Metal-directed affinity labeling of zinc(II), cobalt(II) and cadmium(II) horse liver alcohol dehydrogenases

The active site metal in horse liver alcohol dehydrogenase has been studied by metal-directed affinity labeling of the native zinc(II) enzyme and that substituted with cobalt(II) or cadmium(II). Reversible binding of bromoimidazolyl propionic acid to the cobalt enzyme blueshifts the visible absorption band originating from the catalytic cobalt atom at 655 to 630 nm. Binding of imidazole to the cobalt(II) enzyme redshifts the 655 nm band to 667 nm. Addition of bromoimidazolyl propionic acid blueshifts this 667 nm band back to 630 nm. This proves direct binding of the label to the active site metal in competition with imidazole. The affinity of the label for the reversible binding site in the three enzymes follows the order Zn ? Cd ? Co. After reversible complex formation, bromoimidazolyl propionic acid alkylates cysteine-46, one of the protein ligands to the active site metal. The nucleophilic reactivity of this metal-mercaptide bond in each reversible complex follows the order Co ? Zn ? Cd.     

Divalent metal derivatives of the hamster dihydroorotase domain.

Dihydroorotase (DHOase, EC 3.5.2.3) is a zinc enzyme that catalyzes the reversible cyclization of N-carbamyl-L-aspartate to L-dihydroorotate in the third reaction of the de novo pathway for biosynthesis of pyrimidine nucleotides. The recombinant hamster DHOase domain from the trifunctional protein, CAD, was overexpressed in Escherichia coli and purified. The DHOase domain contained one bound zinc atom at the active site which was removed by dialysis against the chelator, pyridine-2,6-dicarboxylate, at pH 6.0. The apoenzyme was reconstituted with different divalent cations at pH 7.4. Co(II)-, Zn(II)-, Mn(II)-, and Cd(II)-substituted DHOases had enzymic activity, but replacement with Ni(2+), Cu(2+), Mg(2+), or Ca(2+) ions did not restore activity. Atomic absorption spectroscopy showed binding of one Co(II), Zn(II), Mn(II), Cd(II), Ni(II), or Cu(II) to the enzyme, while Mg(II) and Ca(II) were not bound. The maximal enzymic activities of the active, reconstituted DHOases were in the following order: Co(II) --> Zn(II) --> Mn(II) --> Cd(II). These metal substitutions had major effects upon values for V(max); effects upon the corresponding K(m) values were less pronounced. The pK(a) values of the Co(II)-, Mn(II)-, and Cd(II)-substituted enzymes derived from pH-rate profiles are similar to that of Zn(II)-DHOase, indicating that the derived pK(a) value of 6.56 obtained for Zn-DHOase is not due to ionization of an enzyme-metal aquo complex, but probably a histidine residue at the active site. The visible spectrum of Co(II)-substituted DHOase exhibits maxima at 520 and 570 nm with molar extinction coefficients of 195 and 210 M(-1) cm(-1), consistent with pentacoordination of Co(II) at the active site. The spectra at high and low pH are different, suggesting that the environment of the metal binding site is different at these pHs where the reverse and forward reactions, respectively, are favored.     

Active site-specifically reconstituted nickel(II) horse liver alcohol dehydrogenase: Optical spectra of binary and ternary complexes with coenzymes,coenzyme analogues,substrates, and inhibitors

Insertion of nickel ions into the empty catalytic site of horse liver alcohol dehydrogenase yields an active enzyme with 65% metal substitution and about 12% intrinsic activity. The electronic absorption spectrum is characterized by bands at 357 nm (2900 M^?1 cm^?1, 407 nm (3500 M^?1 cm^?1), 505 nm (300 M^?1 cm^?1), 570 nm (?130 M^?1 cm^?1), and 680 nm (?80 M^?1 cm^?1). The absorption and CD spectra are similar to those of nickel(II) azurin and nickel(II) aspartate transcarbamoylase and prove coordination of the nickel(II) ions to sulfur in a distorted tetrahedral coordination geometry. Changes of the spectra upon ligand binding at the metal or conformation changes of the protein induced by coenzyme, or both, indicate alterations of the metal geometry.The chromophoric substrate trans-4-(N, N-dimethylamino)-cinnamaldehyde forms a ternary complex with Ni(II) liver alcohol dehydrogenase and the coenzyme analogue 1,4,5,6-tetrahydronicotinamide-adenine-dinucleotide, stable between pH 6 and 10. The corresponding ternary complex with NADH is only stable at pH > 9.0. The spectral redshifts induced in the substrate are 11 nm larger than those found in the zinc enzyme. We suggest direct coordination of the substrate to the catalytic metal ion which acts as a Lewis acid in both substrate coordination and catalysis.     

A spectroscopic study of metal ion and ligand binding to β-lactamase II

β-Lactamase II has two metal-binding sites. The electronic spectra of Cd(II)- and Co(II)-substituted β-lactamase II have been investigated. It is suggested that a thiol ligand is involved in metal binding at the first site. The stoichiometric dissociation constants for Co(II) binding to β-lactamase II were estimated to be 0.13 and 2.66 mM (pH 6.0, 4°C, 1 M NaCl) by equilibrium dialysis. Competition between Zn(II) and Co(II) for the first metal binding site suggests a value of 0.7 μM (pH 6.0, 30°C, 1 M NaCl) for the dissociation constant o Zn(II).The electronic spectra of the Co(II) enzyme lead to the suggestion that the coordination geometries around the metal ions in the first and second sites are related to those of a distorted tetrahedron and octahedron, respectively.     

Reaction of N,N-diethyldithiocarbamate and other bidentate ligands with Zn, Co and Cu bovine carbonic anhydrases. Inhibition of the enzyme activity and evidence for stable ternary enzyme-metal-ligand complexes

The reactions with N,N-diethyldithiocarbamate (DDC) of zinc, cobalt and copper carbonic anhydrase from bovine erythrocytes were investigated. The native zinc enzyme was inhibited by DDC, but no removal of zinc could be detected even at a very high [ligand]/[protein] ratio. At identical pH values a larger inhibitory effect was found for the cobalt enzyme. The metal was removed by DDC from the protein at pH less than 7.0. No cobalt removal occurred at pH 10, where a stable ternary complex with the enzyme-bound Co(II) was detected. Its optical and EPR spectra are indicative of five-coordinate Co(II). The reaction of the Cu(II) enzyme with stoichiometric chelating agent was marked by the appearance of an electronic transition at 390 nm (epsilon = 4300 M-1 X cm-1). Metal removal from the copper enzyme readily occurred as the ligand was in excess over the metal, with parallel appearance of a band at 440 nm, which was attributed to the free Cu(II)-DDC complex. Also, in the case of the copper enzyme an alkaline pH was found to stabilize the ternary adduct with the diagnostic 390 nm band. EPR spectra showed that the ternary adduct is a mixture of two species, both characterized by the presence in the EPR spectrum of a superhyperfine structure from two protein nitrogens and by a low g parallel value, indicative of coordination to sulfur ligands. It is suggested that the two species contain the metal as penta- and hexacoordinated, respectively. Measurements of the longitudinal relaxation time, T1, of the water protons suggested that water coordination is retained in the latter case. Hexacoordination with retention of water is also proposed for the Cu(II) derivatives with the bidentate oxalate and bicarbonate anions, unlike the corresponding Co(II) derivatives, which are pentacoordinated. Different coordination of Co(II) and Cu(II) adducts may be relevant to the difference of activity of the two substituted enzymes.     

Effects of two ionizing groups on the active site of human carbonic anhydrase B.

Investigation of some pH-dependent properties of human erythrocyte carbonic anhydrase B indicate that the active site is influenced by at least two charged groups. The properties studied include the pH dependence of inhibition of native, monocarboxamidomethyl, and monocarboxymethyl enzymes by iodide ion and the pH dependence of the visible spectra of the cobalt derivatives of these enzymes. One ionizing group has a pKa of about 7.3 in the native enzyme, 8.2 in the carboxyamidomethyl enzyme, and 9.0 in the carboxymethyl enzyme. It has a major influence on activity and anion inhibition, and on the visible spectra of the cobalt enzymes. A second group has a pKa of about 6.1 in native and modified enzymes. When zinc is at the active site, the secondary group in its acidic form decreases the Ki for I-. With the carboxyamidomethyl and carboxymethyl enzymes, the Ki decreases by about an order of magnitude. However, if cobalt is substituted for zinc in the modified enzymes, this group does not influence the Ki for I- and the binding of I- does not influence the pKa of the spectral transitions caused by ionization of this secondary group. In the case of nonalkylated Co2+-enzyme, another ionizing group with a pK of about 6.2 prevents the binging of I- at low pH. These results show that the active site is altered when cobalt is substituted for zinc in carbonic anhydrase B.     

Studies of Zn(II) and Co(II) complexes of imidazole and n-methylimidazole with regard to the activity related ionization in carbonic anhydrase.

Mixed aquo-N-methylimidazole complexes of Co(II) have been studied as a function of pH to gain a fuller understanding of the metal-binding site in Co(II)-carbonic anhydrase. The inherent affinity of N-methylimidazole for Co(II) has been calculated along with a species distribution for the stepwise addition of ligand to the metal ion. From these studies, it is apparent that the occurrence of Zn(II) rather than Co(II) in native carbonic anhydrase can be explained by the stronger affinity of Zn(II) for imidazole and the preference of Zn(II) for a tetrahedral geometry as offered by the enzyme. Octahedral Co(II) fails to ionize metal bound water. However, at high pH, Co(II)-N-methylimidazole complexes interact directly with the hydroxide ion, generating species with visible spectra very similar to that of Co(II)-carbonic anhydrase. Tentative structures have been proposed for these species.     

Chloride binding to alkaline phosphatase. 113Cd and 35Cl NMR

Chloride binding to alkaline phosphatase from Escherichia coli has been monitored by 35Cl NMR for the native zinc enzyme and by 113Cd NMR for two Cd(II)-substituted species, phosphorylated Cd(II)6 alkaline phosphatase and unphosphorylated Cd(II)2 alkaline phosphatase. Of the three metal binding sites per enzyme monomer, A, B, and C, only the NMR signal of 113Cd(II) at the A sites shows sensitivity to the presence of Cl-, suggesting that Cl- coordination occurs at the A site metal ion. From the differences in the chemical shift changes produced in the A site 113Cd resonance for the covalent (E-P) form of the enzyme versus the noncovalent (E . P) form of the enzyme, it is concluded that the A site metal ion can assume a five-coordinate form. The E-P form of the enzyme has three histidyl nitrogens as ligands from the protein to the A site metal ion plus either two water molecules or two Cl- ions as additional monodentate ligands. In the E . P form, there is a phosphate oxygen as a monodentate ligand and either a water molecule or a Cl- ion as the additional monodentate ligand. The shifts of the 113Cd NMR signals of the unphosphorylated Cd(II)2 enzyme induced by Cl- are very similar to those induced in the E-P derivative of the same enzyme, supporting the conclusion that the phosphoseryl residue is not directly coordinated to any of the metal ions. Specific broadening of the 35Cl resonance from bulk Cl- is induced by Zn(II)4 alkaline phosphatase, while Zn(II)2 alkaline phosphatase is even more effective, suggesting an influence by occupancy of the B site on the interaction of monodentate ligands at the A site. A reduction in this quadrupolar broadening is observed upon phosphate binding at pH values where E . P is formed, but not at pH values where E-P is the major species, confirming a specific interaction of Cl- at the A site, the site to which phosphate is bound in E . P, but not in E-P. For the zinc enzyme, a significant decrease in phosphate binding affinity can be shown to occur at pH 8 where one monomer has a higher affinity than the other.     

2,2-dipyridyl binding to metal substituted horse liver alcohol dehydrogenase

The binding of 2,2-dipyridyl to metal substituted horse liver alcohol dehydrogenase was measured by spectrophotometric titrations. Large changes in the visible absorption spectra were seen for the Co2+, Cu2+ and Ni2+ hybrids upon coordination of 2,2-dipyridyl, due to a change in coordination number. The formation constants for binding to the Co2+ and Cd2+ hybrids are of the order 10(6) M-1, which means that these hybrids have a 500-fold higher affinity for 2,2-dipyridyl than the native Zn2+ enzyme. 2,2-dipyridyl has a 100-fold higher affinity for enzyme bound Cd2+ than for aqueous Cd2+ ions, while for Cu2+ and Zn2+ the opposite is the case. None of the substituted metal ions were removed from the active site during titration with the chelator 2,2-dipyridyl.     

The copper-enzyme dopamine beta-monooxygenase: studies on the ability of several metals to inhibit the enzyme activity and to replace the copper

The ability of several metals to inhibit dopamine beta-monooxygenase was measured and compared with their ability to compete with the binding of 64Cu to the water-soluble form of the bovine adrenal enzyme at pH 6.0. In the presence of an optimal concentration of copper (0.5 microM in the present assay system), an inhibition was observed upon addition of Hg(II), Zn(II), or Ni(II). Only a small fraction of the inhibition with these metals may be due to uncoupling of electron transport from hydroxylation. Preincubation of these metals with the Cu-depleted apoenzyme before addition of copper, revealed a stronger inhibition than if copper was added before the other metals. Hg(II), Zn(II), and Ni(II) also compete with the binding of 64Cu(II) to the protein. Hg(II) was the most effective and Ni(II) the least effective of these metals, both with respect to inhibition of the enzyme activity and to prevent the binding of 64Cu(II). Competition experiments on the binding of Zn(II) and 64Cu in the presence and absence of ascorbate, indicated i) a similar affinity of Cu(I) and Cu(II) to the native enzyme, and ii) a more rapid binding of Cu(I) than Cu(II) to the Cu-depleted and Zn-containing enzyme. Al(III), Fe(II), Mg(II), Mn(II), Co(II), Cd(II), and Pb(II) neither inhibited the enzyme activity nor competed with the binding of 64Cu(II) to the protein (Fe(II) was not tested for binding). Of those metals cited above only Cu(II)/Cu(I) was able to reactivate the apoenzyme.     

Colorimetric and fluorimetric assays to quantitate micromolar concentrations of transition metals

Transition metal ions, although maintained at low concentrations, play diverse important roles in many biological processes. Two assays useful for the rapid quantification of a range of first-row transition metal ions have been developed. The colorimetric assay extends the 4-(2-pyridylazo)resorcinol assay of Hunt et al. (J. Biol. Chem. 255, 14793 (1984)) to measure nanomole quantities of Co(2+), Ni(2+), and Cu(2+) as well as Zn(2+). The fluorimetric assay takes advantage of the coordination of a number of metal ions (Mn(2+), Co(2+), Ni(2+), Cu(2+), Zn(2+), Cd(2+)) by Fura-2 and can also be used to measure nanomole quantities of these ions. The assays developed here have the advantage of not requiring the extensive sample preparation necessary for other methodologies, such as atomic absorption spectroscopy and inductively coupled plasma emission spectroscopy (ICPES), while being comparable in accuracy to the detection limits of ICPES for the first-row transition metal ions. To demonstrate the effectiveness of these assays, we determined the affinity of carbonic anhydrase II (CA II), a prototypical zinc enzyme, for Ni(2+) and Cd(2+). These data indicate that CA II binds transition metals with high affinity and is much more selective for Zn(2+) over Ni(2+) or Cd(2+) than most small-molecule chelators or other metalloenzymes.     

Zn(II)-113Cd(II) and Zn(II)-Mg(II) hybrids of alkaline phosphatase. 31P and 113Cd NMR

Methods have been developed for the addition of different metal ion species to the three distinct pairs of metal sites (A, B, and C) found in the dimer of apoalkaline phosphatase. This allows the preparation of hybrid alkaline phosphatases in which A and B sites of each monomer contain two different species of metal ion or the A and B sites of one monomer contain the same species of metal ion, while the adjacent monomer contains a second species. The following hybrids have been characterized in detail: (Zn(II)ACd(II)B)2 alkaline phosphatase, (Zn(II)AMg(II)B)2 alkaline phosphatase, (Cd(II)AZn(II)B)2 alkaline phosphatase, and (Zn(II)AZn(II]B)(Cd(II)ACd(II)B) alkaline phosphatase. 31P and, where appropriate, 113Cd NMR have been used to monitor the behavior of the covalent (E-P) and noncovalent (E X P) phosphointermediates and of the A and B metal ions. From the pH dependencies of the E-P in equilibrium E X P in equilibrium E + Pi equilibria, it is clear that A site metal is the dominant influence in dephosphorylation of E-P and may have a coordinated water molecule, which ionizes to ZnOH- at a low pH providing the nucleophile for dephosphorylation. A site metal also serves to coordinate phosphate in the E X P complex. B site metal has a much smaller effect on dephosphorylation rates, although it does dramatically alter the Pi dissociation rate, which is the rate-limiting step for the native enzyme at alkaline pH, and is probably important in neutralizing the charge on the phosphoseryl residue, thus potentiating the nucleophilic attack of the OH- bound at A site. Phosphate dissociation is slowed markedly by replacement of B site zinc by cadmium. There is clear evidence for long range effects of subunit-subunit interactions, since metal ion and phosphate binding at one active center alters the environments of A and B site metal ions and phosphoserine at the other active site.     

Characterization of an inhibitory metal binding site in carboxypeptidase A

The specificity of metal ion inhibition of bovine carboxypeptidase A ([(CPD)Zn]) catalysis is examined under stopped-flow conditions with use of the fluorescent peptide substrate Dns-Gly-Ala-Phe. The enzyme is inhibited competitively by Zn(II), Pb(II), and Cd(II) with apparent KI values of 2.4 x 10(-5), 4.8 x 10(-5), and 1.1 x 10(-2) M in 0.5 M NaCl at pH 7.5 and 25 degrees C. The kcat/Km value, 7.3 x 10(6) M-1 s-1, is affected less than 10% at 1 x 10(-4) M Mn(II) or Cu(II) and at 1 x 10(-2) M Co(II), Ni(II), Hg(II), or Pt(IV). Zn(II) and Pb(II) are mutually exclusive inhibitors. Previous studies of the pH dependence of Zn(II) inhibition [Larsen, K. S., & Auld, D. S. (1989) Biochemistry 28, 9620] indicated that [(CPD)Zn] is selectively inhibited by a zinc monohydroxide complex, ZnOH+, and that ionization of a ligand, LH, in the enzyme's inhibitory site (pKLH 5.8) is obligatory for its binding. The present study allows further definition of this inhibitory zinc site. The ionizable ligand (LH) is assigned to Glu-270, since specific chemical modification of this residue decreases the binding affinity of [(CPD)Zn] for Zn(II) and Pb(II) by more than 60- and 200-fold, respectively. A bridging interaction between the Glu-270-coordinated metal hydroxide and the catalytic metal ion is implicated from the ability of Zn(II) and Pb(II) to induce a perturbation in the electronic absorption spectrum of cobalt carboxypeptidase A ([(CPD)Co]).(ABSTRACT TRUNCATED AT 250 WORDS)     

Interaction of the unique competitive inhibitor imidazole and related compounds with the active site metal of carbonic anhydrase: linkage between pH effects on the inhibitor binding affinity and pH effects on the visible spectra of inhibitor complexes with the cobalt-substituted enzyme

Previous studies on the interaction of carbonic anhydrase (CA) with the unique CO2 competitive inhibitor imidazole and related compounds were all interpreted as showing that an ionizable water ligand on the metal of this zinc metalloenzyme is not displaced by inhibitor binding. Internal inconsistencies in the pH dependence of binding and the pH dependence of the visible spectra of complexes with cobalt-substituted enzyme prompted us to reinvestigate this binding. Visible spectroscopy was used to measure the binding of imidazole and 1,2,4-triazole to Co(II)-substituted human CA I and active site carboxymethylated human CA I (CmCA I) and the binding of 1,2,4-triazole to bovine CoIICA II. The limiting visible spectra for these enzyme-inhibitor adducts were also computed and examined for pH dependence. It was shown that the pKa of visible spectral changes can be independently predicted from studies on the pH dependence of binding. After consideration of possible contributions from effects of His-200 ionization in CA I and CmCA I, and His-64 in CA II, the pH effects on binding affinity and spectra were found to be of the correct magnitude to establish linkage between binding and an ionization. It was also shown, however, that pH effects on binding and spectra cannot distinguish whether neutral imidazole binds to both ionization forms of the enzyme (Zn-OH2 and Zn-OH) or whether neutral imidazole and its anion both bind to only the acid form of the enzyme, presumably after displacing the water. These findings have implications to the crystallographic interpretations on the imidazole-enzyme complex and to the catalytic mechanism of CO2 hydration.     

Cadmium-109 as a probe of the metal binding sites in horse liver alcohol dehydrogenase.

The noncatalytic and catalytic zinc atoms of horse liver alcohol dehydrogenase, [(LADH)Zn2Zn2] or LADH, have been replaced differentially with 109Cd by equilibrium dialysis, resulting in two new enzymatically active species, [(LADH)109Cd2Zn2] and [(LADH)109Cd2109Cd2]. The UV difference spectra of the cadmium enzymes vs. native [(LADH)Zn2Zn2] reveal maxima at 240 nm with molar absorptivities, delta epsilon 240, of 1.6 X 10(4) M-1 cm-1 per noncatalytic 109Cd atom and 0.9 X 10(4) M-1 cm-1 per catalytic 109Cd atom, consistent with coordination of the metals by four and two thiolate ligands, respectively, strikingly similar to the 250-nm charge-transfer absorbance in metallothionein. Carboxymethylation of the Cys-46 ligand to the catalytic metal in LADH presumably lowers the overall stability constant of the coordination complex and results in loss of catalytic 109Cd or catalytic cobalt but not catalytic zinc from the enzyme.     

Proton magnetic resonance studies of carbonic anhydrase. II. Group controlling catalytic activity.

The seven resonances observed in the histidine region of the proton magnetic resonance (pmr) spectrum of human carbonic anhydrase B and reported in the preceding paper are studied in the presence of sulfonamide, azide, cyanide, and chloride inhibitors and in metal-free, cadmium substituted, cobalt substituted, and carboxymethylated forms of the enzyme. Results indicate that the two resonances that move-downfield with increasing pH and the two that do not move with pH reflect residues located at the active site. The first two resonances are assigned to the same titratable histidine whose pK value of 8.24 corresponds to that of the group controlling catalytic activity. Addition of anions or sulfonamides, removal of zinc, or substitution of cadmium for zinc at the active site, procedures known to abolish enzymatic activity, prevent titration of this residue. Partial inhibition of carbonic anhydrase by chloride slectively increases the pK value of the group controlling catalytic activity and of the histidine with pK equals 8.24. Experiments with metal-free and cadmium carbonic anhydrases and comparisons with model systems suggest that this histidine is bound to the metal ion at high pH; at low pH this complex appears to dissociate as protons compete with the metal for the imidazole group. It is proposed that ionization of the group controlling catalytic activity represents loss of the pyrrole proton of this neutral ligand when it binds to Zn(II), forming an imidazolate anion and juxtaposing a strong base and a powerful Lewis acid at the active site. When bound to zinc as an anion, this histidine can act as a general base catalyst in the hydration of carbon dioxide and be replaced as a metal ligand by an oxygen of the substrate in the course of the reaction. The histidine-metal complex is thought to exist in a strained configuration in the active enzyme so that its imidazole-metal bond is readily broken on addition of substrates or inhibitors. This model is consistent with the available data on the enzyme and is discussed in relation to alternative proposals.     

In-vitro biological evaluation of some ONS and NS donor Schiff's bases and their metal complexes

Complexes of Mn(II), Co(II), Ni(II), Cu(II), Zn(II) and Cd(II) with the Schiff bases salicylidene-o-aminothiophenol (H2L) and thiophene-o-carboxaldeneaniline (SB) have been synthesized and characterized by elemental analyses, magnetic measurements, thermogravimetric analyses as well as infrared spectra and reflectance spectra. The nature of the bonding has been discussed on the basis of IR spectral data. Magnetic susceptibility measurements and electronic spectral data suggest a six-coordinated octahedral structure for these complexes. The complexes of Mn(II), Co(II), Ni(II), Cu(II) are paramagnetic, while Zn(II) and Cd(II) are diamagnetic in nature. The complexes were tested for their antimicrobial activities against Salmonella typhi, Escherichia coli and Serratia marcescens using the "Disc Diffusion Method". The results are compared with the standard drug (tetracycline) and show moderate activity.     

Carbonic Anhydrase Inhibitors: Schiff's Bases of Aromatic and Heterocyclic Sulfonamides and their Metal Complexes

Schiff's bases were obtained from aromatic/heterocyclic sulfonamides and amino-sulfonamide derivatives, such as sulfanilamide, homosulfanilamide, 4-aminoethyl-benzenesulfonamide and 5-amino-1,3,4-thiadiazole-2-sulfonamide. Metal complexes of some of these Schiff's bases, incorporating Zn(II), Co(II), Ni(II) and Cu(II) ions, were also prepared and tested as inhibitors of the zinc enzyme carbonic anhydrase (CA), and more specifically the red blood cell isozymes I and II. The Schiff's bases behaved as medium potency CA I and CA II inhibitors, whereas their metal complexes showed a highly enhanced potency, with several low nanomolar CA II inhibitors detected.     

Preference of Cd(II) and Zn(II) for the two metal sites in Bacillus cereus beta-lactamase II: A perturbed angular correlation of gamma-rays spectroscopic study

Cd-substituted forms of the Bacillus cereus metallo-beta-lactamases (BCII) were studied by perturbed angular correlation of gamma-rays (PAC) spectroscopy. At very low [Cd]:[apo-beta-lactamase] ratios, two nuclear quadrupole interactions (NQI) were detected. For [Cd]:[apo-beta-lactamase] ratios between 0.8 and 3.0, two new NQIs appear, and the spectra show that up to 2 cadmium ions can be bound per molecule of apoenzyme. These results show the existence of two interacting Cd-binding sites in BCII. The relative populations of the two NQIs found at low [Cd]:[apo-beta-lactamase] ratios yielded a 1:3 ratio for the microscopic dissociation constants of the two different metal sites (when only one cadmium ion is bound). X-ray diffraction data at pH 7.5 demonstrate that also for Zn(II) two binding sites exist, which may be bridged by a solvent molecule. The measured NQIs could be assigned to the site with three histidines as metal ligands (three-His site) and to the site with histidine, cysteine, and aspartic acid as metal ligands (Cys site), respectively, by PAC measurements on the Cys168Ala mutant enzyme. This assignment shows that cadmium ions preferentially bind to the Cys site. This is in contrast to the preference of Zn(II) in the hybrid Zn(II)Cd(II) enzyme, where an analysis of the corresponding PAC spectrum showed that Cd(II) occupied the Cys site, whereby Zn(II) occupied the site with three histidines. The difference between Zn(II) and Cd(II) in affinity for the two sites is combined with the kinetics of hydrolysis of nitrocefin for different metal ion substitutions (Zn(2)E, ZnE, Cd(2)E, CdE, and ZnCdE) to study the function of the two metal ion binding sites.     

Environment and conformation dependent sensitivity of the arsanilazotyrosine-248 carboxypeptidase A chromophore.

Reaction of carboxypeptidase A crystals with diazotized arsanilic acid uniquely modifies Tyr-248 to form a monazo derivative, which-in solution-forms an intramolecular inner-sphere coordination complex in the active site zinc atom. tarsanilazocarboxypeptidase exhibits spectral properties that are closely similar to those of the model complex, tetrazolylazo-N-carbobenzoxytyrosine Zn2+, with a distinctive maximum at 510 nm. In addition, its circular dichroic spectrum reveals a negative extremum at this wavelength, also characteristic of this complex. Both spectra are exquisitely responsive to pth changes and serve to monitor formation and dissociation of the metal-azophenol complex. Two pKapp at 7.7 and 9.5 delineate the pH range over which the probe characteristics most effectively gauge conformational features of the active center of arsanilazcarboxypeptidase. Other environmental parameters, e.g., substrates and inhibitors, as well as crystallization of the enzyme also critically influence the formation and dissociation of the complex; the response of the probe suggests that they induce conformational movement of the azoTyr-248 residue away from the zinc atom. tthe now available chemical, functional, structural data bearing on the spatial relationships of Tyr-248 and Zn, both thought critical to catalysis, are evaluated, based on spectra of arsanilazo- and nitrocarboxypeptidase crystals and solutions as well as on detailed kinetic analyses of the native enzyme in both physical states and based on the X-ray structure analysis of the native enzyme and its Gly-L-Tyr complex. Collectively all of the data show that the conformation of carboxypeptidase in crystals differs from that in solution. Moreover, reexamination of the original X-ray maps reported in 1968 and thought to preclude a Tyr-248-Zn interaction now leads to the conclusion that in up to 25 per cent of the molecules in the crystals ttyr-248 interacts with the active site zinc atom (W.D. Lipscomb (1973), Proc. Nat. Acad. Sci U.S. 70, 3797). Thus, even in the crystals the enzyme exists in at least two different conformations. In one of these Tyr-248 is near while in the other it is far from the zinc atom. The spectral effects of Gly-L-Tyr and beta-phenylpropionate on solutions of arsanilazo- and of nitrocarboxypeptidase demonstrate that during the catalytic process Tyr-248 moves away from the zinc atom. This implies a mechanistic role for Tyr-248 different from that postulated on the basis of X-ray crystallographic analysis. Indeed, the proximity of ttyr-248 to the zinc atom, when altered by substrates and inhibitor, may reflect certain of the properties characteristic of the entatic, active site.