzymes, with the exception of the yeast synthase, by sequence analysis of FdUMP-ternary complex containing peptides. Comparison of dTMP synthase from E. coli with those induced by T2- and T4phage. When the synthase activities from T2-phage andE. coli were compared distinct physical and activity differences between them were noted, consistent with the differences in their amino acid compositions and molecular weights (59,000 daltons for E. coli vs 64,000 daltons for T2-phage) (40). As a consequence of t.~ ese differences, it was not surprising that the 2 enzymes did not cross react with one another's antibody or that their respective genes did not cross-hybridize in Southern blots (17), although antibody to T2-dTMP synthase did cross react with the T4-dTMP synthase. One of the distinguishing features of these enzymes was the finding that pteroylhexaglutamate could almost completely inhibit the T2-phase enzyme at 5 × 10-5 M in the absence of added Mg 2+, but barely affected the E. coli synthase at this concentration. However, on addition of M f + a reversal of the inhibitory characteristics of this folate derivative was noted (41). These remarkable differences prompted us to undertake the sequence analysis of the T4-enzyme due to the availability of its gene (38), in the hope that this information might be used to more rationally explain some of the characteristic kinetic responses of the T-even phage and E. coil synthases. By employing techniques similar to those described for the E. coli synthase, the nucleotide sequence of the T4-synthase gene and the amino acid sequence of its product have been completed recently. The data generated by these studies enabled Purohit and Mathews (42), who had been sequencing T4dihydrofolate reductase (FH2R), to establish that the reductase and synthase genes overlap in a rather unique four base sequence, A T G A . It appears from their findings that the nucleotides encoding the terminal lysine and the opal stop codon of FH2R overlap with the methionine or start codon of the dTMP synthase (TS). This relationship is indicated below: FH2R--

Lys -- Stop

-AAArGAAAMet -- Lys --TS

It is of interest to note that the FH2R and dTMP synthase of certain parasites are fused together as a single polypeptide chain (43, 44). Small sequence alterations in the stop codon region, which eliminate its function, might effect a similar fusion of the T4-reductase and synthase genes. Whether the case of the parasite fusion polypeptide is fortuitous or bears any relation to the comparable phage genes remains to be determined.

dTMP SYNTHASE A N D dCMP D E A M I N A S E

423

Deoxycytidylate Deaminase Subunit composition. As indicated in the introduction, the phage induced dCMP deaminase is an oligomeric protein similar to that found for most, if not all, allosteric enzymes, but in this case it is composed of 6 identical subunits (14, 45). The chick embryo dCMP deaminase* has also been shown to be a hexamer and more recently similar results were reported for the donkey spleen deaminase (46). In contrast, the human spleen enzyme has been claimed to be a dimer consisting of 110,000 dalton subunits (47). However, these results are suspect in view of the fact that the specific activity of t h e ' p u r e spleen enzyme' is one sixtieth that of the highest specific activities reported for homogeneous dCMP deaminases from several diverse sources. Recent equilibrium dialysis studies (48) with T2-dCMP deaminase are consistent with earlier kinetic studies on the pH dependence of the allosteric response of this enzyme to its positive effectors (14). In agreement with the latter it is seen in Figure 8 that the binding of H M - d C T P is much more pH dependent than dCTP. It was also somewhat reassuring to note from a pragmatic, as well as from a physiologic, point of view that the enzyme was much more effectively regulated by its natural effector, HM-dCTP, than by dCTP. This effect is complemented by the apparent inability of the phage enzyme to deaminate H M - d C M P whether in the presence of H M - d C T P or dCTP, although the animal deaminase can (11). If H M - d C M P was deaminated by the phage enzyme, the ability of the phage to synthesize D N A would be impaired since this nucleotide is substituted for dCMP in T-even phage DNA.

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FIG. 8. Effect of pH on binding of dCTP and H M - d C T P by T2-dCMP deaminase. The ligand cells contained initially varying concentrations of [2-]4C]dCTP (20 d p m / p m o l ) or [2-~4C]HM dCTP (3.1 dpm/pmol). The protein cells contained 188 gg (152 pmol) of enzyme. Both cells contained 20 # 1 of buffer A in a final vol of 50,u 1. Ligand binding was measured as follows: dCTP pH 6 (O) and pH 10 (0); HM-dCTP, pH 6 (el) and pH 10 (11). Reproduced from (48). *G. Maley, unpublished data.

FRANK MALEY, et al.

424

While binding parameters could easily be obtained for d C T P and H M dCTP, little or no binding was obtained for d T T P under conditions similar to those employed for its positive effector counterparts, that is until it was recognized that the binding o f d T T P was much more sensitive to salt than was the binding o f d C T P (Table 2). Once this problem was resolved, it could be shown that the n u m b e r o f binding sites for d T T P was 6, which is consistent with the n u m b e r of enzyme subunits/mol. This value was identical to that obtained for dCTP, H M - d C T P , as well as that for d C M P and its competitive inhibitors (48). The non-linearity of the binding plots (48) was not unanticipated in view of the cooperative nature of the kinetic responses o f the enzyme to the effectors (14). Since allosteric reactions are usually associated with conformational shifts in protein structure, which can be measured by changes in protease susceptibility or functional group reactivity as already shown for the chick e m b r y o deaminase (49), a more physical a p p r o a c h was sought to confirm this effect for the T2-deaminase. O f those techniques employed, such as UV difference spectra or changes in fluorescence, the most sensitive appeared to be circular dichroism (CD). As seen in Figure 9, the deaminase showed a marked negative cotton effect in the 280 nm region when concentrations of d C T P as low as 5 to 10 #M were present and reached a m a x i m u m at 30/~M. A l t h o u g h d T T P does not elicit C D changes directly, it was f o u n d to reverse the negative shifts effected by dCTP, in keeping with its properties as an antagonist of dCTP. Attempts at locating the allosteric binding sites. Since it appears that d C M P deaminase is regulated by heterotrophic interactions a m o n g its subunits in response to its substrate and allosteric effectors, identifying their binding sites within the enzyme could eventually aid in clarifying the mechanism o f this interplay. Support for the importance o f subunit interactions as a regulatory

TABLE 2. EFFECT OF BUFFERS ON LIGAND BINDING TO T2-dCMP DEAMINASE AS MEASURED BY EQUILIBRIUM DIALYSIS dCTP HM-dCTP dTTP Buffer

mM

APB* Phosphate Tris-HC1

50 50 40

pmol bound 515 546 511

702 685 670

0 245 205

The protein cell contained 152 pmol of enzyme. The ligand cell contained [2-14C]dCTP, 1.8 nmol (22 dpm/pmol), [2-14C]HM-dCTP,5.7 nmol (3.1 dpm/pmol), or [2-14CldTTP, 14 nmol (5.6 dpm/pmol); from (48). *APB, acetate, phosphate, borate universal buffer.

dTMP S Y N T H A S E A N D d C M P D E A M I N A S E

425

80 40 '? 0w x

o

0

-40 7 -80

% u

-120

-o -16C 2o

-200 -240

i

-280 240

I

i

i

260

280

i

i 300

om

F I G . 9. C D changes associated with binding ofdCTP to T2-dCMP deaminase. The basic solution on which the C D analyses were performed contained 0.2 M potassium phosphate, p H 7.1, 10 mM 2-mercaptoethanol, 1 mM MgCI2, 4.34 #M d C M P deaminase, and, as indicated, 0, 10, 20, or 30#1 of 4.08 mM dCTP. The total vol of the solution was 3.0 ml. The C D patterns represent the difference spectra solutions containing identical components except for the omission of enzyme from the second cell. Reproduced from (48).

process for the deaminase was provided by the interesting observation that freezing the enzyme into an oligomer with glutaraldehyde eliminated the need for a positive effector, without impairing inhibition by dTTP (46). While chemically reactive derivatives o f dCTP, dTTP and dCMP can be prepared, a simpler means of identifying nucleotide binding sites was suggested from studies (50, 51) indicating that unmodified nucleotides can be photofixed to their respective enzymes. Of particular interest was the observation of Ericksson et al. (52) that dTTP, an allosteric modifier of ribonucleotide reductase, proportionately inactivates this enzyme as it is photofixed to it. When a similar study was conducted with the allosteric effectors ofT2-phage deaminase, it was found that an extremely rapid fixation of dTTP occurred within 60 to 90 sec, which was both concentration and enzyme dependent (Fig. 10), but which surprisingly did not inhibit the deaminase (53). In contrast, dCTP was not photofixed under these conditions, but did prevent the fixation of dTTP. Even more fascinating was the finding that dCTP could under photolysis conditions affect the release of dTTP from enzyme to which it had been apparently irreversibly fixed. Chromatographic analysis of the released c o m p o u n d indicated that it was dTTP.

426

FRANK MALEY, et al. A

1.5

123 Z

o co

/-

B

1.0

13._ pF--

0.5 E C I

50

I

IO0

SECONDS

I

I

150

20

I

40

I

60

I

80

nmol d T T P

FIG. 10. Fixation of dTTP to T2-deoxycytidylate deaminase as a result of exposure to UV light at 254 nm. (A) rate of incorporation. In this experiment 50 nmol of [a3:P]dTTP was used with 4 nmol of deaminase subunit in a final vol of 0.2 ml. Aliquots of 20 #1 were taken at the indicated times. (B) determination of maximal amount of dTTP fixed per subunit of enzyme. The deaminase subunit concentration was maintained constant at 2 nmol/0.1 ml of reaction mixture, with increasing amounts of dTTP as indicated. Aliquots of 10 #1 were removed for assay after 3 min of irradiation at 254 nm. Reproduced from (53). T o locate the d T T P b i n d i n g region, the enzyme was p h o t o f i x e d with [a32p]dTTP a n d cleaved into 7,000 a n d 13,000 d a l t o n p e p t i d e s with c y a n o g e n b r o m i d e . Since these peptides are easily s e p a r a t e d by S D S - a c r y l a m i d e gel electrophoresis, the label was shown by a u t o r a d i o g r a p h y to be a s s o c i a t e d with the larger o f the two peptides (Fig. 11). T h e task o f l o c a t i n g the a m i n o acids involved in the fixation o f d T T P s h o u l d be a i d e d greatly by o u r recent clarification (25) o f the a m i n o acid sequence o f the T 2 - p h a g e d C M P d e a m i n a s e (Fig. 12). Since the cleavage by c y a n o g e n b r o m i d e is affected at methionine-125, it w o u l d a p p e a r that d T T P is p h o t o f i x e d s o m e w h e r e within the a m i n o t e r m i n a l peptide. Studies are currently u n d e r w a y to d e t e r m i n e its precise location. Finally, after n u m e r o u s fruitless a t t e m p t s to crystallize the d e a m i n a s e we have m a n a g e d to a c c o m p l i s h this feat t h r o u g h the use o f p o l y e t h y l e n e glycol (Fig. 13). I f these crystals p r o v i d e useful x-ray diffraction patterns, it s h o u l d be possible to m o r e clearly define the c o n f o r m a t i o n a l transitions in the d e a m i n a s e structure affected by the allosteric modifiers.

SUMMARY M e t h o d s are described for p r e p a r i n g a n d structurally a n a l y z i n g two enzymes involved in the f o r m a t i o n o f d T M P , d e o x y c y t i d y l a t e d e a m i n a s e a n d t h y m i d y l a t e synthase. In the latter case, it has been possible t h r o u g h the use o f r e c o m b i n a n t D N A techniques with an a m p l i f i c a t i o n p l a s m i d to o b t a i n sufficient a m o u n t s o f theE. c o l i a n d T 4 - p h a g e synthases to c o m p l e t e the entire sequence o f b o t h enzymes by e m p l o y i n g a c o m b i n a t i o n o f p r o t e i n a n d D N A

m

0d

~D (D

>-

I-0 <~ o C3 <~ rr Ld <~ ._3 Ld rr 0

I

2

3

4

5

cm FIG. 11. Location of [a32p]dTTP in CNBr fragments, CN-1 and CN-2, from T2-deoxycytidylate deaminase. The horizontal Coomassie blue-stained gel is compared with the densitometric trace of the gel-exposed x-ray film. Reproduced from (53).

FIG. 13. Crystals of deoxycytidylate deaminase. Crystals were obtained using the hanging dropvapor phase diffusion technique (54). A cover slip containing 1 drop of enzyme solution (2 m g / m t of deaminase in 0.1 M potassium phosphate pH 7.1, plus 50 mM 2-mercaptoethanol) which was 4.5% in polyethylene glycol (Mr 1300 to 1600) was placed over 1 ml of 9% of the same polyethylene glycol in a multiwelled dish. After about 2 weeks in the cold, crystals began to form. The length of the bar is 10 #M. Reproduced from (25).

427

dTMP SYNTHASE A N D d C M P D E A M I N A S E 10 H•MET-LY••ALA•SER•THR•VAL•LE••GLN•ILE•ALA•TYR•LEU•VAL•SER-GLN•GLU-SER•LYS-CYS•CYS•

20

30 40 SER•TRP-LYS-VAL-GLY-ALA-VAL-ILE-GLU-LYS-ASN-GLY-ARG-ILE-ILE-SER-THR-GLY-TYR-AsN50 60 GLY-SER-PR•-ALA-GLY•GLY-VAL-AsN-CYs-AsP-AsN-TYR-ALA-ALA-ILE-•LU-GLY•TRP-LEU-LEU• 70 80 ASN•LY•-PR•-LY•-HIS-THR-ILE-ILE-GLN•GLY-HI•-LY••PR•-GLU-CYS-VAL-SER-PHE-GLY-THR90 1O0 SER-ASP-ARG-PHE-VAL-LEU-ALA•LYS-GLU-HIS-ARG-SER-ALA-HIS-SER•GLU•TRP-SER-sER-LY•• 110 120 ASN-GLU-ILE-HIs•ALA-GLu-LEU-A•N-ALA•ILE-LEU-PHE-ALA•ALA-ARG-AsN-GLY-SER-SER-ILE• 130 140 GLU-GLY•ALA-THR•MET-TYR-VAL-THR-LEU-SER-PR•-CY•-PR•-ASP-CYS-ALA-LYS-ALA-ILE-ALA150 160 GLN-SER-GLY-ILE-LYS-LYS-LEU-VAL•TYR-CYS-GLU•THR•TYR-AsP-LYS-ASN•LYs-PR•-GLY•TRP170 180 AsP-ASP-ILE-LE•-ARG•ASN-ALA-GLY-ILE-GLU-VAL-PHE-ASN-VAL•PR•-LYS-LEu-ASN-TRP-GLUASN-ILE-SER-GLu-PHE-CYs-GLY-GLu-0H

FIG. 12. Amino acid sequence of deoxycytidylate deaminase. Reproduced from (25).

sequencing methods. A comparative analysis of the L. casei and E. coli synthases has revealed a 62% conservation of sequences but an even greater homology in their hydrophobic active site regions (82%), which are primarily hydrophobic in nature. The homology between these enzymes becomes apparent by deleting a 51 amino acid segment (residues 89-139) from the L. casei synthase, which accounts for the difference in size between these enzymes. Methods for obtaining the binding sites of both substrates are described, one being the activation of the carboxyls of folate with a water soluble carbodiimide and the other, the activation of dUMP by ultraviolet light. The D N A and protein sequence of the T4-phage synthase has recently been clarified by us and is in preparation. Of great interest is the finding by Purohit and Mathews (42), based on our sequence data for the synthase, that the gene segment for the carboxyl terminal end of dihydrofolate reductase overlaps with the amino end of the gene for thymidylate synthase. The complete amino acid sequence ofT2-phage deoxycytidylate deaminase has been elucidated by conventional protein sequencing methods. The binding characteristics of this enzyme for its positive allosteric effectors and substrates, as determined by equilibrium dialysis, are consistent with the cooperative nature of its kinetic responses. Consistent with these findings was the demonstration that each of the enzyme's six subunits bound an equivalent amount of substrate or allosteric modifier. Similarly the deaminase showed a marked negative change in ellipticity at 280 nm in response to increasing concentrations of dCTP, changes which could be reversed by dTTP. F r o m the information on the enzyme's primary sequence, it should be possible to define

428

FRANK MALEY, et al.

the substrate and allosteric binding regions within the deaminase with the appropriately activated compounds. A start in this direction has been initiated by the finding that dTTP is rapidly and apparently covalently fixed to the amino terminal cyanogen bromide peptide of the enzyme in the presence of ultraviolet light.

ACKNOWLEDGEMENTS

We would like to express our appreciation to D o n Guarino, Judith Reidl and Joan Pedersen-Lane for their excellent technical assistance during various aspects of this work. In addition we would like to thank Drs. W. Samsonoff and N. Perrins for the scanning electron micrographs of the deaminase and synthase crystals. This work was supported in part by Public Health Service grants from the National Institutes of General Medical Science, P H S / D H H S , GM26387 and GM26645 and National Science Foundation grants PCM8118368 and PCM7908960. REFERENCES 1.

2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13.

F. MALEY and G. F. MALEY, Mechanisms of enzyme modulation involving deoxycytidylate deaminase and thymidylate synthetase, Advances in Enzyme Regulation 8, 55-71 (1970). F. MALEY and G. F. MALEY, The regulatory influence of allosteric effectors on deoxycytidylate deaminase, Current Topics in Cellular Regulation 5, 177- 228 (1972). A. LARSSON and P. REICHARD, Enzymatic reduction of ribonucleotides, Progress in Nucleic Acid Research and Molecular Biology 7, 303-347 (1967). D . H . IVES, P. A. MORSE, Jr. and V. R. POTTER, Feedback inhibition of thymidine kinase by thymidine triphosphate, J. Biol. Chem. 238, 1467-1474 (1963). R . J . BONNEY and F. MALEY, Effect of methotrexate on thymidylate synthetase in cultured parenchymal cells isolated from regenerating rat liver, CancerRes. 31, 1950-1956 (1975). R.B. DUNLAP, N. G. L. HARDING and F. M. HUENNEKENS, Thymidylate synthetase from amethopterin-resistant Lactobacillus casei, Biochemistry 10, 88-97 (1971). R. P. LEARY and R. L. KISLIUK, Crystalline thymidylate synthetase from dichloromethotrexate resistant Lactobacillus casei, Prep. Biochem. 1, 47-54 (1971). W. RODE, K. J. SCANLON, J. HYNES and J. R. BERTINO, Purification of mammalian tumor (L1210) thymidylate synthetase by affinity chromatography on a stable biospecific adsorbent, J. Biol. Chem. 254, 11538-11543 (1979), G. F. MALEY and F. MALEY, The purification and properties of deoxycytidylate deaminase from chick embryo extracts, J. Biol. Chem. 239, 1168-1176 (1964). F. MALEY and G. F. MALEY, Tetrahydrodeoxyuridylate: a potent inhibitor of deoxycytidylate deaminase, Arch. Biochem. Biophys. 144, 723-729 (1971). G . F . MALEY and F. MALEY, The significance of the substrate specificity of T2r+-induced deoxycytidylate deaminase, J. Biol. Chem. 241, 2176-2177 (1966). J . H . GALIVAN, G. F. MALEY and F, MALEY, The effect of substrateanalogs on the circular dichroic spectra of thymidylate synthetase from Lactobacillus caseL Biochemistry ' 14, 3338~3344 ~(1975). A . J . WAHBA and M. FRIEDKIN, Direct spectrophotometric evidence for the oxidation of tetrahydrofolate during the enzymatic synthesis of thymidylate, J. Biol. Chem. 236, PC11-12 (1961).

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