1 Dipartimento di Scienze Veterinarie, Università di Camerino, via Circonvallazione 9395, 62024 Matelica, Italy, 3 Départment de Chimie, Université Montpellier 2, 34095 Montpellier, France, 4 Department of Biological Chemistry, University of Copenhagen, 1307 Copenhagen K, Denmark, 5 Dipartimento di Scienze Morfologiche e Biochimiche Comparate and 6 Dipartimento di Biologia MCA, Università di Camerino, 62032 Camerino, Italy
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Abstract |
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Keywords: cytidine deaminase/mutagenesis/NBS titration/tryptophan fluorescence
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Introduction |
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In order to be able to design more potent inhibitors of the human enzyme and/or new cytosine nucleoside analogs not deaminated by CDA, a knowledge of the structure of the active site and the catalytic mechanism is required.
Human cytidine deaminase is a tetrameric enzyme of identical 16 kDa subunits each containing an essential zinc atom in the active site (Vincenzetti et al., 1996). Recent studies by site-directed mutagenesis and by chemical modifications have indicated that the zinc atom is coordinated with three cysteines (C65, C99 and C102) and that the glutamic acid residue 67 plays an important role in catalysis (Cambi et al., 1998
).
In the present work, we focused on the possible role of residues F36 and F137 in human CDA catalysis. These two residues are highly conserved in CDAs from various sources and crystallographic studies of the dimeric Escherichia coli enzyme have shown that F71 and F565 (from the other subunit), corresponding to F137 and F36 in human CDA respectively, interact with the pyrimidine ring of the ligand (Betts et al., 1994). Furthermore, F71 of E.coli CDA may also influence the access of the substrate to and from the active site (Betts et al., 1994
). F36 and F137 in human CDA, were investigated by site-directed mutagenesis, by kinetic characterization and by fluorescence analysis of the mutant enzymes. Preliminary experiments showed that the intrinsic fluorescence of human CDA was only weakly quenched in the presence of ligands such as tetrahydrouridine (THU) or uridine (Vincenzetti et al.,1997
), suggesting that the only tryptophan residue of the enzyme (W113) is probably not involved in catalysis. Therefore, to study the possible interaction of F36 and F137 with the pyrimidine ring of the substrate, the double mutants F36W/W113F and F137W/W113F of human CDA were constructed and the affinities of the mutant enzymes for 1-ß-(ribofuranosyl)-5-fluoropyrimidin-2-one (5-fluorozebularine, FZEB) were determined using either their intrinsic tryptophan fluorescence or the extrinsic fluorescence of the ligand.
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Materials and methods |
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Isopropyl-1-thio-ß-D-galactopyranoside (IPTG), N-bromosuccinimide (NBS), nucleosides, nucleotides, tris(hydroxymethyl)-aminomethane (Trizma base), 2-mercaptoethanol, dithiothreitol (DTT), sodium dodecyl sulfate (SDS) and iminodiacetic acid Sepharose 6B were obtained from Sigma Chemical (St. Louis, MO). Other chemicals were of reagent grade from J.T. Baker (Deventer, The Netherlands). PM10 membranes were purchased from Amicon (Beverly, MA) and protein markers and Affi-gel 102 from Bio-Rad (Richmond, CA). Oligodeoxyribonucleotide primers were synthesized by DNA Technology (Aarhus, Denmark). The 42 bp His-tag linker, cloned into the pET-3d NcoI site, and the pET-3d plasmid were a kind gift from Dr Giuseppa Levantino (University of Pisa, Italy). Restriction nucleases were obtained from either Promega (Madison, WI) or New England Biolabs (Boston, MA). Plasmid DNA was isolated using the Quiagen DNA kit and PCR products were purified with the Quiagen Purification kit. Zebularine and 5-fluorozebularine were a kind gift from Professor Victor E.Marquez (National Institutes of Health, USA).
Bacterial strains and growth media
E.coli DH5 was used as host for all clonings (Sambrook et al., 1989
). For complementation tests, the pyrimidine requiring the cytidine deaminase negative derivative of MC1061, SØ5201 (MC1061cdd::Tn10 pyrD::Kan), was employed. The E.coli strain BL21 (DE3) (F ompT rB mB) was used as host for cloning and expression of pET-3d derived plasmids (Studier and Moffatt, 1986
; Grodberg and Dunn, 1988
)
Phosphate-buffered AB medium (Clark and Maaløe, 1967) supplemented with 0.2% glucose and 0.2% vitamin-free casamino acid, was used as minimal medium. When required, this medium was supplemented by thiamine, 1 µg/ml; uracil, 20 µg/ml; deoxycytidine (CdR), 40 µg/ml; ampicillin, 100 µg/ml; kanamicin, 30 µg/ml; tetracycline, 10 µg/ml. L-broth was used as a rich medium (Miller, 1972
).
Plasmids and site-directed mutagenesis
The expression vector pTrc99-A (Pharmacia) was used for most clonings. In certain instances pET-3d(His6) containing a histidine hexapeptide was employed (Cambi et al., 1998). pTrcHUMCDA, containing the human CDA cDNA on a 446 bp NcoIBamH1 fragment in pTrc99-A, has been described previously (Vincenzetti et al., 1996
). pTrcHUMCDAW113F, pTrcHUMCDAF36W/W113F and pTrcHUMCDAF137W/W113F containing mutations in the CDA cDNA were constructed by site-directed mutagenesis in three steps using the megaprimer method. A pair of complementary primers, the 3' and 5' internal primers, harboring the desired mutations were used in separate PCR reactions together with a specific 5' vector-specific primer, corresponding to a region 5' of the NcoI cloning site of pTrc99-A and a 3' vector-specific primer, corresponding to a region 3' of the BamHI cloning site of the vector, respectively. pTrcHUMCDA or pTrcHUMCDAW113F was used as a template. The two megaprimers produced in each pair of reactions were used in a third round of PCR amplification in the presence of the two vector-specific primers. The resulting DNA fragment was digested with NcoI and BamHI and cloned into pTrc99-A. The sequences of the primers used are shown in Table I
together with the templates employed in each case. The primary structure of the insert was confirmed by specific restriction endonuclease analysis and DNA sequencing. Endonuclease digestion and ligation of DNA were done according to the suppliers and the procedure for transformation was as described by Sambrook et al. (1989). DNA sequence analysis was performed by the chain-terminating method of Sanger et al. (1977).
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Expression and purification of mutant enzymes
Cell were grown at 37°C in L-broth supplemented with 100 µg/ml ampicillin and CDA expression was induced during late exponential growth (A436 = 1.0) by the addition of 0.6 mM IPTG. After 19 h of shaking at 37°C, the cell were harvested by centrifugation at 5000 g and washed with 0.9% NaCl. The cells were disrupted in a French pressure cell as described previously (Vincenzetti et al., 1996). The mutant proteins W113F and F137W/W113F were purified on an affinity column containing the cytidine analog CV6 bound to an agarose resin, Affi-Gel 102, as described by Cacciamani et al. (1991).
Since the F36W/W113F double mutant CDA did not bind to the CV6 affinity column, it was necessary to purify it as a His-tag fusion protein by immobilized metal affinity chromatography. Strain BL21(DE3)/pET-3d(His6)HUMCDAF36W/W113F was grown at 37°C in L-broth supplemented with 200 µg/ml ampicillin. Expression of His6-CDA was induced during late exponential growth (A600 = 0.6) by the addition of 1 mM IPTG. After 3 h of vigorous shaking at 37°C, the cells were harvested by centrifugation at 5000 g and washed with 0.9% NaCl. The cell pellet was treated as described by Van Dyke et al. (1992) and the mutated CDA was purified by metal chelate affinity chromatography on iminodiacetic acid Sepharose 6B charged with nickel (Ni2+-IDA) as described previously (Cambi et al., 1998). Pooled fractions containing CDA were dialyzed and concentrated by ultrafiltration on PM-10 membranes (Amicon) against 50 mM TrisHCl pH 7.5, 5 mM ß-mercaptoethanol, 1 mM EDTA and analyzed by SDSPAGE.
CDA assay and enzyme stability
CDA activity was measured spectrophotometrically as described previously (Cacciamani et al., 1991). In kinetic studies with inhibitors, deoxycytidine (0.0180.085 mM) was used as substrate. One enzyme unit is defined as the amount of enzyme that catalyzes the deamination of 1 µmol of cytidine per minute at 37°C.
The thermal stability of the wild-type and mutant CDAs was determined by measuring the residual activity after incubation of the enzyme at various temperatures for different lengths of time. The remaining activities were measured spectrophotometrically as described. The stability of wild-type and mutant CDAs was also investigated in the presence of different concentrations of urea and the residual activities were measured after 15 min of incubation of the enzymes with the denaturing agent.
Circular dichroism (CD) spectroscopy
CD measurements were made on a Jasco J-710 spectropolarimeter in the far-UV (190240 nm) and near-UV (240320 nm) ranges. All measurements were carried out at 25°C in 10 mM potassium phosphate pH 7.0; the protein concentration was 2 µM in the far-UV range (cell length 2 mm) and 10 µM in the near-UV range (cell length 10 mm). Analysis and estimation of protein secondary structures were carried out according to Yang et al. (1986).
Oxidation of tryptophan residue by N-bromosuccinimide
Oxidation of tryptophan to the corresponding oxindole derivative was performed by titrating 0.50.8 ml of a solution 1030 µM of wild-type or mutant enzymes with 35 µl aliquots of 1 mM NBS. When titration was performed under native conditions, the buffer used was 0.1 M citrate buffer pH 6.0. When titration was performed under denaturing conditions (8 M urea), 0.1 M acetate buffer pH 4.0 was employed. Following each addition, the mixture was incubated at 25°C for 5 min and the absorbance was measured at 280 nm. The number (n) of tryptophan residues per mole of protein was calculated from the following expression (Spande and Witkop, 1967):
where 1.31 is a correction factor for the absorption change at 280 nm of the oxindole moiety, 5500 is the molar absorptivity at 280 nm for tryptophan using a 1 cm pathlength cuvette and C is the molar concentration of the protein based on a subunit molecular weight of 16 000.
Fluorescence studies
The experiments were performed on an SLM-Smart 8000 spectrofluorimeter equipped with a 450 W lamp. Experiments using the fluorescence of FZEB were carried out at 25°C in a fluorescence buffer containing 50 mM TrisHCl pH 7.5, 100 mM KCl, 1 mM DTT, 5 mM MgCl2 and 10% glycerol. When FZEB was present the samples were excited at 325 nm and the emission was measured at 396 nm with a spectral bandpass of 2 or 4 nm for excitation or emission, respectively. Following incubation for 2 h in the fluorescence buffer, a solution of FZEB (0.7 µM) was titrated with CDA up to a concentration of 1.5 µM of the tetrameric enzyme. The observed decrease in fluorescence was corrected for dilution and for internal quenching as described previously (Elalaloui et al., 1994). Curve fitting was performed with the program Grafit (Erithacus Software, Ltd) using Equation 1, which assumes the existence of one binding site or enzyme state for each subunit:
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The tryptophan intrinsic fluorescence was excited at 292 nm to minimize the substrate inner-filter effect and the emission was measured at 352 nm with a spectral bandpass of 2 or 4 nm for excitation or for emission, respectively. The same buffer and the same experimental procedure were used as in extrinsic fluorescence experiments to obtain the emission spectra of the enzymes. The proteins were unfolded by overnight incubation in 8 M urea at 37°C.
Other analytical procedures
Protein concentration was determined by the method of Bradford (1976) and the molecular mass and the purity of the mutant proteins were estimated by SDSPAGE (Laemmli, 1970) using 15% acrylamide; the markers used were low-range from Bio-Rad. Proteins were stained with Coomassie Brilliant Blue or silver.
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Results |
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Multiple alignments of the deduced amino acid sequences of cytidine deaminases from various sources (Vincenzetti et al., 1999) identified two Phe residues (corresponding to F36 and F137 in human CDA) that are highly conserved. From the crystal structure of the larger homodimeric E.coli CDA (Betts et al., 1994
), it was apparent that the homologous residues (F71 and F565) were involved in interactions with the pyrimidine ring in the active site.
To investigate whether F36 and F137 of the smaller tetrameric human CDA might have similar functions, we have employed site-directed mutagenesis to change them into tryptophan residues susceptible to fluorescence quenching. At the same time, the tryptophan residue present in human CDA (W113) was mutated into a phenylalanine in order to have the signal of a unique and strategically located tryptophan in fluorescence studies. The W113F mutant enzyme was used as a control.
The mutated cDNAs were cloned into pTrc99-A as described in Materials and methods and the plasmids were transformed into SØ5201. The resulting strains were tested for ability to grow with deoxycytidine as sole pyrimidine source in the presence of IPTG. Owing to the cdd::Tn10 mutation present in SØ5201, this strain cannot satisfy its pyrimidine requirement by deoxycytidine. Since all the plasmid-containing strains grew with deoxycytidine plus IPTG, it was concluded that all mutant CDAs possessed CDA activity.
W113F and F137W/W113F mutant proteins were purified by the CV-6 affinity chromatography as described for the wild-type CDA (Vincenzetti et al., 1996). Since the F36W/W113F mutant enzyme did not bind to the affinity column, to facilitate its purification the corresponding cDNA was subcloned from pTrc99-A into pET-3d(His6) which carries a 42 bp synthetic linker encoding a histidine hexapeptide cloned into the NcoI site (Cambi et al., 1998
). The plasmid obtained, pET-3d(His6)HUMCDAF36W/W113F, was transformed into E.coli BL21(DE3) and expression was induced by IPTG. The fusion protein was purified using an Ni2+-IDA Sepharose 6B column as described previously (Cambi et al., 1998
). According to analysis by SDSPAGE, all the CDAs purified were judged to be more then 98% pure (data not shown).
It was shown previously that there was no difference in the enzymatic characteristics of His-tagged and non-His-tagged wild-type CDA (Cambi et al., 1998), and therefore a wild-type CDA His-tagged was used as control to demonstrate that the presence of a His-tag on F36W/W113F mutant protein did not affect the results of the experiments performed.
The thermal stability of W113F was found to be identical with that of the wild-type enzyme, while the two double mutants were less stable at temperatures above 70°C. In the presence of different concentrations of urea as denaturing agent, the stability of the wild-type and W113F CDA was similar, whereas the two double mutants F137W/W113F and F36W/W113F, at concentrations of urea above 3 M, showed greater inactivation compared with the wild-type enzyme (data not shown).
The CD spectra of the mutant enzymes in the far- and near-UV regions, performed as described under Materials and methods, were nearly identical with those of the wild-type enzyme (data not shown), indicating that the secondary structure of the enzyme is not greatly affected by the mutations.
Kinetic characterization of mutant CDAs
The kinetic parameters of the purified mutant and wild-type enzymes with respect to both its natural substrates cytidine and deoxycytidine and the analog cytosine arabinoside are given in Table II. The results show that changing the Trp residue at position 113 to Phe did not significantly affect the Km and Vmax values. In contrast, both F137W/W113F and F36W/W113F showed a 45-fold increase in Km values with F137W/W113F having in addition a strongly decreased Vmax value.
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Tryptophan residues in proteins can be oxidized selectively with NBS, allowing a specific analysis of their functions. The reaction is rapid, irreversible and converts the indole side chain of a Trp residue that absorbs at 280 nm to an oxindole, a much weaker chromophore at this wavelength.
The oxidation of Trp residues was carried out to study the accessibility of residues W113 (wild-type), W137 (F137W) and W36 (F36W) of human CDA. The results of the NBS titrations are shown in Table IV. Under non-denaturing conditions, F36W/W113F and wild-type CDA were insensitive to NBS whereas they were titrated under denaturing conditions, suggesting that the residues W36 and W113 are `buried' in the tertiary structure of the molecule. In contrast, titration of the Trp residue at position 137 in F137W/W113F under non-denaturing conditions suggested that it is located near the surface of the molecule. As expected, W113F CDA did not react with NBS under either native or denaturing conditions.
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Intrinsic tryptophan fluorescence
The intrinsic tryptophan fluorescence of the wild-type and mutant enzymes was excited at 292 nm and the corresponding emission spectra were recorded. The emission spectrum of wild-type CDA gave a maximum that was weak and difficult to locate with certainty (between 320 and 340 nm). Addition of saturating concentrations (5 µM) of FZEB did not induce a sufficient change of the emission spectrum to determine a dissociation constant. However, unfolding the protein with 8 M urea gave a well defined maximum of emission at 358 nm.
Similarly, the emission spectrum of CDA F137W/W113F gave a weak maximum of emission at 342 nm which was red-shifted to 358 nm upon denaturation with 8 M urea and incubation. Addition of saturating FZEB to the active enzyme resulted in no apparent change in the emission spectrum. The excitation at 292 nm acted also on FZEB and resulted in a strong FZEB emission at 393 nm which complicated the interpretation of the overall emission spectrum.
The emission spectrum of CDA F36W/W113F gave a poorly defined maximum at 344353 nm which was red-shifted to 358 upon denaturation in 8 M urea. Addition of 5 µM FZEB to the active enzyme resulted in a quenching of ~20% of the fluorescence intensity with also a strong FZEB emission maximum at 393 nm. An attempt was made to use this quenching of Trp fluorescence to titrate the mutant with FZEB. However, the curve of the observed fluorescence as a function of the total concentration of ligand could not be fitted to Equation 1, probably because the fluorescence emission of free FZEB overlapped that of the tryptophan residue. Under the same conditions, there was no tryptophan emission from the W113F mutant, as expected.
Experiments using the fluorescence of FZEB
Another way to characterize the enzymes using fluorescence was to monitor the titration of FZEB with the proteins by following the changes in the ligand fluorescence, as was done previously with E.coli CDA (Carlow et al., 1996) and B.subtilis CDA (Carlow et al., 1999
). Therefore, the fluorescence of FZEB was excited at 325 nm and the emission recorded at 396 nm. The variations in relative fluorescence intensity with the total concentration of added enzyme are presented in Figures 1 and 2
.
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From the data shown, the dissociation constants, Kd, were determined by non-linear regression analysis of the plots of the observed (relative) fluorescence intensities against the concentration of total enzyme (Table V).
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In all cases, the binding of FZEB to proteins decreased the relative fluorescence of the ligand to a maximum of 6070% depending on the protein and in the presence of saturating amounts of FZEB (Figures 1 and 2). This allowed the calculation of the corresponding dissociation constants Kd (Table V
). The Kd values showed that the mutant CDA W113F had the highest affinity for FZEB, followed by wild-type enzyme and F36W/W113F. The mutant F137W/W113F appeared to have a relatively weak affinity for FZEB.
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Discussion |
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Furthermore, in the E.coli CDA (Betts et al., 1994) it has been shown that the pyrimidine ring of the substrate interacts with three aromatic side chains highly conserved in CDAs from a variety of organisms: Phe71 and Tyr126 are located in the active site of the molecule and F565 from the `broken' active site of the other monomer. During the transition state of the enzymatic reaction, these hydrophobic interactions render the hydroxylic group at C-4 more acidic so that they increase the strength of the hydrogen bonding to the carboxylate group of E104.
We therefore specifically mutated Phe36 or Phe137 of human CDA, corresponding to the E.coli F71 and F565, respectively, into Trp residues and, at the same time, changed the Trp residue 113 into a Phe residue. All the resulting mutant proteins, W113F, F137W/W113F and F36W/W113F, showed a stability similar to that of the wild-type enzyme. Furthermore, the CD spectra of wild-type and mutant CDAs in both the far- and near-UV ranges were found to be similar. All these observations imply that the secondary structure of the human enzymes has not been significantly modified by the mutations; therefore, the observed differences in the activity of the mutants do not involve unfolding or evident secondary structure rearrangements.
More specifically, the study of the binding properties of FZEB with respect to the W113F mutant using the intrinsic fluorescence of the protein or the extrinsic fluorescence of the ligand indicates that the replacement of Trp113 by Phe does not result in major changes in the enzyme properties since the Kd value of the mutant is only slightly altered compared with the wild-type enzyme and the steady-state kinetic constants obtained for W113F and wild-type CDA are similar. It is therefore likely that this residue does not interfere with the enzyme mechanism, as already shown by preliminary fluorescence studies on human placenta CDA (Vincenzetti et al.,1997).
Concerning the replacement of Phe137 with Trp, we also observed convergent results in equilibrium and kinetic experiments. Using the fluorescence of FZEB, the titration of the ligand with the F137W/W113F mutant allowed us to calculate a Kd constant which was rather high compared with that obtained from the wild-type enzyme: the kinetic constants Km and Vmax of different substrates with respect to the mutant enzyme were also negatively affected compared with the wild-type CDA. The fact that there is almost no quenching of the Trp intrinsic fluorescence upon FZEB binding suggests that W137 does not interfere significantly with the binding of the ligand. The small changes in intrinsic Trp fluorescence observed prevented studies of the accessibility of the Trp groups to quenchers. Nevertheless, the NBS titration experiments showed that W137 is readily accessible to the reagent and therefore suggests that this residue may be located near the surface of the enzyme (Spande and Witkop, 1967). These data suggest that there exists a lack of a stabilizing interaction of W137 with the ligand in the mutant enzyme active site and therefore that F137 of the wild-type CDA may be involved in the hydrophobic interaction with the pyrimidine ring, like the homologous F565 residue of the E.coli enzyme (Betts et al., 1994
). This difference between wild-type human CDA and the F137W/W113F mutant enzyme could be attributed to the larger steric requirements of the Trp side chain compared with Phe.
In the F36W/W113F CDA, W36 is apparently not accessible to NBS titration under non-denaturing conditions, suggesting that this residue is buried in a hydrophobic environment. The 20% quenching of F36W/W113F Trp fluorescence in the presence of FZEB indicates that the W36 residue interacts with the binding of the ligand. The higher Kd value and the increased Ki value with respect to FZEB confirmed the low affinity binding between F36W/W113F and the ligand. Finally, it is remarkable that F36W/W113F CDA is not inhibited by cytidine analogs with a reduced pyrimidine ring such as 6-[3-(5-cytidyl)acryloylamino]hexanoic acid (CV6), 4-hydroxy-1-ß-ribofuranosylpiperidin-2-one (CV7) and 5,6-dihydrouridine. This may be explained by the absence of favorable interaction with the active site of the enzyme due to the suppression of aromaticity and, in the case of CV6 analogs, also by the bulk of the lateral chain.
The covalently hydrated FZEB (FZEB-H2O) with a hydroxyl group at position C-4 and formed in the active site has been shown to be a better ligand of E.coli CDA than 5,6-dehydrozebularine or tetrahydrouridine (THU) that presents a fully reduced pyrimidine ring with a hydroxyl group at position C-4 (Xiang et al., 1995). The high affinity of E.coli CDA for FZEB-H2O was explained by the presence of both the 4-OH group and the 5,6-double bond in the bound ligand, allowing good interactions with F71, F565 and Tyr126 (Betts et al., 1994
). The favorable interactions of THU and FZEB with the homologous F36 and/or F137 residues of the human enzyme, as suggested by the increased Ki values for these inhibitors towards the mutant enzymes, may likewise be explained by the presence of either the hydroxyl group or the 5,6-double bond in these molecules. The absence of 5,6-double bond and also of a 4-OH group results in a weak stabilizing interaction and justifies the poor inhibition of wild-type CDA and the mutants observed with cytidine analogs with a reduced pyrimidine ring.
In conclusion, in this paper we have presented evidence that the homologous residues F71/Phe36 and F565/Phe137 of the E.coli and the human CDA, respectively, may have similar functions in the interaction with the pyrimidine ring of the ligand. These results, together with our previous studies on the role exerted by four amino acid residues in catalysis (Cambi et al., 1998), suggest that the structure of the active site and therefore the catalytic mechanism of the two enzymes is very similar.
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Notes |
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7 To whom correspondence should be addressed. E-mail: vita{at}cambio.unicam.it
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Received June 23, 2000; revised September 6, 2000; accepted September 28, 2000.