Functional properties of subunit interactions in human cytidine deaminase

Silvia Vincenzetti1,2, Giampiero De Sanctis3, Stefano Costanzi4, Gloria Cristalli4, Pierluigi Mariani1, Giampiero Mei5, Jan Neuhard6, Paolo Natalini7, Valeria Polzonetti7 and Alberto Vita1

1Dipartimento di Scienze Veterinarie, 3Dipartimento di Biologia MCA, 4Dipartimento di Scienze Chimiche, 7Dipartimento di Scienze Morfologiche e Biochimiche Comparate, Università di Camerino, 5Dipartimento di Medicina Sperimentale e Scienze Biochimiche, Università di Roma Tor Vergata and INFM, Italy and 6Institute of Molecular Biology, University of Copenhagen, Denmark

2 To whom correspondence should be addressed. e-mail: silvia.vincenzetti{at}unicam.it


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
An intersubunit interactions study related to the active site has been performed on the wild-type cytidine deaminase (CDA) and on the mutant enzyme F137W/W113F. F137 is the homologous to the Bacillus subtilis CDA F125 involved in the subunit interactions. In the presence of SDS, wild-type human CDA dissociates into enzymatically inactive monomers without intermediate forms via a non-cooperative transition. Extensive dialysis or dilution of the inactivated monomers restores completely the activity. Circular dichroism measurements show that the secondary/tertiary structure organization of each subunit is unaffected by the SDS concentration, while the mutation Phe/Trp causes weakening in quaternary structure. The presence of the strong human CDA competitive inhibitor 5-fluorozebularine disfavours dissociation of the tetramer into subunits in the wild-type CDA, but not in mutant enzyme F137W/W113F. The absence of tyrosine fluorescence and the much higher quantum yield of the double mutant protein spectrum suggest the occurrence of an energy transfer effect between the protein subunits. This assumption is confirmed by the crystallographic studies on B.subtilis in which it is shown that three different subunits concur with the formation of each of the four active sites and that F125, homologous to the human CDA F137, is located at the interface between two different subunits contributing to the formation of active site.

Keywords: cytidine deaminase/modelling/subunit dissociation


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Human cytidine deaminase (CDA) catalyses the deamination of cytidine and deoxycytidine to uridine and deoxyuridine, respectively. In addition to the natural substrates, CDA recognizes several cytosine nucleoside-based drugs used as antineoplastic and antiviral agents, causing the loss of their therapeutic efficiency (Chabot et al., 1983Go; Bouffard et al., 1993Go; De Clerq, 2001Go). Therefore, high CDA levels may be responsible of the short half-life of these analogues (Ho, 1973Go).

The molecular structure of the dimeric CDA from Escherichia coli has been established (Betts et al., 1994Go) as has the crystal structure of the tetrameric CDA from Bacillus subtilis (Johansson et al., 2002Go). The overall structure as well as the mechanism of catalysis of the dimeric and the tetrameric enzyme are very similar. The dimeric CDA from E.coli (Betts et al., 1994Go) contains one active site per 31.5 kDa subunit and is composed of a small N-terminal alpha-helical domain, with no connections to the active sites, a catalytic domain containing the active site with a zinc atom co-ordinated by two cysteine residues and one histidine residue, and a C-terminal domain containing a cavity described as a ‘broken’ active site. The two larger core domains have nearly identical tertiary structures and are related by approximate two-fold symmetry, but lack amino acid sequence homology. Furthermore, the overall structure of the dimer shows that the active site in one subunit is completed by contribution from the ‘broken’ active site of the other subunit. The homotetrameric CDA from B.subtilis (Johansson et al., 2002Go) contains four active sites; the monomer 14.9 kDa presents significant similarities with the catalytic domain of the E.coli enzyme. The zinc ion in the active site is co-ordinated with three negatively charged cysteine residues. Furthermore, residues from three subunits contribute to each active site through complicated intersubunit interactions.

Human CDA is a tetrameric enzyme of identical 15 kDa subunits, each containing an essential zinc atom in the active site. The substrate binding site on each subunit is independent (Vincenzetti et al., 1996Go). Further studies by site-directed mutagenesis and chemical modification (Cambi et al., 1998Go) of amino acid residues involved in catalysis have indicated that the zinc atom is co-ordinated by three cysteines (C65, C99, C102) and that a glutamic acid residue in position 67 plays an important role in catalysis. Recently, it has been shown by site-directed mutagenesis and fluorescence studies (Vincenzetti et al., 2000Go) that the residues F137 and F36 may be important in stabilizing hydrophobic interactions between the ligand and the enzyme, and thereby facilitate the catalytic process. The dissociation of an oligomeric protein into monomers usually leads to changes in their functional properties (Perutz, 1989Go). Therefore, identification of the residues involved in the intersubunit contacts and their eventual role in the assembly of the active site(s) is fundamental to understand the structure–function relationship of an enzyme. In this work, we have studied the dissociation and re-association of the four subunits of human CDA. The dissociation process induced by small amount of SDS was studied by following changes in the catalytic activity and in the hydrodynamic properties of the enzyme. Fluorescence and circular dichroism (CD) spectroscopy were also used to reveal conformational changes involving the protein secondary and tertiary structure. With B.subtilis CDA (Johansson et al., 2002Go), it was demonstrated that residue F125 (homologous to F137 of the human CDA) is quite close to the subunit interface of each dimer forming the tetrameric structure. Since amino acid replacement at the subunit interface may affect the enzyme function, we investigated the effect of the mutation F137W on the assembling of the subunits into a catalytically active homotetramer. Considering that the phenylalanine fluorescence is not sensitive to change in the surrounding environment and that the human CDA W113 residue is the only tryptophanyl present in each monomer, we used the double mutant, F137W/W113F to study the subunit dissociation process in the human enzyme.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Chemicals

Cytidine (CR), deoxycytidine (CdR), Tris (hydroxymethyl) aminomethane (Trizma base), dithiothreitol (DTT) and isopropyl 1-thio-ß-D-galactopyranoside (IPTG) were from Sigma Chemical Co. (St Louis, MO). Other chemicals were reagent grade from J.T. Baker Chemicals B.V. (Deventer, The Netherlands). PM10 membranes were from Amicon Corporation; Superdex 75 HR10/30 was from Pharmacia Biotech (Uppsala, Sweden); Affigel 102 and protein markers were obtained from Bio-Rad Laboratories (Hercules, CA).

Enzymes preparation

Escherichia coli SØ5201 (MC 1061cdd::Tn10 pyrD::Kan), containing plasmids pTrcHUMCDA (Vincenzetti et al., 1996Go), pTrcHUMCDAW113F or pTrcHUMCDAF137W/W113F (Vincenzetti et al., 2000Go) were used as sources of wild-type and mutant CDAs. Cells were grown exponentially at 37°C in L-broth supplemented with 100 µg/ml of ampicillin and CDA expression was induced during late exponential growth by the addition of 1 mM IPTG. After 19 h with vigorous shaking, cells were harvested by centrifugation, washed with 0.9% NaCl, resuspended in buffer A (50 mM Tris–HCl pH 7.5; 1 mM DTT, 1 mM EDTA) and lysed by sonic disruption. After centrifugation at 10 000 g for 10 min, the supernatant was heat treated at 70°C for 10 min, rapidly cooled and centrifuged again at 27 000 g for 20 min. The supernatant was applied on an affinity column containing the cytidine analogous CV6 (Cacciamani et al., 1991Go) bound to an agarose resin, Affigel 102, equilibrated with buffer A. The column was washed and eluted as described by Cacciamani et al. (Cacciamani et al., 1991Go). The final preparation of wild-type CDA and F137W/W113F mutant enzyme was judged >98% homogeneous by SDS–PAGE. The purified enzyme, after dialysis against buffer A, was used in all experiments.

Other analytical procedures

The zinc content was determined by inductively coupled plasma optical-emission spectrometry (ICP-OES) using a Jobin Yvon 24R instrument. All measurements were performed under ‘metal-free’ conditions (Vincenzetti et al., 1996Go). Protein concentration was determined by the Bradford protein assay (Bradford, 1976Go).

Enzyme assay and stability in presence of SDS

CDA activity was assayed spectrophotometrically as previously described (Cacciamani et al., 1991Go). The stability of the enzyme in the presence of SDS was determined by incubating the enzyme in presence of the detergent in a final volume of 20 µl in with different SDS:enzyme molar ratios. After 2 h of incubation at 25°C, aliquots were taken and assayed for CDA activity. Renaturation of the enzymes after SDS treatment was initiated by 100-fold dilution of the denatured samples into buffer A or dialysing the mixture by ultrafiltration against buffer A.

Polyacrylamide gel electrophoresis experiments

Polyacrylamide gel electrophoresis (PAGE) was performed as described by Laemmli (Laemmli, 1970Go), using 15% acrylamide and the Mini Protean II apparatus (Bio-Rad, gel size 7 x 8 cm x 0.75 mm). The markers used were Bio-Rad low range. To study the dissociation of CDA into subunits, a series of 15% PAGEs were done in the presence of increasing concentrations of SDS, ranging from 0.35 to 1.73 mM both in the gels and in the running buffer. The samples were prepared as follows: 3.6 µg of each protein sample were incubated in presence of SDS ranging from 0.35 to 1.73 mM in order to reach different molar SDS:enzyme ratios. After 2 h of incubation at 25°C, Bromophenol Blue was added and the sample was loaded on a gel. The electrophoresis was performed at 4°C with a constant voltage of 200 V.

The effect of the strong CDA inhibitor F-ZEB on dissociation was studied as follows: 4.0 µg of enzyme were incubated with F-ZEB at final concentrations ranging from 0.06 to 60 µM. After 3 h at 25°C, SDS was added to a final concentration of 1.73 mM (SDS:enzyme molar ratio = 200). The mixture was incubated for an additional 2 h at 25°C and subsequently subjected to 15% PAGE in the presence of 1.73 mM SDS. W113F mutant enzyme was used as control in all experiments. CDA was visualized on the gel by Coomassie Blue. In certain experiments, the gels were cut into 2 mm slices and each slice extracted for 2 h in buffer A containing the same percentage of SDS as used during electrophoresis. CDA activity in the eluate of each slice was determined as described above.

To measure the relative amount of the tetramer at each SDS:enzyme ratio, both in the wild-type CDA and mutant enzyme, semi-quantitative analyses on the electrophoretic gels were performed. The separating gels (prepared as described above) were scanned and analysed with QUANTISCAN software (BIOSOFT, Ferguson, MO). A graphic presentation of protein zones as peaks was produced and from the calculated peak areas the percentage of the tetrameric and monomeric form was calculated.

Reactivation of CDA

Monomeric CDA eluted from a 15% PAGE gel containing 1.73 mM SDS, as described in the previous section, was dialysed against buffer A under ‘metal-free’ conditions (Vincenzetti et al., 1996Go), analysed for enzymatic activity and zinc content, and run again on 15% PAGE, 0.35 mM SDS to determine the extent of re-association.

Spectroscopic measurements

The CD experiments were performed with a Jasco J710 spectropolarimeter at 25°C in the far (90–250) and in the near (250–320) UV range. Buffer was 0.02 M Tris–HCl pH 7.6, 1 mM DTT; protein concentration was 2 µM in the far UV range (cell length 2 mm) and 20 µM in the near UV range (cell length 10 mm). Molar ellipticity [{theta}] (deg cm2 dmol–1) is expressed on a mean residue concentration basis in the far UV region and on a protein concentration basis in the near UV region. All spectra were corrected by baseline subtraction.

Fluorescence measurements were performed on a Fluoromax 3 (Jobin Yvon) spectrofluorometer, equipped with an external bath circulator at 25°C. The steady-state emission spectra were corrected by blank subtraction in order to eliminate Raman scattering effects. The buffer used in the experiments was 20 mM Tris–HCl pH 7.6 containing 1 mM DTT. The excitation wavelength was 280 nm.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
CDA activity in presence of SDS

A time course of the inactivation of CDA by low concentration of SDS (ranging from 0.35 to 1.73 mM) was performed. The results obtained indicated that the process reached equilibrium after 2 h at 25°C (data not shown). At different SDS:enzyme molar ratios, the inactivation of both wild-type and the F137W/W113F mutant enzyme was non-cooperative as demonstrated in Figure 1a. The same experiment conducted on the W113F mutant enzyme showed an inactivation curve similar to that of the wild-type CDA (data not shown). Both wild-type CDA and the W113F mutant enzyme reached complete inactivation at an SDS:enzyme molar ratio of ~800, whereas the F137W/W113F mutant enzyme seemed more sensitive and was completely inactivated at an SDS:enzyme molar ratio of 250. CDA activity was immediately recovered following a 100-fold dilution of the SDS-treated samples into renaturation buffer (see Materials and methods).



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Fig. 1. (a) Enzyme activity remaining after incubation with various concentrations of SDS for 2 h at 25°C. Wild-type CDA (triangle), F137W/W113F mutant CDA (square). The experiment was performed as described in Materials and methods. (b) 15% PAGE of human CDA at different SDS concentrations. Electrophoresis was performed as described in Materials and methods. Each lane represents separate gels with different concentrations of SDS. The SDS:enzyme concentration ratios in the individual lanes were as follows: lane 1, 61; lane 2, 123; lane 3, 185; lane 4, 616; lane 5, 31; lane 6, 62; lane 7, 92; lane 8, 154. Lanes 1–4, wild-type CDA; lanes 5–8, F137W/W113F mutant CDA; St, standard LMW (Bio-Rad). The arrows indicate the tetrameric form (Rm = 0.19; MW = 62 000) and the monomeric form (Rm = 0.72; MW = 15 500).

 
SDS-mediated dissociation of homotetrameric CDA

The dissociation of human CDA into subunits was studied by 15% PAGE performed at different SDS concentrations. As shown in Figure 1b, the wild-type CDA was a tetramer with a relative migration (Rm) of 0.19 up to an SDS:enzyme molar ratio of 61; at an SDS:enzyme ratio of 123, it started to dissociate into monomers with an Rm of 0.72 (lane 2). The dissociation was completed at an SDS:enzyme ratio of 616 (lane 4). Intermediate forms such as trimers and/or dimers were not observed. With the F137W/W113F mutant enzyme, the dissociation process was significantly faster. Figure 1b shows that at an SDS:enzyme ratio of 31, the enzyme was present only in the tetrameric form (lane 5), whereas it existed predominantly as monomer at an SDS:enzyme ratio of 62 (lane 6). The dissociation process in the mutant enzyme was completed at an SDS:enzyme ratio of 154 (lane 8). Measurements of the CDA activity eluted from 2 mm gel slices following electrophoresis showed that only the tetrameric form of CDA retained activity, while the monomer was completely inactive (data not shown). The inactive monomeric enzyme extracted from the gel was reactivated after extensive dialysis, under ‘metal-free’ conditions, against buffer without the dissociating agent. ICP analysis on the reactivated CDA revealed the presence of 1 mol zinc per mol of enzyme subunits. Furthermore, the reactivated enzyme showed one single band, corresponding to the tetrameric form of CDA upon PAGE run under native conditions (data not shown). The mutant enzyme W113F was used as control in all the experiments and its dissociation pattern resemble that of the wild-type CDA (data not shown).

Semi-quantitative analysis of the PAGE images allowed the calculation of the SDS:enzyme ratio that caused 50% dissociation of the tetrameric form of the enzyme into monomer. This value was 316 for the wild-type CDA and 69 for the mutant enzyme (data not shown). The presence of the strong CDA competitive inhibitor 5-fluorozebularine (F-ZEB, 5-fluoropyrimidine-2-one ribonucleoside) affected the subunit dissociation of wild-type CDA (Figure 2a, lanes 1–4); in fact, at an SDS:enzyme molar ratio of 200, the wild-type CDA is predominantly present in the monomeric form, but addition of increasing amounts of F-ZEB ranging from 0.06 to 60 µM resulted in the disappearance of the monomer and increased amounts of the tetrameric form. On the contrary, the presence of the inhibitor did not affect the F137W/W113F mutant enzyme subunit dissociation (Figure 2a, lane 5). By scanning the gels, the percentage of the monomer at each F-ZEB concentration was calculated and from the graphic representation of the data (Figure 2b), it was deduced that 1.2 µM of the inhibitor was required to reduce the dissociation of the tetramer to 50%, under the conditions employed.



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Fig. 2. (a) Effect of F-ZEB on SDS-promoted dissociation of tetrameric human CDA. The samples were incubated together with increasing amounts of F-ZEB and subjected 15% PAGE in the presence of SDS 1.75 mM as described in Materials and methods. ST: low molecular weight standards (Bio-Rad); lanes 1–4, wild-type CDA in the presence of 0.06, 0.6, 6 and 60 µM of F-ZEB, respectively; lane 5, mutant enzyme in the presence of 60 µM of F-ZEB. (b) Percentage of monomer at each F-ZEB concentration. The data were obtained by scanning lanes 1–4 of the gel shown in 3(a).

 
Spectroscopic measurements

The CD spectra in the far UV region (190–250 nm) of wild-type and mutant F137W/W113F CDA revealed great similarities in the secondary structure organization of the two proteins (Figure 3), indicating that the double mutation does not affect the overall structure of the protein. In contrast, CD spectra in the near UV region showed clear differences between the two proteins (Figure 3, inset). The spectrum of the mutant protein completely lacked optical activity between 250 and 290 nm. As the CD signal in this region arises from asymmetric interactions of aromatic amino acids with nearby groups (Strickland, 1974Go) provided by the protein matrix, this result and the peak position in wild-type CDA (267 nm) suggest that the substitution of F137 with a larger amino acid side chain (such as a tryptophan) induced a small but detectable perturbation in the local tertiary structure. The presence of increasing concentration of SDS caused a slight decrease in the secondary structure content for both wild-type and mutant CDAs (data not shown). This effect may be ascribed to the SDS-induced dissociation revealed by PAGE and the gel filtration experiments performed under the same conditions. A similar effect has been observed with other proteins (Kelly and Price, 1987Go; Gahn and Roskoski, 1995Go). In Figure 4, the ratio between the ellipticity at 222 and 208 is reported at different SDS concentrations. As shown, the monomerization process differed for the two homotetrameric proteins: the quaternary structural organization of the mutant protein seemed more affected as increasing SDS concentration leads to a steeper decrease in secondary structure content than for the wild type; around an SDS:enzyme molar ratio of 500, the secondary structure content became stable. Surprisingly, when we overcame the critical micellar concentration of SDS (cmc = 2.2 mM) (Takeda et al., 1985Go), CD spectra showed a significant recovery of secondary structure probably due to the formation of a compact micelle that protected the exposed hydrophobic surfaces of each monomer by mimicking the quaternary structure interactions: as a control, no activity was evident for these samples. Tetramer to monomer dissociation of both wild-type and double mutant CDA was also followed by fluorescence spectroscopy. The steady-state fluorescence spectrum of wild-type CDA (Figure 5) peaked at ~335 nm, which is a typical emission wavelength for a partially buried tryptophan residue (Burstein et al., 1973Go). The double mutant spectrum was shifted to longer wavelengths ({approx}348 nm), indicating that the polarity around the tryptophan residue at position 137 is much higher than that at position 113, i.e. its original position in the wild-type protein. Interestingly, the wild-type CDA spectrum exhibits a shoulder at ~309 nm, which is totally absent in the mutated protein. Since the measurements depicted in Figure 5 were done with an excitation wavelength of 280 nm, this contribution in the blue-edge of the fluorescence spectrum may be ascribed to tyrosine fluorescence, which is typically peaked at ~303–305 nm (Lakowicz, 2000Go). This conclusion is also supported by the disappearance of the 309 nm shoulder when the sample was excited at 295 nm (data not shown), where tyrosine emission is negligible. The addition of SDS yielded different results for the two samples. In the case of wild-type CDA, a slight broadening of the spectrum was observed, without any major change in the total fluorescence intensity. On the other hand, a much larger effect was obtained with the double mutant, including a dramatic decrease of the signal at 349 nm upon SDS-induced monomerization, as well as the appearance of a pronounced shoulder at 305 nm.



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Fig. 3. Far UV CD spectra of wild-type (solid line) and mutant (dashed line) CDA. Experimental conditions: 0.02 M Tris–HCl buffer pH 7.6, 1 mM DTT; cell length 2 mm; protein concentration 2 µM; temperature 25°C. The inset shows the near UV CD spectra of wild-type CDA (solid line) and the mutant (dashed line). Protein concentration was 20 µM and cell length 10 mm.

 


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Fig. 4. SDS dependence of the ratio of the dichroic measurements performed at 222 and 208 nm. The spectral data were obtained at increasing concentrations of SDS for wild-type (triangle) and the mutant (square) CDA, respectively. Experimental conditions are the same as described in Materials and methods.

 


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Fig. 5. Fluorescence emission spectra (ex = 280 nm) of wild-type CDA (solid line); wild-type CDA in the presence of SDS (3.5 mM) (short dashed line), double mutant (long dashed line) and double mutant plus SDS (3.5 mM) (dotted line). All spectra were recorded in the same experimental conditions (i.e. temperature and protein concentration) and corrected by blank subtraction.

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The aim of this work was to gain further insight into the molecular structure of human CDA. Since previous studies indicated that residue F137 may be important in stabilizing hydrophobic interactions with the substrate (Vincenzetti et al., 2000Go), and comparisons with the B.subtilis CDA structure (Johansson et al., 2002Go) suggest that this residue may be located close to the subunit interface, the present study involved enzymes from both the wild-type and the F137W/W113F double mutant. The W113F mutation was introduced to enable fluorescence studies on the effect of the F137W mutation, as W113F is the only tryptophan residue in the wild-type monomer. Dissociation of the tetrameric human CDA was achieved by low concentration of SDS, and it occurs without the intermediate formation of trimers or dimers, via a non-cooperative transition (Figure 1). This agrees with earlier kinetic data which indicated that each subunit acts independently of the other (Vincenzetti et al., 2000Go). The absence of cooperativity between subunits is also observed in the B.subtilis CDA, where the active site of the individual subunits involves contributions from three of the four subunits (Johansson et al., 2002Go). Dissociation of the enzyme into monomers results in complete loss of activity, but full retention of the catalytic zinc ion. The dissociation is reversible as extensive dialysis or dilution of the SDS-treated sample with buffer lacking SDS restores full enzyme activity. CD measurements of the enzyme in the presence and absence of SDS indicate that neither the tertiary nor the secondary structure of the monomer is greatly affected by SDS concentration in the range used to dissociate the tetramer. On the other hand, the F137W mutation causes a severe weakening of the quaternary structure (Figure 3). The presence of the strong competitive inhibitor F-ZEB (Ki = 3x10–7 M) counteracts the SDS-induced dissociation of the wild-type tetramer (Figure 2a). This may indicate that a residue(s) involved in creating the active site may also participate in subunit interaction. W113F mutant CDA, used as control, presents the same behaviour in presence of F-ZEB with respect to the wild-type enzyme. With the F137W/W113F double mutant enzyme, the dissociation induced by SDS is not affected by F-ZEB (Ki = 6x10–6 M). F137W/W113F CDA shows a 20-fold higher Ki value for F-ZEB than the wild-type enzyme; furthermore, the kinetic constants Km and Vmax for different substrates were negatively affected by the mutation (Vincenzetti et al., 2000Go). These kinetic parameter alterations may be due to the larger tryptophan side chain compared to phenylalanine, but it is also an indication of the involvement of F137 residue in the active site of human CDA. Therefore, the fact that the presence of F-ZEB inhibitor does not protect F137W/W113F mutant protein from dissociation into monomers may also be due to a lack of interactions between W137 and the ligand because of the steric clutter exerted by tryptophan. On the other hand, preliminary studies performed on F137A mutant protein show a complete loss of the enzymatic activity and a dissociation into monomers at the lower SDS:CDA ratio of 61, indicating a possible involvement of F137 residue in the subunit interaction. The intrinsic fluorescence of most proteins is generally dominated by the tryptophan residues. When a single tryptophan is present in the protein structure, tyrosine emission may be negligible, due to the so-called energy transfer effect. In this case, tyrosines act as donors, transferring the excitation energy directly to the tryptophan residue, via a non-radiative dipole–dipole interaction (Lakowicz, 2000Go). The absence of tyrosine emission and the much higher quantum yield of the double mutant protein (Figure 5) demonstrate that a more efficient energy transfer process occurs in the mutant than in the wild-type CDA. Since the most relevant parameter that affects dipole–dipole interaction is the distance between the donor(s) and the acceptor, these findings indicate that, on average, W137 is closer to the surrounding tyrosines in the mutant protein than W113 is to its nearest tyrosines in the wild-type enzyme. The large decrease in emission at 348 nm following SDS-induced monomerization of the double mutant demonstrates that a large part of the tyrosines involved the energy transfer process do not belong to the same subunit containing the tryptophan residue. In contrast, only marginal changes characterize the dissociation of the wild-type protein. A possible correlation between the protein structure and its intrinsic fluorescence that may explain these observations is schematically illustrated in Figure 6.



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Fig. 6. Models of fluorescence energy transfer processes in CDA. (Top) Scheme of the energy transfer process in the wild-type CDA. The tyrosines in each subunit are assumed to interact only with the tryptophan residue of the same subunit (A, C and D). Upon dissociation into monomers (B), no changes are observed. (Bottom) Fluorescence transfer model for the double mutant. In this case, the tyrosines may interact also with the tryptophan residue of a different subunit (A and C) unless dissociation occurs (B and D).

 
The crystal structure of B.subtilis CDA (Johansson et al., 2002Go) shows that F125 residue, corresponding to the human CDA F137, is located between A and B subunits, and contributes to the generation of the uridine-binding pocket. In this way, a tetramer contains four independent active sites, while no complete active site is found within a single monomer. This is in good agreement with our experimental data obtained for human CDA and for the mutated proteins F137W/W113F, and F137A evidencing that in human CDA, F137 residue may be located at the interface between subunits and may contribute to the stabilization of the CDA quaternary structure. From B.subtilis CDA structure (Johansson et al, 2002Go), it seems evident that other amino acid residues, highly conserved among homotetrameric CDAs, are located between A and B subunits: F24, R90, Q91 and L121, corresponding to the human CDA F36, R103, Q104 and L133, respectively. Instead the residues Y21 and R56, corresponding to the human Y33 and R68, respectively, are located between subunits A and D. Human CDA Y60 is located in a conserved region in some of the homotetrameric CDAs and in B.subtilis CDA its corresponding Y48 of the D subunits makes a hydrogen bond with the 5'-hydroxylic group of THU.

Site-directed mutagenesis is in progress to modify the highly conserved residues Y60 (corresponding to the E.coli Y633 and B.subtilis Y48) and Y33 in order to better clarify how this residue may participate in the intersubunit interactions together with the F137 residue.


    Acknowledgements
 
The authors thank Professor Victor E.Marquez (National Institutes of Health, USA) for the generous gift of 5-fluorozebularine and Mrs Natalina Cammertoni for the excellent technical assistance.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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Received February 10, 2003; revised October 14, 2003;; accepted October 21, 2003





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