Limiting rates of ligand association to alcohol dehydrogenase.

Detailed stopped-flow kinetic studies of the association of 2,2-bipyridine, 1,10-phenanthroline, and 5-chloro-1,10-phenanthroline to the zinc ion at the active site of alcohol dehydrogenase have demonstrated that a process with a limiting rate constant of about 200 s^?1 restricts the binding of the bidentate chelating agents to the free enzyme. The formation of the enzyme-ligand complexes has been followed by means of the characteristic absorption spectra of the resulting complexes or by the displacement of the fluorescent dye, auramine O. Monodentate ligands, upon binding to the free enzyme or enzyme-NAD⁺ and enzyme-NADH complexes, do not exhibit a comparable limiting rate. In analogy with simple inorganic systems, these observations have been interpreted in terms of the rate limiting dissociation of an inner sphere water molecule following the rapid formation by the bidentate ligand of an outer sphere complex. The displacement of a water molecule from the zinc ion by 1,10-phenanthroline has been observed in crystallographic studies which have also established that the zinc ion in the enzyme-1,10-phenanthroline complex is pentacoordinate. Monodentate ligands, which are substrate analogs, do not exhibit limiting rates because displacement of water is not required for their addition to a coordinate position which is apparently vacant in the free enzyme. If a water molecule remains bound to the zinc ion in the kinetically competent ternary complex, it could play an essential role in the proton transfer reaction accompanying catalysis.     

The metalloenzymic nature of collagenase-like peptidase of the rat testis.

Collagenase-like peptidase, an enzyme degrading synthetic collagenase substrate (PZ-pentapeptide), was purified from rat testes and its properties were examined. Its activity was strongly inhibited by chelating agents, such as EDTA and 1,10-phenanthroline. By chelation and exhaustive dialysis it was possible to obtain this enzyme in its inactive, metal-free form. The activity of the metal-free enzyme was partly recovered by treatment with zinc or manganese ions, while a combined zinc and manganese treatment resulted in complete recovery of enzyme activity.     

Nucleotide pyrophosphatase from yeast. The presence of bound zinc.

Nucleotide pyrophosphatase from yeast was inhibited by thiols, o-phenanthroline, 8-hydroxyquinoline, EDTA, and 8-hydroxyquinoline-5-sulfonic acid. The inhibition by chelating agents was time and concentration dependent. Inhibition by EDTA was decreased by complexing the EDTA with metal ions before addition to the enzyme. The effectiveness of the metal ions in preventing inhibition by EDTA paralleled the stability constants of the EDTA-metal complexes. Partial recovery of EDTA-inhibited enzyme activity was achieved with Zn²⁺, Co²⁺, Fe²⁺, and Mn²⁺. Analyses for zinc in the purified enzyme by atomic absorption spectroscopy and by titration with 8-hydroxyquinoline-5-sulfonic acid revealed the presence of approximately 1 g atom/mol of enzyme (M_r 65,000). The data indicate that yeast nucleotide pyrophosphatase is a metalloenzyme in which the zinc plays some role in activity.     

Inhibition of E. coli DNA polymerase I by 1,10-phenanthroline.

A 1,10-phenanthroline-cuprous ion complex is a potent reversible inhibitor of $\text{E. coli}$ DNA polymerase I yielding 50% inhibition in the micromolar concentration range. The 2:1 1,10-phenanthroline-cuprous ion complex is most probably the inhibitory species. Complexes of cupric ion and 1,10-phenanthroline have no apparent kinetic effect. The previously reported inhibition of the enzyme by 1,10-phenanthroline (1,2) is most likely due to the formation of this complex from thiols normally added to the assay mixtures and trace amounts of cupric ion invariably present notwithstanding reasonable precaution. The reversible and instantaneous 1,10-phenanthroline inhibition observed for other polymerases may be due to this unique inhibitory species and not coordination of a catalytically important zinc ion at the active site by the chelating agent.     

Evidence for mammalian collagenases as zinc ion metalloenzymes.

Collagenases (EC 3.4.24.3) from human skin, rat skin and rat uterus were inhibited by the chelating agents EDTA, 1,10-phenanthroline and tetraethylene pentamine in the presence of excess Ca2+, suggesting that a second metal ion participates in the activity of the enzyme. Collagenase inhibition by 1,10-phenanthroline could be both prevented and reversed by a number of transition metal ions, specifically Zn2+, Co2+, Fe2+ and Cu2+. However, Zn2+ is effective in five-fold lower molar concentrations (1-10(-4) M) than the other ions. Furthermore, Zn2+ was the only ion tested able to prevent and reverse the inhibition of collagenase by EDTA in the presence of excess Ca2+. Atomic absorption analysis of purified collagenase for Zn2+ showed that Zn2+ was present in the enzyme preparations, and that the metal co-purifies with collagenase during column chromatography.     

Leukotriene A4 hydrolase: a zinc metalloenzyme

Purified human leukotriene A4 hydrolase is shown to contain 1 mol of zinc per mol of enzyme, as determined by atomic absorption spectrometry. The enzyme is inhibited dose-dependently by the chelating agents 8-hydroxy-quinoline-5-sulfonic acid, and 1,10-phenanthroline with KI values of about 2 and 8 x 10(-4) M, respectively, whereas dipicolinic acid and EDTA are ineffective in this respect. The inhibition by 1,10-phenanthroline is time-dependent, and at a concentration of 5 mM, 50% inhibition of enzyme (3 x 10(-7) M) occurs after about 15 min. The zinc atom of leukotriene A4 hydrolase can be removed by dialysis against 1,10-phenanthroline which results in loss of enzyme activity. The catalytic activity is almost completely restored by the addition of stoichiometric amounts of Zn2+ or Co2+.     

Stimulation of the activity of prolyl hydroxylase in 3T3 fibroblasts by 1,10-phenanthroline.

In confluent cultures of 3T3 fibroblasts, incubated for 24 h with 1,10-phenanthroline at 10(-5)--10(-9) M, the activity of prolyl hydroxylase was significantly increased. 1,10-Phenanthroline was inhibitory at concentrations greater than 10(-4) M. The stimulatory effect of 1,10-phenanthroline manifests itself after 6 h incubation and increased with time up to 48 h. 2,2'-dipyridyl and 5,6-dimethyl-1,10-phenanthroline were also stimulatory; a nonchelating analog, 1,7-phenanthroline had no effect. Cycloheximide did not modify the 1,10-phenanthroline effect. The stimulatory effect does not seem to depend on the shift of an inactive precursor of prolyl hydroxylase to an active form because 1,10-phenanthroline was shown to be ineffective in logarithmically growing cells. While dialysis of washed and homogenized cells significantly increased prolyl hydroxylase activity in cell extracts, undialyzed 1,10-phenanthroline treated samples exhibited higher prolyl hydroxylase activity than dialyzed controls. These data suggested to us that 1,10-phenanthroline and other chelating agents may be forming complexes with certain metal ions or protein-metal ions which are inhibitory towards prolyl hydroxylase.     

Inhibition of enzymes by metal ion-chelating reagents. The action of copper-chelating reagents on diamine oxidase

1. The inhibition of diamine oxidase has been studied by using the following copper-chelating reagents: 1,10-phenanthroline; 2,2'-bipyridyl; 8-hydroxyquinoline (oxine); diethyldithiocarbamate and dithio-oxamide (rubeanic acid). 2. Addition of chelating reagent caused a rapid inhibition of enzyme to a degree dependent solely on the final inhibitor concentration. Addition of substrate gave linear initial rates of reaction showing that under these conditions the inhibition was not being rapidly reversed. 3. The inhibition has been investigated by using new graphical methods and has been found in all cases to involve the chelating agents completely removing two Cu(2+) ions from the enzyme. An alternative possibility, involving ligand substitution, was eliminated. 4. A value of K=8.0x10(-33)m(-2) has been found for the enzyme in equilibrium with 2 Cu(2+) ions (i.e. beta(2), the stability constant for diamine oxidase/two Cu(2+), is 32.1).     

1,10-Phenanthroline-cuprous ion complex, a potent inhibitor of DNA and RNA polymerases.

The inhibition by 1,10-phenanthroline of $\text{E. coli}$ DNA polymerase I has recently been attributed to the formation in the assay mixtures of a unique and effective inhibitor, the 2:1 1,10-phenanthroline-cuprous ion complex (1). We have now found that this coordination complex is also an effective inhibitor of $\text{E. coli}$ DNA dependent RNA polymerase, Micrococcus luteus DNA dependent DNA polymerase, and T-4 DNA dependent DNA polymerase. This conclusion is based either on the requirement of a thiol for 1,10-phenanthroline inhibition or on the reversal of 1,10-phenanthroline inhibition by the non-inhibitory cuprous ion specific chelating agent 2,9-dimethyl-1,10-phenanthroline. 2,2′,2″-Terpyridine is also very effective at relieving 1,10-phenanthroline inhibition. The reversal of 1,10-phenanthroline inhibition should be attempted before it is claimed that 1,10-phenanthroline inhibits any polymerases by coordinating a zinc ion at the active site.     

Metal complexes of salicylhydroxamic acid (H2Sha), anthranilic hydroxamic acid and benzohydroxamic acid. Crystal and molecular structure of [Cu(phen)2(Cl)]Cl x H2Sha, a model for a peroxidase-inhibitor complex

Stability constants of iron(III), copper(II), nickel(II) and zinc(II) complexes of salicylhydroxamic acid (H2Sha), anthranilic hydroxamic acid (HAha) and benzohydroxamic acid (HBha) have been determined at 25.0 degrees C, I=0.2 mol dm(-3) KCl in aqueous solution. The complex stability order, iron(III) > copper(II) > nickel(II) approximately = zinc(II) was observed whilst complexes of H2Sha were found to be more stable than those of the other two ligands. In the preparation of ternary metal ion complexes of these ligands and 1,10-phenanthroline (phen) the crystalline complex [Cu(phen)2(Cl)]Cl x H2Sha was obtained and its crystal structure determined. This complex is a model for hydroxamate-peroxidase inhibitor interactions.     

Stimulation of the activity of prolyl hydroxylase in 3T3 fibroblasts by 1,10-phenanthroline

In confluent cultures of 3T3 fibroblasts, incubated for 24 h with 1,10-phenanthroline at 10^?5–10^?9 M, the activity of prolyl hydroxylase was significantly increased. 1,10-Phenanthroline was inhibitory at concentrations greater than 10^?4 M. The stimulatory effect of 1,10-phenanthroline manifets itself after 6 h inhubation and increased with time up to 48 h. 2,2′-dipyridyl and 5,6-dimethyl-1-1,10-phemamthroline were also stimulatory; a nonchelating analog, 1,7-phenanthroline had no effect.Cycloheximide did not modify the 1,10-phenanthroline effect. The stimulatory effect does not seem to depend on the shift of an inactive precursor of prolyl hydroxylase to an active form because 1,10-phenanthroline was shown to be ineffective in logarithmically growing cells.While dialysis of washed and homogenized cells significantly increased prolyl hydroxylase activity in cell extracts, undialyzed 1,10-phenanthroline treated samples exhibited higher prolyl hydroxylase activity than dialyxed controls.These data suggested to us that 1,10-phenanthroline and other chelating agents may be forming complexes with certain metal ions or protein-metal ions which are inhibitory towards prolyl hydroxylase.     

Yeast RNA polymerase III: A zinc metalloenzyme

Atomic absorption spectroscopy has been used to demonstrate that zinc is associated with yeast RNA polymerase III. The enzyme purified by DNA-Sepharose chromatography gives a single predominant protein band in polyacrylamide gel electrophoresis and contains 0.7 gram-atoms of zinc per 100,000 grams of protein. The zinc is tightly associated with the enzyme and cannot be removed by passing the protein through a column of Chelex-100 resin under conditions where free zinc is quantitatively removed. Inhibition by the chelating agent 1,10-phenanthroline demonstrates that the zinc is essential to the catalytic process. The enzyme is inhibited 50% at 0.1 mM and 100% at 1 mM 1,10-phenanthroline.     

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.     

Interaction of cobalt(II) bovine carbonic anhydrase with pyridine-2,6-dicarboxylate ion and other chelating ligands

The removal of cobalt from cobalt(II) bovine carbonic anhydrase by pyridine-2-carboxylate, pyridine-2,6-dicarboxylate and 5-methyl-1,10-phenanthroline occurs via formation of an intermediate. This is presumed to be a ternary adduct of cobalt(II) enzyme with the ligand. In this, metal-protein bonds are loosened, probably via distortion of the normal geometry, resulting in accelerated breakdown of the adduct to apoprotein, compared with the behavior of the cobalt(II) enzyme alone. With 2-carboxy-1,10-phenanthroline, removal of metal is very rapid but no adduct is observed. Values of stability constants of the adducts and rate constants for their decomposition to apoprotein and their formation from apoprotein and cobalt(II) complex were measured at pH 5.5 and 25°C. Formation and dissociation rate constants for the adduct of cobalt carbonic anhydrase with pyridine-2,6-dicarboxylate could be measured from pH 5 to 7 and 10° to 25°C by stopped flow. Values of thermodynamic parameters for the various reactions agreed well with those estimated from the kinetic data.     

Zinc and cadmium in 5-aminolevulinic acid dehydratase. Equilibrium,kinetic, and 113Cd-nmr-studies

5-Aminolevulinic acid dehydratase (ALAD) from bovine liver contains zinc that is partially lost during the isolation of the enzyme. ALAD has its maximal activity at 10^?5 M ZnCl₂. It binds 7.4 Zn per octameric protein with an association constant of 5.3 × 10⁶ M^?1. ALAD is inactivated by 1,10-phenanthroline or ethylenediaminetetraacetic acid (EDTA) but not by monodentate anions like cyanide or sulfide. After removal of zinc by chelating agents, the enzyme activity may be restored by Zn²⁺ or Cd²⁺. Removal or zinc by EDTA increases K_M 60-fold and decreases V_max to about $\text{1}\text{2}$ of its original value. The ¹¹³Cd nuclear magnetic resonance spectrum of the enzyme reconstituted with ¹¹³Cd-acetate exhibits a single sharp resonance signal at 79 ppm. It does not change by the addition of substrate but disappears when the inhibitor lead acetate is added. Therefore, an immediate interaction between the metal ion of the enzyme and the substrate is excluded, whereas lead changes the environment of cadmium and probably of zinc too.     

Crayfish carboxypeptidase. Affinity chromatography, characterization and amino-terminal sequence.

In an effort to trace the evolutionary history of the pancreatic metalloexopeptidases, carboxypeptidase has been isolated from the cardia of the crayfish Astacus fluviatilis. The isolation procedure included affinity chromatography on a column of potato carboxypeptidase inhibitor covalently linked to Sepharose. Approximately 25 mg of pure enzyme can be obtained by the present procedure from 50 ml of cardia fluid. The pure enzyme resembles bovine carboxypeptidase B in specificity and is inhibited both by 3-phenyllactate and by 6-aminohexanoate. The pH optimum of activity is about pH 6.5, and the isoelectric point,pH 4.0. Inhibition by typical metal chelating agents (i.e. ethylenediamine tetraacetate and 1,10-phenanthroline) and neutron activation analysis indicate that, like the mammalian enzyme, crayfish carboxypepetidase is a zinc metalloenzyme. The purified enzyme migrates as a single band in cellulose acetate, disc gel and sodium dodecylsulfate gel electrophoresis. The amino acid composition is similar to that of pancreatic carboxypeptidases except for a higher content of acidic amino acid residues. The amino acid sequence of the first 19 amino-terminal residues reveals significant homology to that of pancreatic carboxypeptidases A and B.     

Zinc metalloenzyme properties of active and latent collagenase from rabbit bone.

1. Inhibition of collagenase from rabbit bone cultures by the chelating agents 1,10-phenanthroline and EDTA is almost completely reversed by Zn2+; other metal cations are less effective in reversing the inhibition. Optimal restoration of activity is achieved at Zn2+ concentrations below that of the chelator, but excess of Zn2+ is inhibitory. 2. Prolonged incubation of collagenase with either chelator causes irreversible inactivation. This inactivation is prevented by Zn2+ at the same concentrations needed to reverse the primary inhibition. 3. Collagenase incorporates 65Zn by exchange when incubated with 1,10-phenanthroline and Zn2+ containing this radioactive isotope. The 65Zn2+ can be removed from its binding site in collagenase by 1,10-phenanthroline or EDTA. Irreversible inactivation of collagenase by chelators destroys its ability to incorporate 65Zn2+. 4. Latent collagenase, the inhibited form in which collagenase first appears in culture, behaves similarly to the active enzyme in 65Zn2+-exchange experiments, but is resistant to irreversible inactivation by chelators. 5. It is concluded that collagenase is a zinc metalloenzyme that forms an inactive and unstable apoenzyme on treatment with chelators. The bound inhibitor component of latent collagenase evidently stabilizes the apoenzyme.     

A novel 35 kDa frog liver acid metallophosphatase

The lower molecular weight (35 kDa) acid phosphatase from the frog (Rana esculenta) liver is a glycometalloenzyme susceptible to activation by reducing agents and displaying tartrate and fluoride resistance. Metal chelators (EDTA, 1,10-phenanthroline) inactivate the enzyme reversibly in a time- and temperature-dependent manner. The apoenzyme is reactivated by divalent transition metal cations, i. e. cobalt, zinc, ferrous, manganese, cadmium and nickel to 130%, 75%, 63%, 62%, 55% and 34% of the original activity, respectively. Magnesium, calcium, cupric and ferric ions were shown to be ineffective in this process. Metal analysis by the emission spectrometry method (inductively coupled plasma-atomic emission spectrometry) revealed the presence of zinc, iron and magnesium. The time course of the apoenzyme reactivation, the stabilization effect and the relatively high resistance to oxidizing conditions indicate that the zinc ion is crucial for the enzyme activity. The presence of iron was additionally confirmed by the visible absorption spectrum of the enzyme with a shoulder at 417 nm and by the electron paramagnetic resonance line of high spin iron(III) with geff of 2.4. The active center containing only zinc or both zinc and iron ions is proposed. The frog liver lower molecular weight acid phosphatase is a novel metallophosphatase of lower vertebrate origin, distinct from the mammalian tartrate-resistant, purple acid phosphatases.     

Evidence for metalloenzyme character of tRNA nucleotidyl transferase.

Previous studies (1–5) have shown that several nucleotidyl transferases are metalloenzymes and in a few cases (1–3) the metal has been identified as zinc. In all instances these enzymes are specifically inhibited by incubation with the chelating agent 1,10-phenanthroline but are not affected by the structurally similar 1,7-phenanthroline which does not chelate metals. We report here that tRNA nucleotidyl transferase from E. coli is inhibited by 1,10-phenanthroline and that only the initial rate of incorporation of AMP is affected; CMP incorporation is not specifically inhibited by this chelator. This finding is in conflict with a previous study (5) in which it was claimed that tRNA nucleotidyl transferase from Rous sarcoma virus and from yeast was unaffected by 1,10-phenanthroline and suggests that the E. coli tRNA nucleotidyl transferase is a metalloenzyme.     

Proteins of the kidney microvillar membrane. Enzymic and molecular properties of aminopeptidase W.

Aminopeptidase W is a newly discovered enzyme of the renal and intestinal brush borders, having been first isolated as a 130 kDa glycoprotein recognized by a monoclonal antibody [Gee & Kenny (1985) Biochem. J. 230, 753-764]. It is particularly effective in the hydrolysis of dipeptides, Glu-Trp (Km 0.57 mM; kcat. 6770 min-1) being a favoured substrate. Dipeptides with tryptophan, phenylalanine or tyrosine in the P1 position were rapidly hydrolysed, but the requirements in respect of the P1 residue were not stringent. The activity of aminopeptidase W is markedly influenced by ionic conditions. The highest activity was observed in 100 mM-Tris/HCl, pH 8; phosphate ions were strongly inhibitory. Activity was also greatly affected by bivalent metal ions, and the magnitude and direction of the effects depended on the nature of the buffer anions and on pH. The most effective inhibitors were amastatin and bestatin. Some thiols also inhibited, but other chelating agents, EDTA and 1,10-phenanthroline, had no effect over the concentration range 1-10 mM. Other group-specific inhibitors, for cysteine, serine or aspartic peptidases, were also ineffective. Some molecular properties were studied. Deglycosylation by treatment with N-glycanase diminished the apparent subunit Mr from 130,000 to 90,000. The enzyme contained zinc, 1.2 atoms/subunit, and in spite of the atypical properties of this enzyme in respect of chelating agents, a zinc-catalysed mechanism is the most probable. Its roles in digestion and in renal function are not yet clear.