Coordination geometries of metal ions in d- or l-captopril-inhibited metallo-beta-lactamases

d- and l-captopril are competitive inhibitors of metallo-beta-lactamases. For the enzymes from Bacillus cereus (BcII) and Aeromonas hydrophila (CphA), we found that the mononuclear enzymes are the favored targets for inhibition. By combining results from extended x-ray absorption fine structure, perturbed angular correlation of gamma-rays spectroscopy, and a study of metal ion binding, we derived that for Cd(II)1-BcII, the thiolate sulfur of d-captopril binds to the metal ion located at the site defined by three histidine ligand residues. This is also the case for the inhibited Co(II)1 and Co(II)2 enzymes as observed by UV-visible spectroscopy. Although the single metal ion in Cd(II)1-BcII is distributed between both available binding sites in both the uninhibited and the inhibited enzyme, Cd(II)1-CphA shows only one defined ligand geometry with the thiolate sulfur coordinating to the metal ion in the site composed of 1 Cys, 1 His, and 1 Asp. CphA shows a strong preference for d-captopril, which is also reflected in a very rigid structure of the complex as determined by perturbed angular correlation spectroscopy. For BcII and CphA, which are representatives of the metallo-beta-lactamase subclasses B1 and B2, we find two different inhibitor binding modes.     

Zinc Ion-induced Domain Organization in Metallo-β-lactamases: A FLEXIBLE “ZINC ARM” FOR RAPID METAL ION TRANSFER?*

The reversible unfolding of metallo-β-lactamase from Chryseobacterium meningosepticum (BlaB) by guanidinium hydrochloride is best described by a three-state model including folded, intermediate, and unfolded states. The transformation of the folded apoenzyme into the intermediate state requires only very low denaturant concentrations, in contrast to the Zn₂-enzyme. Similarly, circular dichroism spectra of both BlaB and metallo-β-lactamase from Bacillus cereus 569/H/9 (BcII) display distinct differences between metal-free and Zn₂-enzymes, indicating that the zinc ions affect the folding of the proteins, giving a larger α-helix content. To identify the regions of the protein involved in this zinc ion-induced change, a hydrogen deuterium exchange study with matrix-assisted laser desorption ionization tandem time of flight mass spectrometry on metal-free and Zn₁- and Zn₂-BcII was carried out. The region spanning the metal binding metallo-β-lactamases (MBL) superfamily consensus sequence His-X-His-X-Asp motif and the loop connecting the N- and C-terminal domains of the protein undergoes a zinc ion-dependent structural change between intrinsically disordered and ordered states. The inherent flexibility even appears to allow for the formation of metal ion-bridged protein-protein complexes which may account for both electrospray ionization-mass spectroscopy results obtained upon variation of the zinc/protein ratio and stoichiometry-dependent variations of ^199mHg-perturbed angular correlation of γ-rays spectroscopic data. We suggest that this flexible “zinc arm” motif, present in all the MBL subclasses, is disordered in metal-free MBLs and may be involved in metal ion acquisition from zinc-carrying molecules different from MBL in an “activation on demand” regulation of enzyme activity.The production of metallo-β-lactamases (MBLs)² is one of the defense strategies of bacteria against β-lactam antibiotics. MBLs hydrolyze the C-N bond of the β-lactam ring of these compounds using protein-bound zinc ions as cofactors (1). Their emergence in pathogenic bacterial strains and their broad substrate profile make them clinically important (2). Whereas the overall structure of all known MBLs is very similar (3), distinct differences in the set of protein ligands for bound zinc ions led to the classification into subclasses B1–B3 (4). Here we have studied the two subclass B1 enzymes BcII and BlaB from Bacillus cereus strain 569/H/9 and Chryseobacterium meningosepticum, respectively, which show 35.2% identical residues (5). The very similar structure of these enzymes is organized in a αββα sandwich (6, 7). The N- and C-terminal domains are connected by an external loop, and the active site is located in a long channel between the two domains. The binuclear zinc binding site is composed of a 3-His (3H) site and a Asp-Cys-His (DCH) site. Three metal ion ligands are located on the N-terminal domain and constitute the HXHXD motif, which is strictly conserved in proteins of the MBL super family (8). The three remaining metal ligands are located on the C-terminal domain of the proteins. Both for the native and for the cadmium-substituted enzyme, it has been shown that a single metal ion, when bound to BcII, appears to be distributed between the metal binding sites (9–11).The metal ion requirement for catalytic activity of the three subclasses B1–B3 of MBLs is heavily debated. Although most crystal structures of subclass B1 enzymes show binuclear zinc sites (3), it was found that BcII from B. cereus 569/H/9, CcrA from Bacteroides fragilis, BlaB from C. meningosepticum, IMP-1 from Pseudomonas aeruginosa, and L1 from Stenotrophomonas maltophilia are both active as the mono- and di-zinc enzymes (9, 12–15). Recently a study with Co(II)-substituted BcII challenged this view in concluding that only the di-Co-enzyme might be catalytically active (16). The same authors came to the conclusion that also native BcII requires two bound zinc ions for activity (17). A very recent study on the Co(II)-substituted enzyme came to the conclusion that both the Co₁- and the Co₂-enzymes are catalytically active with the DCH site as the primary catalytic site (18).Variable metal loading states of zinc proteins are attracting increasing interest in the field of cellular regulation processes being a key to the understanding of physiological functions of zinc sensors and metallothioneins as well as regulatory functions of zinc ions. The coexistence of zinc proteins in the metal-loaded and the metal-free form, however, requires the regulation of “free” zinc ion concentrations in narrow limits, with nm to pm concentrations in eucaryotes (19) or even much lower concentration in procaryotes (20). The issue, however, that zinc enzymes in their natural environment might be regulated by reversible metal ion binding is infrequently considered.The impact of metal ion binding on structure and stability of MBL superfamily proteins has been studied in some detail. Zinc was found to be required for the folding of glyoxalase II (21) and arylsulfatases (22) into the native state. For CphA from Aeromonas hydrophila, differential scanning calorimetry and fluorescence spectroscopy demonstrated that zinc binding stabilizes the protein against denaturation with urea. The inactive Zn₂-CphA proved to be the most stabile species (23). Crystal structures of metal-free and metal-loaded BcII revealed minor structural changes in the active site of the protein (6). ¹H,¹⁵N heteronuclear single quantum coherence spectra of the backbones amides of BcII resulted in distinct signals for different metal ion/enzyme ratios which allowed discrimination of apoenzyme and metal-loaded states (24). Metal ion binding was considered to be essential for folding of L1 in vivo (25), and variable loading states were described in dependence of the bioavailability of various metal ions (26). We hypothesized earlier that metallo-β-lactamases are most likely in the metal-free apoenzyme state in the absence of substrates, which is because of the moderate affinity of the enzymes for zinc ions and the very low concentration of free zinc in cellular environments (14). We suggested that substrate availability might induce a spontaneous self-activation by direct transfer of zinc via ligand exchange reactions with delivery systems as substrate presence leads to a drastic increase of zinc affinity. The suggested self-activation mechanism, however, requires a direct interaction of the apoenzymes with other zinc carriers to allow a ligand exchange reaction to occur. Because such interactions might be considered as unspecific, it seemed reasonable to postulate a high structural flexibility to allow the transient formation of zinc-bridged complexes. To verify this prediction, we initiated an investigation on the role of zinc ions on folding and stabilization of MBLs with BlaB and BcII as test cases. Our results demonstrate that the systems, when unsaturated with metal ions, cannot be correctly described as being composed of variable fractions of the proteins in the loaded and unloaded state alone. We will present indications of the formation of labile, metal-bridged ternary complexes formed under such conditions. The latter may be considered as important intermediate states for metal ion transfer between identical or different zinc binding molecules in general.     

Dramatic broadening of the substrate profile of the Aeromonas hydrophila CphA metallo-beta-lactamase by site-directed mutagenesis

Among class B beta-lactamases, the subclass B2 CphA enzyme is characterized by a unique specificity profile. CphA efficiently hydrolyzes only carbapenems. In this work, we generated site-directed mutants that possess a strongly broadened activity spectrum when compared with the WT CphA. Strikingly, the N116H/N220G double mutant exhibits a substrate profile close to that observed for the broad spectrum subclass B1 enzymes. The double mutant is significantly activated by the binding of a second zinc ion under conditions where the WT enzyme is non-competitively inhibited by the same ion.     

The binding of zinc to angiotensin-converting enzyme

The equilibrium constant for the dissociation of zinc ion from angiotensin-converting enzyme (ACE) was measured using zinc ion buffers of zinc chloride and nitrilotriacetic acid (NTA). The dissociation constant is 6.4 X 10(-10) M. The fraction of active enzyme at equilibrium is independent of the presence of substrate which indicates that hippuryl-histidylleucine binds equally well to the holoenzyme and apoenzyme. The rate constant for the dissociation of zinc from ACE was measured as 0.68 min-1 for the free enzyme; the rate constant for the enzyme substrate complex was roughly 0.18 min-1. The association of zinc ion and ACE is very fast; the rate constant is 1.06 X 10(9) M-1 min-1. Ethylenediaminetetraacetic acid (EDTA) and NTA rapidly remove zinc from ACE with rate constants of 1.27 X 10(3) and 2.2 X 10(3) M-1 min-1. The equilibrium constant for the reaction of NTA with ACE was measured as 4.6 X 10(-2) and was calculated for EDTA as 3.8 X 10(3).     

Purification and properties of aryl acylamidase from Pseudomonas fluorescens ATCC 39004

Aldehyde binding to liver alcohol dehydrogenase in the absence and presence of coenzymes has been characterized by spectrometric equilibrium methods, using auramine O and bipyridine as reporter ligands. Free enzyme shows a significant affinity for aldehydes, and equilibrium constants for dissociation of the binary complexes formed with typical aldehyde substrates are reported. Binary-complex formation does not lead to any detectable inner-sphere coordination of aldehydes to the catalytic zinc ion of the enzyme subunit. Complex formation with NAD+ or NADH increases the affinity of the enzyme for aromatic aldehydes by a factor of 1.8 - 3.5 and 6-17, respectively. Benzaldehyde and dimethylaminocinnamaldehyde binding to the enzyme . NAD+ complex is not detectably associated with inner-sphere coordination of the aldehyde to zinc. It is concluded that binding of NADH is required to induce catalytically adequate bonding interactions between enzyme and aromatic aldehydes. The effect of reduced coenzyme in this respect is attributed to hydrophobic interactions leading to dehydration of the active-site region, which allows aldehyde substrates to compete successfully with water for inner-sphere coordination to the catalytic zinc ion. Oxidized coenzyme is proposed to have a similar promoting effect on metal coordination of aldehydes which function as substrates for the dismutase activity of the enzyme.     

Mutational analysis of the zinc- and substrate-binding sites in the CphA metallo-beta-lactamase from Aeromonas hydrophila

The subclass B2 CphA (Carbapenemase hydrolysing Aeromonas) beta-lactamase from Aeromonas hydrophila is a Zn(2+)-containing enzyme that specifically hydrolyses carbapenems. In an effort to evaluate residues potentially involved in metal binding and/or catalysis (His(118), Asp(120), His(196) and His(263)) and in substrate specificity (Val(67), Thr(157), Lys(224) and Lys(226)), site-directed mutants of CphA were generated and characterized. Our results confirm that the first zinc ion is in interaction with Asp(120) and His(263), and thus is located in the 'cysteine' zinc-binding site. His(118) and His(196) residues seem to be interacting with the second zinc ion, as their replacement by alanine residues has a negative effect on the affinity for this second metal ion. Val(67) plays a significant role in the binding of biapenem and benzylpenicillin. The properties of a mutant with a five residue (LFKHV) insertion just after Val(67) also reveals the importance of this region for substrate binding. This latter mutant has a higher affinity for the second zinc ion than wild-type CphA. The T157A mutant exhibits a significantly modified activity spectrum. Analysis of the K224Q and N116H/N220G/K224Q mutants suggests a significant role for Lys(224) in the binding of substrate. Lys(226) is not essential for the binding and hydrolysis of substrates. Thus the present paper helps to elucidate the position of the second zinc ion, which was controversial, and to identify residues important for substrate binding.     

Inhibitors of the FEZ-1 metallo-beta-lactamase

Metallo-beta-lactamases (MBLs) catalyze the hydrolysis of beta-lactams including penicillins, cephalosporins and carbapenems. Starting from benzohydroxamic acid (1) structure-activity studies led to the identification of selective inhibitors of the FEZ-1 MBL, e.g., 2,5-substituted benzophenone hydroxamic acid 17 has a K(i) of 6.1+/-0.7microM against the FEZ-1 MBL but does not significantly inhibit the IMP-1, BcII, CphA or L1 MBLs.     

Coordination chemical studies on metalloenzymes. II. Kinetic behavior of various types of chelating agents towards bovine carbonic anhydrase.

In order to investigate the kinetics and mechanism of the removal of zinc ions from bovine carbonic anhydrase [EC 4.2.1.1] (BCA), several chelating agents with various stability constants were used to remove zinc from BCA. The second-order rate constants (kaap) of zinc removal from BCA were found to be in the following order; 2,6-pyridinedicarboxylic acid greater than 2-pyridinecarboxylic acid greater than 2,4-pyridinedicarboxylic acid greater than 2,3-pyridinedicarboxylic acid greater than or approximately 1,10-phenanthroline greater than or approximately 5-methyl-1,10-phenanthroline greater than 2,2'-bipyridine. With similar chelating agents the greater the stability constant, the faster was the rate of removal of zinc ions from BCA. With EDTA, trans-1,2-cyclohexanediaminetetraacetic acid, and nitrilotriacetic acid, the rate of zinc ion removal from the native enzyme was governed by the rate of spontaneous dissociation of zinc enzyme. The rate constants for the removal of zinc ions from BCA were governed by the affinity of the chelating agents for the metal ion and the conformation of the chelating agents. Based on these findings, reaction pathways for various chelating agents are proposed.     

Crystal Structure of Serratia fonticola Sfh-I: Activation of the Nucleophile in Mono-Zinc Metallo-β-Lactamases

Metallo-β-lactamases (MBLs) or class B β-lactamases are zinc-dependent enzymes capable of inactivating almost all classes of β-lactam antibiotics. To date, no MBL inhibitors are available for clinical use. Of the three MBL subclasses, B2 enzymes, unlike those from subclasses B1 and B3, are fully active with one zinc ion bound and possess a narrow spectrum of activity, hydrolyzing carbapenem substrates almost exclusively. These remain the least studied MBLs. Sfh-I, originally identified from the aquatic bacterium Serratia fonticola UTAD54, is a divergent member of this group. Previous B2 MBL structures, available only for the CphA enzyme from Aeromonas hydrophila, all contain small molecules bound in their active sites. In consequence, the mechanism by which these enzymes activate the water nucleophile required for β-lactam hydrolysis remains to be unambiguously established. Here we report crystal structures of Sfh-I as a complex with glycerol and in the unliganded form, revealing for the first time the disposition of water molecules in the B2 MBL active site. Our data indicate that the hydrolytic water molecule is activated by His118 rather than by Asp120 and/or zinc. Consistent with this proposal, we show that the environment of His118 in B2 MBLs is distinct from that of the B1 and B3 enzymes, where this residue acts as a zinc ligand, and offer a structure-based mechanism for β-lactam hydrolysis by these enzymes.     

On the competition for available zinc

Extended x-ray absorption fine structure (EXAFS) spectroscopy was combined with thermodynamic and kinetic approaches to investigate zinc binding to a zinc finger (C2H2) and a tetrathiolate (C4) peptide. Both peptides represent structural zinc sites of proteins and rapidly bind a single zinc ion with picomolar dissociation constants. In competition with EDTA the transfer of peptide-bound zinc ions proved to be 6 orders of magnitude faster than predicted for a dissociation-association mechanism thus requiring ligand exchange mechanisms via peptide-zinc-EDTA complexes. EXAFS spectra of C2H2 showed the expected Cys2His2-ligand geometry when fully loaded with zinc. For a 2-fold excess of peptide, however, the existence of zinc-bridged peptide-peptide complexes with dominating sulfur coordination could be clearly shown. Whereas zinc binding kinetics of C2H2 appeared as a simple second order process, the suggested mechanism for C4 comprises a zinc-bridged Zn-(C4)2 species as well as a Zn-C4 species with less than 4 metal-bound thiolates, which is supported by EXAFS results. A rapid equilibrium of bound and unbound states of individual ligands might explain the kinetic instability of zinc-peptide complexes, which enables fast ligand exchange during the encounter of occupied and unoccupied acceptor sites. Depending on relative concentrations and stabilities, this results in a rapid transfer of zinc ions in the virtual absence of free zinc ions, as seen for the zinc transfer to EDTA, or in the formation of zinc-bridged complexes, as seen for both peptides with excess of peptides over available zinc.     

Characterization of purified New Delhi metallo-β-lactamase-1

New Delhi metallo-β-lactmase-1 (NDM-1) has recently emerged as a global threat because of its ability to confer resistance to almost all clinically used β-lactam antibiotics, its presence within an easily transmissible plasmid bearing a number of other antibiotic resistance determinants, its carriage in a variety of enterobacteria, and its presence in both nosocomial and community-acquired infections. To improve our understanding of the molecular basis of this threat, NDM-1 was purified and characterized. Recombinant NDM-1 bearing its native leader sequence was expressed in Escherichia coli BL21 cells. The major processed form found to be released into culture media contains a 35-residue truncation at the N-terminus. This form of NDM-1 is monomeric and can be purified with 1.8 or 1.0 equiv of zinc ion, depending on the experimental conditions. Treatment of dizinc NDM-1 with EDTA results in complete removal of both zinc ions, but the relatively weaker chelator PAR chelates only 1 equiv of zinc ion from folded protein but 1.9 equiv of zinc ion from denatured protein, indicating different affinities for each metal binding site. UV-vis spectroscopy of the dicobalt metalloform along with molecular dynamics simulations of the dizinc metallo form indicates that the dinuclear metal cluster at the active site of NDM-1 is similar in structure to other class B1 metallo-β-lactamases. Supplementation of excess zinc ions to monozinc NDM-1 has differential effects on enzyme activity with respect to three different classes of β-lactam substrates tested, penems, cephems, and carbapenems, and likely reflects dissimilar contributions of the second equivalent of metal ion to the catalysis of the hydrolysis of these substrates. Fits to these concentration dependencies are used to approximate the K(d) value of the more weakly bound zinc ion (2 μM). NDM-1 achieved maximal activity with all substrates tested when supplemented with approximately 10 μM ZnSO(4), displaying k(cat)/K(M) values ranging from 1.4 × 10(6) to 2.0 × 10(7) M(-1) s(-1), and a slight preference for cephem substrates. This work provides a foundation for an improved understanding of the molecular basis of NDM-1-mediated antibiotic resistance and should allow more quantitative studies to develop targeted therapeutics.     

The variation of catalytic efficiency of Bacillus cereus metallo-beta-lactamase with different active site metal ions

The kinetics and mechanism of hydrolysis of the native zinc and metal substituted Bacillus cereus (BcII) metallo-beta-lactamase have been investigated. The pH and metal ion dependence of k(cat) and k(cat)/K(m), determined under steady-state conditions, for the cobalt substituted BcII catalyzed hydrolysis of cefoxitin, cephaloridine, and cephalexin indicate that an enzyme residue of apparent pK(a) 6.3 +/- 0.1 is required in its deprotonated form for metal ion binding and catalysis. The k(cat)/K(m) for cefoxitin and cephalexin with cadmium substituted BcII is dependent on two ionizing groups on the enzyme: one of pK(a1) = 8.7 +/- 0.1 required in its deprotonated form and the other of pK(a2) = 9.3 +/- 0.1 required in its protonated form for activity. The pH dependence of the competitive inhibition constant, K(i), for CdBcII with l-captopril indicates that pK(a1) = 8.7 +/- 0.1 corresponds to the cadmium-bound water. For the manganese substituted BcII, the pH dependence of k(cat)/K(m) for benzylpenicillin, cephalexin, and cefoxitin similarly indicated the importance of two catalytic groups: one of pK(a1) = 8.5 +/- 0.1 which needs to be deprotonated and the other of pK(a2) = 9.4 +/- 0.1 which needs to be protonated for catalysis; the pK(a1) was assigned to the manganese-bound water. The rate was metal ion concentration dependent at the highest manganese concentrations used (10(-)(3) M). The metal substituted species have similar or higher catalytic activities compared with the zinc enzyme, albeit at pHs above 7. Interestingly, with cefoxitin, a very poor substrate for ZnBcII, both k(cat) and k(cat)/K(m) increase with increasing pK(a) of the metal-bound water, in the order Zn < Co < Mn < Cd. A higher pK(a) for the metal-bound water for cadmium and manganese BCII leads to more reactive enzymes than the native zinc BcII, suggesting that the role of the metal ion is predominantly to provide the nucleophilic hydroxide, rather than to act as a Lewis acid to polarize the carbonyl group and stabilize the oxyanion tetrahedral intermediate.     

Excess zinc ions are a competitive inhibitor for carboxypeptidase A

The mechanism for inhibition of enzyme activity by excess zinc ions has been studied by kinetic and equilibrium dialysis methods at pH 8.2, I = 0.5 M. With carboxypeptidase A (bovine pancreas), peptide (carbobenzoxyglycyl-L-phenylalanine and hippuryl-L-phenylalanine) and ester (hippuryl-L-phenyl lactate) substrates were inhibited competitively by excess zinc ions. The Ki values for excess zinc ions with carboxypeptidase A at pH 8.2 are all similar [Ki = (5.2-2.6) X 10(-5) M]. The apparent constant for dissociation of excess zinc ions from carboxypeptidase A was also obtained by equilibrium dialysis at pH 8.2 and was 2.4 X 10(-5) M, very close to the Ki values above. With arsanilazotyrosine-248 carboxypeptidase A ([(Azo-CPD)Zn]), hippuryl-L-phenylalanine, carbobenzoxyglycyl-L-phenylalanine, and hippuryl-L-phenyl lactate were also inhibited with a competitive pattern by excess zinc ions, and the Ki values were (3.0-3.5) X 10(-5) M. The apparent constant for dissociation of excess zinc ions from arsanilazotyrosine-248 carboxypeptidase A, which was obtained from absorption changes at 510 nm, was 3.2 X 10(-5) M and is similar to the Ki values for [(Azo-CPD)Zn]. The apparent dissociation and inhibition constants, which were obtained by inhibition of enzyme activity and spectrophotometric and equilibrium dialysis methods with native carboxypeptidase A and arsanilazotyrosine-248 carboxypeptidase A, were almost the same. This agreement between the apparent dissociation and inhibition constants indicates that the zinc binding to the enzymes directly relates to the inhibition of enzyme activity by excess zinc ions. Excess zinc ions were competitive inhibitors for both peptide and ester substrates.(ABSTRACT TRUNCATED AT 250 WORDS)     

Subunit interactions of nerve growth factor: Sedimentation analysis of the dissociation of the 7 S oligomer promoted by salt and ethylenediaminetetraacetate

The dissociation of the 7 S oligomer of nerve growth factor prepared from mouse submaxillary gland has been studied by sedimentation velocity as a function of added NaCl and/or EDTA at pH 6.8 in phosphate buffer. Dilution with or without EDTA results in a symmetrical dissociation to the 4.5 S protomer, in agreement with previous work. In the presence of increasing NaCl concentration the 7 S nerve growth factor oligomer undergoes limited dissociation which is characterized by complex boundary formation and the presence of a stable intermediate (weight-average s_{20, w} for the system of 4. 1 S at 2 n NaCl). The dissociation mode is probably asymmetrical in NaCl with the system resulting in an equilibrium mixture of γ and α₂β complex (s_20,w about 4.7 S). The removal of zinc ion by EDTA causes only a small change in the native equilibrium but destabilizes the complex with respect to salt-mediated dissociation, leading to complete dissociation to subunits at relatively low concentrations of NaCl. Zinc ion also promotes reassociation of mixtures of isolated α + β or β + γ subunits. Thus, a structural role of zinc ion in stabilizing subunit interactions, probably α ? β or β ? γ, is proposed. The specificity of the interactions with zinc ion and the specificity of the ionic interactions stabilizing the oligomer are further evidence for a biological specificity, if not function, of the oligomer.     

Calcium-binding properties of wild-type and EF-hand mutants of S100B in the presence and absence of a peptide derived from the C-terminal negative regulatory domain of p53

S100B is a dimeric Ca(2+)-binding protein that undergoes a 90 +/- 3 degrees rotation of helix 3 in the typical EF-hand domain (EF2) upon the addition of calcium. The large reorientation of this helix is a prerequisite for the interaction between each subunit of S100B and target proteins such as the tumor suppressor protein, p53. In this study, Tb(3+) was used as a probe to examine how binding of a 22-residue peptide derived from the C-terminal regulatory domain of p53 affects the rate of Ca(2+) ion dissociation. In competition studies with Tb(3+), the dissociation rates of Ca(2+) (k(off)) from the EF2 domains of S100B in the absence and presence of the p53 peptide was determined to be 60 and 7 s(-)(1), respectively. These data are consistent with a previously reported result, which showed that that target peptide binding to S100B enhances its calcium-binding affinity [Rustandi et al. (1998) Biochemistry 37, 1951-1960]. The corresponding Ca(2+) association rate constants for S100B, k(on), for the EF2 domains in the absence and presence of the p53 peptide are 1.1 x 10(6) and 3.5 x 10(5) M(-)(1) s(-)(1), respectively. These two association rate constants are significantly below the diffusion control ( approximately 10(9) M(-)(1) s(-)(1)) and likely involve both Ca(2+) ion association and a Ca(2+)-dependent structural rearrangement, which is slightly different when the target peptide is present. EF-hand calcium-binding mutants of S100B were engineered at the -Z position (EF-hand 1, E31A; EF-hand 2, E72A; both EF-hands, E31A + E72A) and examined to further understand how specific residues contribute to calcium binding in S100B in the absence and presence of the p53 peptide.     

Analysis of the non-covalent interaction between metal ions and the cysteine-rich domain of protein kinase C eta by electrospray ionization mass spectrometry

Effect of zinc and other metal ions on the folding of the protein kinase C (PKC) surrogate peptide (PKCeta-C1B) was analyzed intact under neutral conditions by electrospray ionization mass spectrometry (ESI-MS). ESI-MS spectrum of 64ZnCl(2)-folded PKCeta-C1B clearly showed that PKCeta-C1B coordinates specifically two atoms of zinc, and that the two thiol protons are lost in each zinc ion coordinate center. 113CdCl(2)-folded PKCeta-C1B also showed stoichiometry of two cadmium atoms that was proved by addition of EDTA. The dissociation constants of zinc- and cadmium-folded PKCeta-C1B in the phorbol 12,13-dibutyrate binding (PDBu) were similar (0.66 and 0.81 nM) with different B(max) values (46.4 and 71.4%). The difference would reflect higher coordination potency of cadmium ion that was demonstrated by ESI-MS when PKCeta-C1B was folded by 1:1 mixture of zinc and cadmium ions. In contrast, 63CuCl(2)-treated PKCeta-C1B did not show any copper-coordinated peak, instead a molecular mass less than 6 mass units smaller than that of apo-PKCeta-C1B was observed. The multiple charge mass envelope of copper-treated PKCeta-C1B shifted to that of the lower mass charge state like zinc-treated PKCeta-C1B. These data suggest that the copper treatment formed three intramolecular S-S bonds to abolish the PDBu binding of PKCeta-C1B.     

Loss of enzyme activity during turnover of the Bacillus cereus β-lactamase catalysed hydrolysis of β-lactams due to loss of zinc ion

Metallo-beta-lactamases are zinc-ion-dependent and are known to exist either as mononuclear or as dinuclear enzymes. The kinetics and mechanism of hydrolysis of the native zinc Bacillus cereus metallo-beta-lactamase (BcII) have been investigated under pre-steady-state conditions at different pHs and zinc-ion concentrations. Biphasic kinetics are observed for the hydrolysis of cefuroxime and benzylpenicillin with submicromolar concentrations of enzyme and zinc. The initial burst of product formation far exceeds the concentration of enzyme and the subsequent slower rate of hydrolysis is attributed to a branched kinetic pathway. The pH and metal-ion dependence of the microscopic rate constants of this branching were determined, from which it is concluded that two enzyme species with different metal-to-enzyme stoichiometries are formed during catalytic turnover. The dizinc enzyme is responsible for the fast route but during the catalytic cycle it slowly loses the less tightly bound zinc ion via the branching route to give an inactive monozinc enzyme; the latter is only catalytic following the uptake of a second zinc ion. The rate constant for product formation from the dinuclear enzyme and the branching rate constant show a sigmoidal dependence on pH indicative of important ionizing groups with pK (a)s of 9.0 +/- 0.1 and 8.2 +/- 0.1, respectively. The rate constant for the regeneration of enzyme activity depends on zinc-ion concentration. This unusual behaviour is attributed to an intrinsic property of metallo hydrolytic enzymes that depend on a metal bound water both as a ligand for the second metal ion and as the nucleophile which is consumed during hydrolysis of the substrate and so has to be replaced to maintain the catalytic cycle.     

Nicotinamide adenine dinucleotide-dependent and nicotinamide adenine dinucleotide-independent lactate dehydrogenases in homofermentative and heterofermentative lactic acid bacteria

Three homofermentative (Lactobacillus plantarum B38, L. plantarum B33, Pediococcus pentosaceus B30) and three heterofermentative (Leuconostoc mesenteroides 39, L. oenos B70, Lactobacillus brevis) lactic acid bacteria were examined for the presence or absence of nicotinamide adenine dinucleotide (NAD)-dependent and NAD-independent d- and l-lactate dehydrogenases. Two of the six strains investigated, P. pentosaceus and L. oenos, did not exhibit an NAD-independent enzyme activity capable of reducing dichlorophenol indophenol. The pH optima of the lactic dehydrogenases were determined. The NAD-dependent enzymes from homofermentative strains exhibited optima at pH 7.8 to 8.8, whereas values from 9.0 to 10.0 were noted for these enzymes from heterofermentative organisms. The optima for the NAD-independent enzymes were between 5.8 and 6.6. The apparent Michaelis-Menten constants determined for both NAD and the substrates demonstrated the existence of a greater affinity for d- than l-lactic acid. A comparison of the specific NAD-dependent and NAD-independent lactate dehydrogenase activities revealed a direct correlation of the d/l ratios of these activities with the type of lactic acid produced during the growth of the organism.     

The role of zinc in Bacillus subtilis cytidine deaminase

Cytidine deaminase (CDA) from Bacillus subtilis is a zinc-containing enzyme responsible for the hydrolytic deamination of cytidine to uridine and 2'-deoxycytidine to 2'-deoxyuridine. Titration of the cysteinyl groups of the enzyme with p-hydroxymercuriphenyl sulfonate (PMPS) resulted in release of one zinc ion per subunit. Addition of EDTA to chelate the zinc and dithiothreitol (DTT) to remove PMPS, followed by removal of the low molecular weight compounds by gel filtration, resulted in an apoenzyme with no enzymatic activity. The apoenzyme was almost fully reactivated by addition of zinc chloride, indicating that the zinc ion played a central role in catalysis, in keeping with what has been observed with Escherichia coli CDA [Betts, L., Xiang, S., Short, S. A., Wolfenden, R., and Carter, C. W. J. (1994) J. Mol. Biol. 235, 635-656]. Addition of Cd(2+) or Co(2+) caused partial reactivation of the apoenzyme. Zinc reconstitution of the apoenzyme was strictly dependent on the presence of reducing agents, suggesting that the zinc-ligating cysteines, when unligated, participated in disulfide bond formation. An enzymatically active isoform of the tetrameric CDA protein, containing an extension of 13 amino acids at the C-terminus of each subunit, was used in conjunction with the wild-type CDA in subunit-subunit dissociation studies to show that the zinc ion does not assist in the thermodynamic refolding of the protein. After treatment with PMPS and EDTA, the enzyme existed as unfolded unassociated subunits. Immediately following DTT addition to remove PMPS, the subunits refolded into a tetrameric structure, independent of the presence of zinc.     

Metal binding to angiotensin converting enzyme: implications for the metal binding site

Angiotensin converting enzyme interacts with the chelator, 1,10-phenanthroline (OP) to form an OP-Zn-ACE ternary complex, which subsequently dissociates to OP-Zn and apoenzyme. The association and dissociation rate constants for the reaction OP + Zn-ACE in equilibrium OP-Zn-ACE have been determined and compared with those of known OP-metal complexes. Such constants were also used to calculate the rate constant for formation of the OP-Zn complex from OP-Zn-ACE. The rate of dissociation of zinc from ACE has been measured in the presence of EDTA (which acts only as a metal scavenger) as a function of chelator concentration, at different pH values, and with different buffers. The stability constant for the binding of zinc to apoACE log Kc = 8.2, determined by equilibrium dialysis using atomic absorption spectroscopy to assess metal concentration, is much smaller than that for Zn-carboxypeptidase A. Zn-thermolysin, or Zn-carbonic anhydrase. This weak binding is attributable to the zinc dissociation rate constant of ACE, 7.5 X 10(-3) sec-1 at pH 7.0, which is much greater than that of the other zinc metalloenzymes. These results lead to inferences regarding the metal binding site of ACE.