Exchange inert Co (III)-carboxypeptidase A: a catalytically inactive metallocarboxypeptidase.

Exposure of cobalt (II) carboxypeptidase A_α, [(CPD)Co(II)], to small molar excesses of the oxidizing agent m-chloroperbenzoate rapidly destroys (< 30 sec) both its peptidase and esterase activities in parallel. Concomitantly, the characteristic Co(II) electron paramagentic resonance (EPR) signal is abolished. [(CPD)Co(III)], isolated from the reaction mixture, has the same molecular weight and amino acid composition as [(CPD)Co(II)], contains 0.95 g-atom of Co and 0.01 g-atom of Zn per mole of protein, does not exhibit an EPR spectrum and is catalytically completely inactive towards both peptide and ester substrates. Identical treatment of the native zinc enzyme affects neither its catalytic activity nor its metal content. The reaction of m-chloroperbenzoate with [(CPD)Co(II)] follows saturation kinetics and is prevented by the inhibitor β-phenylpropionate. Furthermore, under the conditions found to oxidize [(CPD)Co(II)] effectively, there is no reaction with Co(II) $\text{E. coli}$ alkaline phosphatase. Thus, m-chloroperbenzoate has the characteristics of an active-site directed oxidizing reagent for [(CPD)Co(II)].     

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)     

Carboxypeptidase A: mechanism of zinc inhibition

Zinc ions competitively inhibit carboxypeptidase A from bovine pancreas. The state(s) of hydroxylation of zinc and their possible site(s) of interaction with the enzyme have been investigated by determining the strength of zinc inhibition over pH range 4.6-10.5. The inhibition kinetics were recorded under stopped-flow conditions using the alpha-Val isozyme and the peptide substrate Dns-Gly-Ala-Phe in 0.5 M NaCl at 25 degrees C. The pH dependence of pKI follows a pattern which indicates that the enzyme is selectively inhibited by zinc monohydroxide, ZnOH+ (KI = 7.1 X 10(-7) M). The formation of the inhibitory ZnOH+ complex from fully hydrated Zn2+ is characterized by an ionization constant of 9.05, and the consecutive conversion of ZnOH+ to Zn(OH)2, Zn(OH)3-, and Zn(OH)4(2-) complexes takes place with ionization constants of 9.75, 10.1, and 10.5, respectively. Ionization of a ligand, LH, in the enzyme's inhibitory site (pKLH 5.8) is obligatory for binding of the ZnOH+ complex. The enzymatic activity (kcat/Km) is influenced by three ionizable groups: pKEH2 5.78, pKEH 8.60, and pKE 10.2. Since the values of pKLH and pKEH2 are virtually identical, it is possible that the inhibitory ZnOH+ complex interacts with the group responsible for pKEH2. Previous studies have suggested that pKEH2 reflects the ionization of Glu-270 and its interaction with a water molecule coordinated to the catalytic zinc ion. It is proposed that the inhibitory zinc ion binds to the carboxylate of Glu-270 and that the inhibition process is specific for zinc monohydroxide because it allows the formation of a stabilizing hydroxide bridge between the inhibitory and catalytic zinc ions.     

Spectral properties of cobalt carboxypeptidase A. Interaction of the metal atom with anions

At pH greater than 7 the absorption and magnetic circular dichroic spectra of cobalt carboxypeptidase A are insensitive to anions [Latt, S. A., & Vallee, B. L. (1971) Biochemistry 10, 4263-4270], but at pH less than 6 chloride and other anions perturb them in a manner specific for each anion. Lowering of the pH apparently facilitates the entry of an anion into the metal coordination sphere, suggesting that an acidic group normally stabilizes a metal-coordinated water molecule against displacement. The lack of sensitivity to anions at pHs between 7 and 9--when the enzyme is maximally active--and its evident abolition upon protonation of an active-site group are consistent with this interpretation. Selective modification of cobalt carboxypeptidase at Glu-270 using a carbodiimide affinity reagent generates sensitivity to anions at pH 7 very similar to that of the unmodified enzyme at pH approximately 5. This suggests that the group stabilizing the metal-coordinated water is the catalytically essential carboxylate of Glu-270. These and related results provide evidence for a mechanistically important interaction of Glu-270 with a metal-bound water molecule.     

Allosteric negative regulation of smt O/P binding of the zinc sensor,SmtB, by metal ions: a coupled equilibrium analysis

The Synechococcus PCC 7942 smt operon is responsible for cellular resistance to excess zinc and consists of two divergently transcribed genes, smtB and smtA. SmtB is the Zn(II)-sensing metal-regulated repressor of the system and binds to a 12-2-12 imperfect inverted repeat in the smtA O/P region. Using fluorescence anisotropy to monitor SmtB-smt O/P multiple equilibria, we show that four SmtB homodimers bind to a 40 bp oligonucleotide containing a single 12-2-12 inverted repeat. The binding affinities of the first two dimers are very tight (K(int) = 2.9 x 10(9) M(-1)) with the affinities of the third and fourth dimers lower by approximately 10- and approximately 30-fold, respectively. A single monomer equivalent of Zn(II), Cd(II), or Co(II) promotes disassembly of the oligomeric complex to a mixture of (P(2)).D and (P(2))(2).D SmtB dimer-DNA complexes with the intrinsic affinity of all SmtB homodimers for DNA greatly reduced by approximately 500-2000-fold. Substitution or derivatization of cysteines which comprise the alpha3N metal binding site (Cys14 and Cys61) [VanZile, M. L., et al. (2002) Biochemistry 41, 9765-9775] has no effect on allosteric negative regulation by Zn(II); in contrast, H106Q SmtB, harboring a single zinc-liganding substitution in the alpha5 metal binding site, is refractory to zinc-induced disassembly of SmtB-DNA complexes. The alpha5 metal binding sites are therefore regulatory for Zn(II) sensing in vitro and in vivo, while the high-affinity alpha3N sites play some other role. This finding for SmtB is the opposite of that previously determined for Staphylococcus aureus pI258 CadC, a Pb(II)/Cd(II)/Bi(III) sensor [Busenlehner, L. S., et al. (2002) J. Mol. Biol. 319, 685-701], thus providing insight into the origin of functional metal ion selectivity in this family of metal sensor proteins.     

Toxic effects of mercury(II), cadmium(II) and lead(II) porphyrins on Trypanosoma brucei brucei growth

The effects of free mercury(II), cadmium(II) and lead(II) ions and their metalloporphyrin-derivatives on Trypanosoma brucei brucei growth in culture were studied. All experiments were conducted in the dark. IC(50) values on growth obtained in 24-h time-course experiments were 1.5 x 10(-7), 2.4 x 10(-6), 4.4 x 10(-6) and 2.6 x 10(-5) M for mercury(II) porphyrin, cadmium(II) porphyrin, lead(II) porphyrin and free base porphyrin, respectively. While the IC50 values for Hg2+, Cd2+ and Pb2+ were 3.6 x 10(-6), 1.5 x 10(-5) and 1.6 x 10(-5) M, respectively. These results clearly indicate that the toxicity of the metalloporphyrin complexes of mercury(II), cadmium(II) and lead(II) to T. b. brucei parasites was much higher compared to their free metal ions and free base porphyrin at low concentrations. It was also observed after 8 h incubation that the metalloporphyrins were effective in inhibiting the division of the parasites at concentrations >1.25 x 10(-7) M for mercury(II) porphyrin, concentrations >1.2 x 10(-6) M for cadmium(II) and lead(II) porphyrins and at concentrations >3.6 x 10(-6) M for Hg2+ ion. These observations were not detected in samples treated with the free metal ions and the free base porphyrin at the same concentrations. Interestingly, trypanosomes treated with metalloporphyrin complexes displayed different morphological features from those cells treated with free base porphyrin or metal ions. The chemotherapeutic potential of the metalloporphyrins of H2TMPyP for treatment of African trypanosomiasis is discussed.     

Spectroscopic properties of the metalloregulatory Cd(II) and Pb(II) sites of S. aureus pI258 CadC

Staphylococcus aureus pI258 CadC is an extrachromosomally encoded metalloregulatory repressor protein from the ArsR superfamily which negatively regulates the expression of the cad operon in a metal-dependent fashion. The metalloregulatory hypothesis holds that direct binding of thiophilic divalent cations including Cd(II), Pb(II), and Zn(II) by CadC allosterically regulates the DNA binding activity of CadC to the cad operator/promoter (O/P). This report presents a detailed characterization of the metal binding and DNA binding properties of wild-type CadC. The results of analytical ultracentrifugation experiments suggest that both apo- and Cd(1)-CadC are stable or weakly dissociable homodimers characterized by a K(dimer) = 3.0 x 10(6) M(-1) (pH 7.0, 0.20 M NaCl, 25.0 degrees C) with little detectable effect of Cd(II) on the dimerization equilibrium. As determined by optical spectroscopy, the stoichiometry of Cd(II) and Pb(II) binding is approximately 0.7-0.8 mol/mol of wild-type CadC monomer. Chelator (EDTA) competition binding isotherms reveal that Cd(II) binds very tightly, with K(Cd) = 4.3 (+/-1.8) x 10(12) M(-1). The results of UV-Vis and X-ray absorption spectroscopy of the Cd(1) complex are consistent with a tetrathiolate (S(4)) complex formed by four cysteine ligands. The (113)Cd NMR spectrum reveals a single resonance of delta = 622 ppm, consistent with an S(3)(N,O) or unusual upfield-shifted S(4) complex. The Pb(II) complex reveals two prominent absorption bands at 350 nm (epsilon = 4000 M(-1) cm(-1)) and 250 nm (epsilon = 41 000 M(-1) cm(-1)), spectral properties consistent with three or four thiolate ligands to the Pb(II) ion. The change in the anisotropy of a fluorescein-labeled oligonucleotide containing the cad O/P upon binding CadC and analyzed using a dissociable CadC dimer binding model reveals that apo-CadC forms a high-affinity complex [K(a) = (1.1 +/- 0.3) x 10(9) M(-1); pH 7.0, 0.40 M NaCl, 25 degrees C], the affinity of which is reduced approximately 300-fold upon the binding of a single molar equivalent of Cd(II) or Pb(II). The implications of these findings on the mechanism of metalloregulation are discussed.     

Prochelators triggered by hydrogen peroxide provide hexadentate iron coordination to impede oxidative stress

Prochelators are agents that have little affinity for metal ions until they undergo a chemical conversion. Three new aryl boronate prochelators are presented that are responsive to hydrogen peroxide to provide hexadentate ligands for chelating metal ions. TRENBSIM (tris[(2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzylidene)-2-aminoethyl]amine), TRENBSAM (tris[(2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzoyl)-2-aminoethyl]amine), and TB (tris[(2-boronic acid-benzyl)2-aminoethyl]amine) convert to TRENSIM (tris[(salicylideneamino)ethyl]amine), TRENSAM (tris[(2-hydroxybenzoyl)-2-aminoethyl]amine), and TS (tris[2-hydroxybenzyl)2-aminoethyl]amine), respectively. The prochelators were characterized by ¹¹B NMR, and the structures of TRENBSAM, TRENBSIM, and the Fe(III) complex of TS were determined by X-ray crystallography. Of the three prochelator/chelator pairs, TB/TS was identified as the most promising for biological applications, as they prevent iron and copper-induced hydroxyl radical generation in an in vitro assay. TB has negligible interactions with metal ions, whereas TS has apparent binding constants (log K′) at pH 7.4 of 15.87 for Cu(II), 9.67 Zn(II) and 14.42 for Fe(III). Up to 1 mM TB was nontoxic to retinal pigment epithelial cells, whereas 10 μM TS induced cell death. TS protected cells against H₂O₂-induced death, but only within a 1-10 μM range. TB, on the other hand, had a much broader window of protection, suggesting that it may be a useful agent for preventing metal-promoted oxidative damage.     

Metal-binding characteristics of the amino-terminal domain of ZntA: binding of lead is different compared to cadmium and zinc

ZntA from Escherichia coli, a P1-type ATPase, specifically transports Pb(II), Zn(II), and Cd(II). Most P1-type ATPases have an N-terminal domain that contains one or more copies of the conserved metal-binding motif, GXXCXXC. In ZntA, the N-terminal domain has approximately 120 residues with a single GXXCXXC motif, as well as four additional cysteine residues as part of the CCCDGAC motif. The metal-binding specificity and affinity of this domain in ZntA was investigated. Isolated proteins, N1-ZntA and N2-ZntA, containing residues 1-111 and 47-111 of ZntA, respectively, were characterized. N1-ZntA has both the CCCDGAC and GXXCXXC motifs, while N2-ZntA has only the GXXCXXC motif. ICP-MS measurements showed that N1-ZntA can bind both divalent metal ions such as Cd(II), Pb(II), and Zn(II) and monovalent metal ions such as Ag(I), with a stoichiometry of 1. N2-ZntA can bind Zn(II) and Cd(II) with a stoichiometry of 1 but not Pb(II). The affinity of N1-ZntA for Zn(II), Pb(II), and Cd(II) was measured by competition titration with metallochromic indicators. Association constants of approximately 10(8) M(-)(1) were obtained for Zn(II), Pb(II), and Cd(II) binding to N1-ZntA. To investigate whether the CCCDGAC sequence has an important role in binding specifically Pb(II), a mutant of ZntA, which lacked the first 46 residues, was constructed. This mutant, Delta46-ZntA, had the same activity as wtZntA with respect to Cd(II) and Zn(II). However, its activity with Pb(II) was similar to the mutant DeltaN-ZntA, which lacks the entire N-terminal domain (Mitra, B., and Sharma, R. (2001) Biochemistry 40, 7694-7699). Thus, binding of Pb(II) appears to involve different ligands, and possibly geometry, compared to Cd(II) and Zn(II).     

Phytic acid-enhanced metal ion exchange reactions: the effect on carboxypeptidase A

On the basis of the known interaction of phytic acid to form soluble or insoluble complexes with cations, the effect of this naturally occurring polydentate ligand on carboxypeptidase A, a zinc-containing metalloenzyme, and its Co(II)-substituted derivative, has been studied. Under conditions of rigorous exclusion of adventitious metal ions, phytate showed no inhibitory effect. However, the addition of Cu(II) ions to form soluble phytate-Cu(II) complexes at pH 7.2 and 25 degrees C caused more than a 95% decrease in activity. The Cd(II) ion was nearly as effective but other ions showed only a small or no effect. In the absence of phytate, incubation of the enzyme with Cu(II) or Cd(II) at the same concentration produced only about a 25% reduction in activity. The decrease in activity followed first-order kinetics, and the rate constant was the same (1.2 x 10(-4) sec-1) as seen upon incubation with EDTA. However, in contrast to that observed upon incubation of the enzyme with phytate and Cu(II), exposure to EDTA produced a complete loss in activity which could be regained by addition of Zn(II) to the assay solution. In the former case, not only was there residual activity left after incubation at pH 7.2 for 24 hrs at 25 degrees C, but the initial activity could not be regained under similar assay treatment. An increase in either the Cu(II) or phytate concentration while the other was kept constant, yielded saturation curves with maximal effect at 3 x 10(-5) M for Cu(II) and at 5 x 10(-5) M for phytate (enzyme at ca. 10(-6) M). At these ratios, all of the cupric ions are completely bound to phytate as determined by ion-selective potentiometry. A preparative scale reaction of phytate and Cu(II) with carboxypeptidase A (kcat 8460 min-1; K'M 0.23 mM with CBZ-glycyl-glycyl-L-phenylalanine as substrate at pH 7.5, 25 degrees C) gave a product isolated in 95% yield but with lower activity (kcat 198 min-1; K'M 0.25 mM). A Cu(II)-carboxypeptidase preparation had similar kinetic parameters (kcat 207 min-1; K'M 0.34 mM). This near identity of constants suggested that a metal exchange reaction had occurred, i.e., incubation of Zn(II)-carboxypeptidase with a phytate-Cu(II) complex resulted in not only the removal of the zinc ion from the active site but also the sequential and rapid incorporation of a cupric ion into the apoenzyme so formed.(ABSTRACT TRUNCATED AT 400 WORDS)     

Transition metal complexes with N, S donor ligands as synthetic antioxidants: synthesis, characterization and antioxidant activity

Transition metal complexes containing bidentate N, S donor ligands i.e., carvone thiosemicarbazone [(RS)-5-isopropenyl-2-methylcyclohex-2-en-1-one thiosemicarbazone (IPMCHTSC)] and carvone N(1)-phenylthiosemicarbazone [(RS)-5-isopropenyl-2-methylcyclohex-2-en-1-one phenylthiosemicarbazone (IPMCHPhTSC)] have been synthesized. All the metal complexes (1-8) have been characterized by elemental analysis, molar conductance measurement and various spectral studies [infrared (IR), electronic, fast-atom bombardment (FAB) mass and NMR (for ligands)] and thermogravimetric analysis. FAB mass spectroscopic studies of (1), (3), (4), (5), (6) (7), and (8) suggest their monomeric nature. Metal complexes are [M(LH)Cl(2)] and [M(LH)(2)Cl(2)] type, where M?=?Fe(III), Co(II), and Cu(II) and LH?=?IPMCHTSC and IPMCHPhTSC. The proposed geometries of the complexes were octahedral for 1:2 complexes, square planar for 1:1 complexes and distorted octahedral for Cu(II) complexes (1:2). The free radical scavenging activity of ligands (IPMCHTSC and IPMCHPhTSC) and their metal complexes have been determined at the concentration range of 10-400 μg/mL by means of their interaction with the stable free radical 2,2'-diphenyl-1-picrylhydrazyl and 5-200 μg/mL by 2,2'-Azinobis-3-ethylbenzothiazoline-6-sulphonic acid. All the compounds have shown encouraging antioxidant activities.     

Metal(II) binding ability of a novel N-protected amino acid. A solution-state investigation on binary and ternary complexes with 2,2'-bipyridine

The binary and ternary systems 2,2'-bipyridine (bpy)-M(II)-NO2psglyH2 (M(II) = Mn(II), Co(II), Ni(II), Cu(II), Zn(II), Cd(II), Pb(II); NO2psglyH2 = N-(2-nitrophenylsulfonyl)glycine) were investigated in aqueous solution by means of potentiometry and electron spectroscopy in order to identify the type, number and stability of the complex species as a function of pH and metal-to-ligand molar ratio. The aim is to evaluate the effect of a substituent on the phenyl ring of the N-sulfonyl amino acids on their coordination properties. The prevailing species in the binary systems is the [ML] (M = Co(II), Ni(II), Cu(II), Cd(II), Pb(II)) where the amino acid molecule is in the dianionic form and coordinates the metal ion through both carboxylic oxygen and deprotonated sulfonamidic nitrogen, while in the Mn(II)- and Zn(II)-containing binary system the only complex species found are those with the amino acid in the monoanionic form. In the ternary 2,2'-bipyridine-containing systems the chelating coordination mode of the dianionic amino acid is maintained with M(II) = Co(II), Ni(II), Cu(II), Cd(II), Pb(II) and the addition of the aromatic base also enables the Zn(II) ion to substitute for the sulfonamide nitrogen-bound hydrogen of NO2psglyH2.     

Reverse Na(+)/Ca(2+)-exchange mediated Ca(2+)-entry and noradrenaline release in Na(+)-loaded peripheral sympathetic nerves

[(3)H]noradrenaline ([(3)H]NA) released from sympathetic nerves in the isolated main pulmonary artery of the rabbit was measured in response to field stimulation (2Hz, 1ms, 60V for 3min) in the presence of uptake blockers (cocaine, 3 x10(-5)M and corticosterone, 5 x10(-5)M). The [(3)H]NA-release was fully blocked by the combined application of the selective and irreversible 'N-type' voltage-sensitive Ca(2+)-channel (VSCC)-blocker omega-conotoxin (omega-CgTx) GVIA (10(-8)M) and the 'non-selective' VSCC-blocker aminoglycoside antibiotic neomycin (3x10(-3)M). Na(+)-loading (Na(+)-pump inhibition by K(+)-free perfusion) was required to elicit further NA-release after blockade of VSCCs (omega-CgTx GVIA+neomycin). In K(+)-free solution, in the absence of functioning VSCCs (omega-CgTx GVIA+neomycin), the fast Na(+)-channel activator veratridine (10(-5)M) further potentiated the nerve-evoked release of [(3)H]NA. This NA-release was significantly inhibited by KB-R7943, and fully blocked by Ca(o)(2+)-removal. However, Li(+)-substitution was surprisingly ineffective. The non-selective K(+)-channel blocker 4-aminopyridine (4-AP, 10(-4)M) also further potentiated the nerve-evoked release of NA in K(+)-free solution. This potentiated release was concentration-dependently inhibited by KB-R7943, significantly inhibited by Li(+)-substitution and abolished by Ca(o)(2+)-removal. It is concluded that in Na(+)-loaded sympathetic nerves, in which the VSCCs are blocked, the reverse Na(+)/Ca(2+)-exchange-mediated Ca(2+)-entry is responsible for transmitter release on nerve-stimulation. Theoretically we suppose that the fast Na(+)-channel and the exchanger proteins are close to the vesicle docking sites.     

Transition metal acetylsalicylates and their anti-inflammatory activity

Mononuclear and binuclear transition metal [Co(II), Cu(II), Ni(II) and Zn(II)] acetylsalicylates of the type [M(L)2], [M(L)2Cl2] and [(M)2(L)4] have been prepared and characterized on the basis of their physical, spectral and analytical data. The complexes have been investigated in an in vivo animal model for anti-inflammatory activity and show a better effect and a more potent action than acetylsalicylic acid.     

Transition Metal Acetylsalicylates and Their Anti-inflammatory Activity

Mononuclear and binuclear transition metal [Co(II), Cu(II), Ni(II) and Zn(II)] acetylsalicylates of the type [M(L) 2], [M(L) 2 Cl 2] and [(M) 2 (L) 4] have been prepared and characterized on the basis of their physical, spectral and analytical data. The complexes have been investigated in an in vivo animal model for anti-inflammatory activity and show a better effect and a more potent action than acetylsalicylic acid.     

Hippuryl-alpha-methylphenylalanine and hippuryl-alpha-methylphenyllactic acid as substrates for carboxypeptidase A. Syntheses, kinetic evaluation and mechanistic implication

(R)- and (S)-Hippuryl-alpha-methylphenylalanine [(R)- and (S)-Hipp-alpha-MePhe] and (S)-hippuryl-alpha-methylphenyllactic acid [(S)-Hipp-alpha-MeOPhe] were synthesized and evaluated as substrates for carboxypeptidase A (CPA) in an effort to shed further light on the catalytic mechanism of the enzyme. The rate of CPA-catalyzed hydrolysis of (S)-Hipp-alpha-MePhe was reduced by 105-fold compared with that of (S)-Hipp-Phe, but the hydrolysis rate of (S)-Hipp-OPhe was lowered by only 6.8-fold by the introduction of a methyl group at the alpha-position. (R)-Hipp-alpha-MePhe failed to be hydrolyzed initially, then started to undergo hydrolysis in about 2 h at a much reduced rate. The results of present study may be envisioned on the basis of the proposition that while peptide substrate is hydrolyzed via a tetrahedral transition state formed by the attack of the zinc-bound water molecule at the peptide carbonyl carbon, ester hydrolysis takes the path that involves an anhydride intermediate generated by the attack of the carboxylate of Glu-270 at the ester carbonyl carbon.     

Binding of divalent cations with low density lipoproteins. A study by ESR

The binding of bivalent metal ions Cu2+, Zn2+, Ca2+, Mg2+ to low-density lipoproteins (LDL) was investigated by the ESR technique. The monitoring of ESR spectra of paramagnetic Mn2+ ions in the presence of above-listed cations made it possible to evaluate the dissociation constants of their complexes with LDL. The effective dissociation constant of the complex Mn(2+)-LDL used for calculations was KD = (1.1 +/- 0.4) x 10(-4) M according to literature data. The investigated cations may be classified into two groups: 1) low dissociation constants were characteristic for Cu2+ ions [KD = (1.3 +/- 0.5) x 10(-4) M], which demonstrated a high oxidative ability, and for Zn2+ [KD = (0.95 +/- 0.45) x 10(-4) M] and Mn2+ ions, which could strongly influence the copper-induced LDL oxidation; 2) Ca2+ and Mg2+ were characterized by higher values of KD [(6 +/- 1) x 10(-4) M and (7.5 +/- 1.5) x 10(-4) M, accordingly] and slightly affected the Cu(2+)-induced oxidation of LDL. The results of the present work reinforced our earlier conjecture that cations may influence the process of lipid peroxidation, binding only to particular binding sites on the surface of LDL.     

Invertase inhibition based electrochemical sensor for the detection of heavy metal ions in aqueous system: Application of ultra-microelectrode to enhance sucrose biosensor's sensitivity

We are reporting fabrication and characterization of electrochemical sucrose biosensor using ultra-microelectrode (UME) for the detection of heavy metal ions (Hg(II), Ag(I), Pb(II) and Cd(II)). The working UME, with 25 microm diameter, was modified with invertase (INV, EC: 3.2.1.26) and glucose oxidase (GOD, EC: 1.1.3.4) entrapped in agarose-guar gum. The hydrophilic character of the agarose-guar gum composite matrix was checked by water contact angle measurement. The atomic force microscopy (AFM) images of the membranes showed proper confinement of both the enzymes during co-immobilization. The dynamic range for sucrose biosensor was achieved in the range of 1 x 10(-10) to 1 x 10(-7)M with lower detection limit 1 x 10(-10)M at pH 5.5 with 9 cycles of reuse. The spectrophotometric and electrochemical studies showed linear relationship between concentration of heavy metal ions and degree of inhibition of invertase. The toxicity sequence for invertase using both methods was observed as Hg(2+)>Pb(2+)>Ag(+)>Cd(2+). The dynamic linear range for mercury using electrochemical biosensor was observed in the range of 5 x 10(-10) to 12.5 x 10(-10)M for sucrose. The lower detection limit for the fabricated biosensor was found to be 5 x 10(-10)M. The reliability of the electrochemical biosensor was conformed by testing the spike samples and the results were comparable with the conventional photometric DNSA method.     

1H NMR spectroscopic characterization of binary and ternary complexes of cobalt(II) carboxypeptidase A with inhibitors

The binding of L- and D-phenylalanine and carboxylate inhibitors to cobalt(II)-substituted carboxypeptidase A, Co(II)CPD (E), in the presence and absence of pseudohalogens (X = N3-, NCO-, and NCS-) has been studied by 1H NMR spectroscopy. This technique monitors the proton signals of histidine residues bound to cobalt(II) and is therefore sensitive to the interactions of inhibitors that perturb the coordination sphere of the metal. Enzyme-inhibitor complexes, E.I, E.I2, and E.I.X, each with characteristic NMR features, have been identified. Thus, for example, L-Phe binds close to the metal ion to form a 1:1 complex, whereas D-Phe binds stepwise, first to a nonmetal site and then to the metal ion to form a 2:1 complex. Both acetate and phenylacetate also form 2:1 adducts stepwise with the enzyme, but beta-phenylpropionate gives a 2:1 complex without any detectable 1:1 intermediate. N3-, NCO-, and NCS- generate E.I.X ternary complexes directly with Co(II)CPD.L-Phe and indirectly with the D-Phe and carboxylate inhibitor 2:1 complexes by displacing the second moiety from its metal binding site. The NMR data suggest that when the carboxylate group of a substrate or inhibitor binds at the active site, a conformational change occurs that allows a second ligand molecule to bind to the metal ion, altering its coordination sphere and thereby attenuating the bidentate behavior of Glu-72. The 1H NMR signals also reflect alterations in the histidine interactions with the metal upon inhibitor binding. Isotropic shifts in the signals for the C-4 (c) and N protons (a) of one of the histidine ligands are readily observed in all of these complexes.(ABSTRACT TRUNCATED AT 250 WORDS)