From the aDepartment of Biochemistry, University of the Saarland, D-66041 Saarbruecken, Germany, the cDepartment of Mathematics and Physics, The Royal Veterinary and Agricultural University, Thorvaldsensvej 40, DK-1871 Frederiksberg C, Denmark, fEuropean Molecular Biology Laboratory-Outstation Hamburg at Deutsches Elektronen Synchrotron, Notkestr. 85, D-22603 Hamburg, Germany, the gDyson Perrins Laboratory, Department of Chemistry, University of Oxford, South Parks Road, Oxford, OX1 3QY, United Kingdom, hDepartment of Medicinal Chemistry MMPD CEDD, GlaxoSmithKline Pharmaceuticals, 3rd Avenue, Harlow, Essex CM19 5AW, United Kingdom, and iBiochemistry and Center for Bioinformatics, University of the Saarland, D-66041 Saarbruecken, Germany
Received for publication, December 10, 2002 , and in revised form, March 17, 2003.
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ABSTRACT |
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INTRODUCTION |
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The structural investigation of D- and gL-captopril binding presented here is based on results obtained from enzyme kinetic and thermodynamic studies. Captopril is known as an angiotensin converting enzyme-blocking agent used in the therapy of blood pressure diseases.
Although different catalytic mechanisms for mono- and binuclear metallo--lactamases have been discussed in the literature, it is still not clearly understood why the enzymes have two conserved metal binding sites (for review, see Ref. 8). The motivation for the present investigation was the demand for a better knowledge of the nature of metal ion binding in the presence of bound ligands. By a combination of extended x-ray absorption fine structure (EXAFS) and perturbed angular correlation of
-rays (PAC) spectroscopy, we studied the nature of captopril interactions with the cadmium-substituted enzymes (detailed descriptions of the methods can be found in Refs. 9 and 10, respectively). Both methods delivered consistent results that are additionally supported by UV-visible spectroscopic results of Co(II)-substituted enzymes. The present investigation contributes new insights with respect to the physiological importance of mono- and binuclear metallo-
-lactamases.
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MATERIALS AND METHODS |
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To produce "metal-free" buffers, buffer solutions in bidistilled water were treated by extensive stirring with Chelex 100 (Sigma). Apo-enzymes were prepared by dialysis of the corresponding enzymes against two changes of 15 mM HEPES, pH 7.0, containing 0.2 M NaCl and 20 mM EDTA over 12 h under stirring. EDTA was removed from the resulting apoenzyme solution by three dialysis steps against the same buffer containing 1 M NaCl and Chelex-100 and finally two dialysis steps against 15 mM HEPES, pH 7.0, containing 0.2 M NaCl and Chelex-100. In all preparations, the residual zinc content did not exceed 5% as determined by atomic absorption spectroscopy.
Synthesis of D-Captopril
To synthesize D-captopril (Scheme 1), we prepared compound 1 according to the procedure described in Ref. 14. Compound 2 was prepared following a method reported by Skiles et al. (15), and the classical hydrolysis reaction to obtain the D-captopril was carried out with NaOH 1 N under an atmosphere of argon (16). As there are two asymmetric centers in the molecule, here D- and L-designations refer to absolute stereochemistry at the prolinyl stereocenter (see Fig. 3).
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Determination of Steady-state Kinetic Parameters and Inhibition Constants
All kinetic measurements were performed at 25 °C with imipenem (kind gift of Merck) as a substrate following the hydrolysis at 300 nm (
(300 nm) = 9000 M1 cm1) in 15 mM HEPES, pH 7.0. The photometric measurements were performed with either a spectrophotometer (CphA) or the stopped-flow system DX-17MV (Applied Photophysics, Leatherhead, UK) in those cases where high concentrations of enzymes (0.11 µM) during the measurements were required to exactly define the reconstitution state of the metallo-enzymes. Under such conditions, it was possible to study Zn(II)1- and Cd(II)1-BcII with apo-enzyme reconstituted with only 0.1 equivalents of metal without the interference of residual zinc in the metal-depleted buffers. The data evaluation was based on the concentration of metal ions added. Effects of residual zinc in the solutions could be minimized, and it was possible to clearly discriminate Me1 and Me2 species. The steady-state parameters Km and kcat and the inhibition constants for D- and L-captopril were determined from initial rates. Standard non-linear regression analysis was used for data evaluation by directly fitting the Michaelis-Menten equation (uninhibited or competitively inhibited) to the data. Activities of binuclear enzymes were studied with enzyme samples in the presence of excess of the respective metal ions. Inhibition constants for D- and L-captopril were determined by the variation of the inhibitor concentration at substrate concentrations fixed in the range of the respective Km values.
UV-visible Spectroscopy of Co(II)-substituted BcII
UV-visible spectra of Co(II)-substituted BcII were recorded with a Lambda9 spectrophotometer (PerkinElmer Life Sciences) and processed with the UV-Winlab software from PerkinElmer Life Sciences. Co(II)1-BcII was prepared by reconstitution of 130 µM apo-enzyme with 120 µM Co(II). Co(II)2-BcII was prepared by preincubation of 118 µM apo-BcII with 500 µM Co(II). To remove traces of precipitated protein, the samples were centrifuged for 10 min at 30,000 x g immediately before the measurement. The D-captopril complexes were obtained by adding 800 µM D-captopril to the sample cells.
Determination of Metal Ion Dissociation Constants
The dissociation constants for a first (Kmono) and second (Kbi) cadmium ion bound to BcII in presence of 25 µM D-captopril and 0.1 M NaCl were obtained from competition experiments with the chromophoric chelator Mag-fura-2 (Molecular Probes, Eugene, OR) in 15 mM HEPES, pH 7.0, as described previously (17, 18).
X-ray Absorption Spectroscopy
X-ray Absorption Spectroscopy (XAS) Sample PreparationThe buffer used during purification has been exchanged for 20 mM bis-Tris, pH 7, by iterative use of Millipore Centricon devices to decrease scattering background. The final protein concentration was 23 mM. The free metal concentration was below 2 µM. Samples have been frozen and stored at 20 °C.
XAS MeasurementsFor XAS, about 100120 µl of enzyme solution were transferred to the EXAFS cuvettes covered with Kapton tape (DuPont) as an x-ray transparent window material, capped, mounted on the sample holder, dropped into liquid nitrogen, and transferred to the beamline cryostat. The Cadmium-K edge (26,711.0 eV) XAS was collected at the beamline D2 at Deutsches Elektronen Synchrotron (European Molecular Biology Laboratory outstation, Hamburg, Germany) running at 4.4 GeV and 70125 mA current in fluorescence mode at 25 K sample temperature. An internal cadmium foil sample was used for calibration.
XAS Data AnalysisStandard EXAFS analysis was performed using the EXPROG software package (developed by C. Hermes and H. F. Nolting at the European Molecular Biology Laboratory-Outstation/Hamburg, Germany) to process the raw data and EXCURV98 (developed by N. Binsted, S. W. Cambell, S. J. Gurman, and P. Stepherson at Science and Engineering Research Council, Daresbury, UK) using exact curve wave scattering theory (19, 20) to analyze the spectra. The energy range was set to 30650 eV above the edge. Phases were calculated ab initio using Hedin-Lundqvist potentials and von Barth ground states (21). Both single and multiple scattering paths up to 4.5 Å from the metal atom were used to identify and quantify imidazole coordination of histidine ligands by using implemented small molecule data base of the program. After eliminating all non-imidazole atoms of the His unit, the complete imidazole ring was simulated by iterating the distance and Debye-Waller factor of the pivotal (directly coordinating) N atom and the angle of the second imidazole N atom for slight distance corrections of the constrained outer shell imidazole atoms. Debye-Waller factors of the outer shell atoms of imidazole rings were constrained assuming the Debye-Waller factors of atoms with similar distance to the absorber to be equal. Since the constraints for the Debye-Waller factors are unique for each parameter set, details are summarized in Tables II and III.
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The fitting process included additional constraints for the following parameters. Two coordination clusters were introduced, each having an integer number of ligands. The fit then determined the fractional occupancy of each cluster for the mononuclear enzyme if the metal ions were distributed between the two metal sites. Theoretical simulations were generated by adding shells of scatterers around the central cadmium atoms and iterating the number of scatterers, bond lengths, and Debye-Waller factors in each shell. Additionally, the Fermi energy Ef (edge position relative to calculated vacuum position) was refined to achieve the best fit to the experimental data. The improvement of the fit after the addition of each shell beyond the first was assessed by comparing the residual R-factor (22).
Perturbed Angular Correlation of -Rays Spectroscopy
111mCd was produced by the Cyclotron Department at the University Hospital in Copenhagen, Denmark. Preparation and purification of 111mCd is described in Ref. 23. The PAC spectrometer is described in Ref. 24 and references therein.
In the case of identical, static, and randomly oriented molecules, the perturbation function G2(t) is shown in Equation 1,
![]() | (Eq.1) |
In the liquid state, the NQI is time-dependent because of the Brownian reorientation of the protein, described by the rotational diffusion time R. This has the consequence that G2(t) converges to 0 as a function of time, representing thermal equilibrium and isotropy in the angular correlation between the two
-rays.
The perturbation function A2G2(t), where A2 is the amplitude, was analyzed by a conventional non-linear least squares fitting routine. Satisfactory fitting was obtained with a relative Gaussian distribution =
0/
0 applied to all the three frequencies. Non-zero values for
indicate that the 111mCd nuclei are located in a distribution of surroundings. An NQI is then described by the parameters
0,
,
, and
R. In cases where more than a single NQI is present, the perturbation function is the sum of the different perturbation functions, where each NQI is weighted by its population (23).
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RESULTS |
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We have investigated the influence of inhibitor binding on metal dissociation constants. In the presence of 25 µM D-captopril and 5 µM BcII, a first Cd(II) ion binds with a KD of 1.6 nM as compared with 8.3 nM in absence of D-captopril (18). A second cadmium ion is bound with a KD of 50 µM as compared with 5.9 µM in the absence of the inhibitor (18). These KD values are macroscopic constants, and in case of the mononuclear enzymes, they do not reflect any binding site assignment (see below).
For CphA, the substitution of cadmium for zinc results in a drastic decrease of Km and kcat. Binding of captopril is much stronger to the cadmium species than to the zinc species with a strong preference for D-captopril (Table I).
UV-visible SpectroscopyBinding of Co(II) to the metal-free enzyme at a [Co(II)]/[enzyme] stoichiometry of 0.8 results in the appearance of a ligand-to-metal charge transfer band at 344 nm and bands in the d-d transition region (400700 nm). In general, the intensity of the ligand-to-metal charge transfer bands is mainly due to Cys (sulfur)-Co(II) interaction, whereas the d-d transitions are caused by the His-Co(II) interaction (17).
Increasing the [Co(II)]/[enzyme] ratio above 1 results in a shift of the charge transfer band to 383 nm (Fig. 1) (17). Besides this difference, the d-d regions are almost identical in shape and intensity at low and high stoichiometry of Co(II) relative to the enzyme. A similar H-site occupancy at low and high stoichiometry reflects a strong preference of Co(II) for the H-site in Co(II)1-BcII (17). Binding of D-captopril to Co(II)-BcII leads to changes both in the charge transfer region and in the d-d region, indicating the binding of an additional ligand and likely changes in the coordination geometry of both sites. The difference spectra between inhibitor-bound and free enzyme at low and high stoichiometry are very similar in the charge transfer region and virtually identical in the d-d region, indicating that the modes of binding for D-captopril to Co(II) are also almost identical at both metal stoichiometries (Fig. 1). It has to be emphasized, however, that even under the conditions used for the Co(II)2-BcII experiments (Fig. 1A), the enzyme was not completely available as the Co(II)2 species. Even at the very high concentration of Co(II) used, a fraction of the enzyme still shows the charge transfer band for the mononuclear enzyme at 348 nm, and thus 1020% of the enzyme still had Co(II) bound only in the DCH site. Thus, a direct quantitative comparison of absorption coefficients of Co(II)1 and Co(II)2 enzyme is difficult and, thus, a quantitative estimate of relative occupancies of both binding sites for the mononuclear enzyme.
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EXAFS SpectroscopyFor EXAFS spectroscopy on BcII and CphA, 0.8 eq of Cd(II)/enzyme were used to minimize contributions from the eventually formed binuclear species. A 3-fold surplus of D-captopril was added to maximize the abundance of the inhibited species. The EXAFS results are given in Tables II and III for BcII and CphA, respectively. The corresponding spectra are shown in Fig. 2.
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To illustrate the fitting procedure used, Table II shows two alternative models used for simulation of the spectra for uninhibited Cd(II)1-BcII, namely a 1-cluster and a 2-cluster model. Since it is well known that a single cadmium ion is distributed between the two available binding sites (10, 18), a typical 1-cluster model necessarily results in an "averaged" ligand sphere. From the coordination numbers resulting for the 1-cluster model, it becomes obvious that neither the 3-His nor the DCH site is fully occupied (NS 0.4; NN/imidazole
2.02). The fractional occupation of the DCH site (
40%) leads to a theoretical value of
2.2 for NN/imidazole, which is in contradiction to the simulated value of NN/imidazole
2.02. The resulting lack of intensity in the theoretical spectrum is compensated by an added broad contribution of oxygen ligands (high NO and very high Debye-Waller factor) in this simple model. If multiple scattering contributions of histidines are not taken into account, the result becomes even more corrupted by not using constrained imidazole ring units since contributions from nitrogen or oxygen ligands are virtually identical (data not shown). Also, second shell contributions were omitted, which is clearly reflected in the lack in intensity at
3.2 Å (Fig. 2C) of the theoretical spectrum.
Since the amino acid ligand geometry is well known for BcII from x-ray crystallographic data (Protein Data Bank code 1BVT
[PDB]
), we could make use of these data by constructing a 2-cluster model. The atomic distribution in the 2 cluster model was accordingly assigned (3-His and DCH site, respectively). Thus, the problem of correlated coordination numbers and Debye-Waller factors could be solved by fixing the ligand coordination numbers according to the structural data derived from the Protein Data Bank file 1BVT
[PDB]
. Thus, the only free remaining coordination number in the 2-cluster model is the one of the cadmium ion itself, which determines the fractional occupation of each cluster. Additionally second shell nitrogen/oxygen ligands with a fixed coordination number were introduced to account for back scattering contributions in the 3.2-Å range. For the 2-cluster model, an occupation of 70% results for the H-site. Interestingly the ligand specific distances found are in good agreement with the values obtained with the 1-cluster model.
All attempts to generate theoretical spectra with 1-cluster models for the D-captopril-inhibited enzyme (with exactly the same method as used for the uninhibited enzyme) failed in the sense that useful data could not be obtained due to mutual dependences of parameters. Although low R-factors could be obtained, Debye-Waller factors and coordination numbers resulted in unrealistic values (data not shown). In the theoretical 2-cluster model for the D-captopril-inhibited enzyme, it proved to be possible to replace the second shell nitrogen/oxygen contributions in the DCH site by introducing a small molecule model of the Asp side chain. Again, all coordination numbers except the coordination number of Cd(II) in both available binding sites were fixed. The resulting distribution of Cd(II) for the inhibited species shows a 60% occupation of the DCH site.
The ligand geometry of Cd(II)1-BcII is roughly similar to the one published for Zn(II)2-BcII (17) (x-ray structure, Protein Data Bank code 1BVT [PDB] (26)). However, instead of one bound OH as found in the zinc EXAFS, the cadmium EXAFS shows two oxygen ligands at 2.17Å ± 0.01 Å. This distance appears too long to be qualified as two hydroxide ions bound, even taking the higher ionic radius of cadmium into account. Together with the three amino acid ligands, these two water molecules lead to a penta-coordinated H-site, whereas the tetra-coordinated DCH site is conserved relative to the zinc enzyme.
The metal distribution found for Cd(II)1-BcII (70% of Cd(II) are located in the H-site; 30% of Cd(II) in the DCH site) compares very well to the preference for the H-site suggested for cobalt but differs from the distribution found for the mono-zinc species where both sites are equally populated (17). The EXAFS spectrum from CphA in the presence of 0.8 eq of Cd(II) relative to the enzyme, here denoted Cd(II)1-CphA, can be fitted with only the DCH site occupied, and the fit gives one additional oxygen ligand very similar to the one found in Cd(II)1-BcII.
Binding of D-captopril to Cd(II)1-BcII shifts the metal occupancy between the two sites to 40% in the H-site and 60% in the DCH site. The sulfur of D-captopril binds to the H-site and replaces the two previously bound water molecules. Both binding spheres appear tetra-coordinated. The additional oxygen ligand found in the DCH site might be the carboxylate from D-captopril. Fig. 3B presents a hypothetical model where D-captopril binds either with its thiolate sulfur to the cadmium ion in the H-site or with its carboxylate oxygen to the cadmium ion when bound in the DCH site. This assumption is based on the significantly shorter cadmium-oxygen distance in the inhibited complex (2.12 Å as compared with the cadmium-oxygen distance of 2.28 Å in the Cd(II)1 uninhibited enzyme).
In the case of Cd(II)1-CphA, D-captopril binds to cadmium in the DCH site with its thiolate sulfur, replacing the previously bound water molecule. There is no indication for any distribution of the cadmium ion between the two sites, supported by the high rigidity (second sphere atoms are detectable with reasonable accuracy). This result is consistent with XAS studies on Zn(II)1-CphA (27).
For both inhibited enzyme species, the Debye-Waller factors of sulfurs in the DCH site are higher than those found for the uninhibited species. A possible explanation is that binding of an additional negatively charged ligand to the DCH site cadmium ion (captopril sulfur for CphA; a very close oxygen for BcII) results in a partial displacement of the bound Cys sulfur due to electrostatic repulsion. Thus, the increased Debye-Waller factors might result either from an overestimation of the coordination number of sulfur, which has been introduced by the chosen model, or by a "true" flexibility concerning the positioning of ligands. Due to the strong correlation of occupation number and Debye-Waller factor, however, an independent fitting of these parameters delivered no reliable results (data not shown).
PAC SpectroscopyPAC experiments on Cd(II)-BcII derivatives with either 0.2 eq Cd(II) (Cd(II)1-BcII) or 1.71.8 eq Cd (Cd2-BcII) in the presence of either 1 eq L-captopril or 1 eq D-captopril show that 2 NQIs with nearly equal abundancy can be detected in all cases (Table IV, Fig. 4). The two NQIs for both the L and D form of captopril as well as the two NQIs for Cd(II)1- and Cd(II)2-BcII are different.
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PAC data for CphA are only shown for [Cd(II)]/[enzyme] stoichiometries below 1 (Table IV). The spectrum of the uninhibited CphA is characterized by two NQIs with an abundance of 74% for the dominating form. To assign the two different NQIs to the two different metal sites, an experiment with moxalactam-modified enzyme was performed. A pretreatment with moxalactam leads to a covalent modification of the cysteine in the DCH site (7), whereby only the H site remains available for metal binding. One NQI with (0 = 130 megarads/s and
= 0.6) now dominates the spectrum. Its close resemblance with the less populated NQI in the free enzyme leads to the conclusion that the DCH site of the uninhibited Cd(II)1 enzyme is populated to about 80%.
For Cd(II)1-CphA in the presence of 1 eq of D-captopril, PAC spectroscopy shows a single sharp NQI, which is different from both NQIs present without the inhibitor. For L-captopril under the same conditions, a single new NQI is detected being different from both the NQI in the presence of D-captopril and the two NQIs detected without inhibitor.
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DISCUSSION |
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Both the cadmium and zinc enzymes of BcII exhibit negative cooperativity with respect to metal binding to the two conserved sites (17, 18). This, however, does not mean that there is a high and a low affinity binding site. We have shown earlier that a single metal ion, when bound to the binuclear site, is distributed between both binding sites (10, 12) and that rapid exchange among binding sites occurs (18). A recent work (28) shows that only mononuclear metallo--lactamases might be physiologically important. These findings pose the question whether strategies to find inhibitors for the binuclear enzymes are the only ones being adequate. A set of structures has been obtained for inhibitors with a mercapto group coordinating with the sulfur to both zinc ions (4, 5). In order for these inhibitors to be pharmaceutically relevant, one needs to ensure that the inhibition constants are low for all metallo-
-lactamases from pathogenic bacteria even at low zinc abundance. Under such conditions, the mononuclear enzymes are the dominating form.
Whereas D- and L-captopril show identical inhibition constants for Zn(II)1- and Zn(II)2-BcII, within experimental error, both inhibitors show a higher efficiency for the Cd(II)1 as compared with the Cd(II)2 species (compare Table I). Since the two macroscopic dissociation constants for cadmium binding to BcII in the presence of D-captopril differ by more than 4 orders of magnitude, we mainly focused on inhibition of the mononuclear enzymes. Both EXAFS and PAC spectroscopy with Cd(II)1-BcII resulted in a distribution of the single metal ion between both binding sites. This has also been observed for Zn(II)1-BcII (17). Surprisingly, binding of D-captopril does not result in a forced location of the metal ion in one of the two binding sites but leads only to a shift in the relative occupation.
The analysis of the EXAFS data (Table II) further shows that the sulfur of D-captopril coordinates to Cd(II) when located at the H-site of BcII. For the fraction where the single cadmium ion is located in the DCH site, the carboxylate group of D-captopril might be a ligand (see "Results"). The spectroscopic results obtained for Co(II)2-BcII (Fig. 1) give further evidence for the binding of the D-captopril sulfur at the H-site. The spectra of Co(II)1- and Co(II)2-BcII-D-captopril complexes both show identical features in the d-d region that are significantly different from those of the uninhibited enzyme. It is likely that a replacement of water by sulfur in the H-site results in the observed changes (compare Ref. 29). The appearance of two additional bands at 310 and 375 nm can be attributed to sulfur-cobalt ligand-to-metal charge transfer due to binding of the captopril sulfur. We did not perform an inhibition study of the Co(II) enzymes since they show a reduced stability as compared with the zinc and cadmium enzyme, which results in unreliable steady-state kinetic data. The weak binding of the second metal ion to the enzyme in the presence of captopril leads to the conclusion that the binuclear species might be of minor relevance as a target for inhibition with captopril-like compounds.
The enzyme from A. hydrophila is a representative of subclass B2 with a strong preference for imipenem as substrate. Furthermore, the activity of the binuclear enzyme is reduced relative to the mononuclear enzyme (13). The main difference in the active site relative to BcII is a substitution of a His residue with an Asn residue in the H-site. The PAC results for Cd(II)1-CphA clearly demonstrate a distribution of the single cadmium ion bound between both binding sites, which are different from the one found for Cd(II)1-BcII (18). However, the DCH site is strongly preferred. PAC spectroscopy detects a single sharp coordination geometry for cadmium in the D-captopril-inhibited enzyme. The binding site of the cadmium ion is clearly identified as the DCH site by EXAFS spectroscopy. In case of the L-captopril-inhibited Cd(II)-CphA, a new unique coordination geometry is found by PAC spectroscopy. A strongly increased Gaussian distribution of the observed frequencies (Table IV) demonstrates a more flexible environment than the one found for the D-captopril-inhibited species. Because of the nearly identical position of the first peak in the Fourier transform (0), it is likely that the same set of ligands composes the first coordination sphere of Cd(II) in L- and D-captopril-inhibited species. The difference in the symmetry parameters (
) (Table IV) is indicative of a modified geometrical arrangement of the ligands. For both enzymes studied, PAC derives a more rigid coordination geometry for the mononuclear cadmium enzyme with D-captopril relative to L-captopril as the inhibitor, consistent with lower inhibition constants for D-captopril as compared with L-captopril (Table I). Cd(II)1- and Zn(II)1-CphA clearly discriminate both diastereomers with a strong preference for D-captopril, whereas the inhibition constants for the binuclear form of all metal-substituted BcII species are very similar for D- and L-captopril (Table I).
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CONCLUSIONS |
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FOOTNOTES |
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b Supported by the Bundesministerium für Bildung und Forschung (contract 05SN8TSA1) and the Deutsche Forschungsgemeinschaft (Ad 152/1-2,3).
d Supported by the Danish Natural Science Research Council.
e Supported by the European research network on metallo--lactamases, within the TMR Program (CT 98-0232).
j To whom correspondence should be addressed. Tel.: 49-681-3022492; Fax: 49-681-3022097; E-mail: hwadolph{at}mx.uni-saarland.de.
1 The abbreviations used are: BcII, metallo--lactamase from B. cereus; CphA, metallo-
-lactamase from A. hydrophila; PAC, perturbed angular correlation of
-rays; EXAFS, extended x-ray absorption fine structure; H-site, zinc binding site composed of three histidine residues in subclass B1; DCH site, zinc binding site composed of asparagine, cysteine, and histidine; NQI, nuclear quadrupole interaction; XAS, x-ray absorption spectroscopy.
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ACKNOWLEDGMENTS |
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REFERENCES |
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