Metal-containing Enzymes Research Articles

Metalloenzymes and signal transduction: the protein serine/threonine phosphatases, a novel class of binuclear metal-containing enzymes

The serine/threonine protein phosphatases are important regulatory enzymes involved in signal transduction pathways in eukaryotic organisms. These enzymes include protein phosphatases 1, 2A, and 2B (also known as calcineurin). Recent structural data have indicated that the serine/threonine protein phosphatases are novel metalloenzymes containing a dinuclear metal ion cofactor at the active site. The dinuclear metal site is situated in a unique protein fold, a β-α-β-α-β motif which provides the majority of ligands to the metal ions. A similar fold is also seen in plant purple acid phosphatases, which also contain a dinuclear iron–zinc cofactor. In these enzymes, the two metal ions are bridged by a solvent molecule and a carboxylate group from an aspartic acid residue, juxtaposing the two metal ions to within 3.0–4.0 A of each other. A similar motif has been identified in a number of other enzymes which exhibit phosphoesterase activity, implicating several of them as metalloenzymes which contain dinuclear metal ion cofactors.

Insights derived from the structures of the Ser/Thr phosphatases calcineurin and protein phosphatase 1.

The crystal structures of serine/threonine phosphatases provide the basis for understanding their inhibition by physiologically relevant compounds such as microcystin, cyclosporin and FK506. The structures also highlight the importance of a common sequence motif found in a large family of metal-containing enzymes involved in phosphate ester hydrolysis.

In vitro effects of aspirin on malondialdehyde formation and on activity of antioxidant and some metal-containing enzymes

The in vitro effects of aspirin in different concentrations on malondialdehyde formation and on the activity of antioxidant and some metal-containing enzymes in rat liver homogenate and erythrocytes were studied. Aspirin showed a biphasic dependence on concentration: low concentrations (to 1.0 mM) stimulated the spontaneously formed malondialdehyde in the liver homogenate and the high concentration (5.0 mM) inhibited it; all aspirin cocentrations tested had no effect on the liposomes; 5.0 mM aspirin inhibited the Fe 2+-induced lipid peroxidation in the liver homogenate but had an opposite effect on the liposomes. Aspirin dose-dependently inhibited nitro-blue tetrazolium reduction and decreased deoxyribose degradation. The higher aspirin concentrations inhibited the activity of antioxidant and some metal-containing enzymes. It is suggested that these aspirin effects are determined by the aspirin-metal complexes formed rather than by aspirin itself.

Superoxide Dismutase in Plants

Abstract Superoxide dismutases (SODs) are metal-containing enzymes that catalyze the dismutation of superoxide radicals to oxygen and hydrogen peroxide. The enzyme has been found in all aerobic organisms examined where it plays a major role in the defense against toxic-reduced oxygen species, which are generated as byproducts of many biological oxidations. The generation of oxygen radicals can be further exacerbated during environmental adversity and consequently SOD has been proposed to be important for plant stress tolerance. In plants, three forms of the enzyme exist, as classified by their active site metal ion: copper/zinc, manganese, and iron forms. The distribution of these enzymes has been studied both at the subcellular level and at the phylogenic level. It is only in plants that all three different types of SOD coexist. Their occurrence in the different subcellular compartments of plant cells allows a study of their molecular evolution and the possibility of understanding why three functionally ...

In vivo effects of indomethacin on the activity of metal-containing enzymes

1. 1. Indomethacin injected subcutaneously at a single “ulcerogenic” dose decreased aminooxidase and leucine aminopeptidase activity and did not change alcohol dehydrogenase and ceruloplasmin activity. 2. 2. Indomethacin administered at an oral “therapeutic” dose inhibited aminooxidase activity in small intestinal mucosa but not in liver and did not change leucine aminopeptidase activity in blood, liver and intestinal mucosa; it, however, increased alcohol dehydrogenase and ceruloplasmin activity. 3. 3. The decreased activity of ceruloplasmin and alcohol dehydrogenase by metal deficiency increased after oral indomethacin treatment, reaching the control values when indomethacin was chelated with copper. 4. 4. The results suggest the participation of endogenous metals in the indomethacin effect.

ChemInform Abstract: Synthesis and Characterization of Metal‐Containing Enzyme Mimics

ChemInformVolume 20, Issue 8 Reviews ChemInform Abstract: Synthesis and Characterization of Metal-Containing Enzyme Mimics C. F. JUN. CARRAHER, C. F. JUN. CARRAHER Dep. Chem., Fla. Atl. Univ., Boca Raton, FL 33431, USASearch for more papers by this authorL. G. TISSINGER, L. G. TISSINGER Dep. Chem., Fla. Atl. Univ., Boca Raton, FL 33431, USASearch for more papers by this authorM. WILLIAMS, M. WILLIAMS Dep. Chem., Fla. Atl. Univ., Boca Raton, FL 33431, USASearch for more papers by this authorI. LOPEZ, I. LOPEZ Dep. Chem., Fla. Atl. Univ., Boca Raton, FL 33431, USASearch for more papers by this author C. F. JUN. CARRAHER, C. F. JUN. CARRAHER Dep. Chem., Fla. Atl. Univ., Boca Raton, FL 33431, USASearch for more papers by this authorL. G. TISSINGER, L. G. TISSINGER Dep. Chem., Fla. Atl. Univ., Boca Raton, FL 33431, USASearch for more papers by this authorM. WILLIAMS, M. WILLIAMS Dep. Chem., Fla. Atl. Univ., Boca Raton, FL 33431, USASearch for more papers by this authorI. LOPEZ, I. LOPEZ Dep. Chem., Fla. Atl. Univ., Boca Raton, FL 33431, USASearch for more papers by this author First published: February 21, 1989 https://doi.org/10.1002/chin.198908336Read the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat No abstract is available for this article. Volume20, Issue8February 21, 1989 RelatedInformation

Interaction of chloride with yeast aminopeptidase I. Equilibrium binding studies.

The effect of chloride on metal binding by yeast aminopeptidase I, as well as the binding of chloride to various enzyme forms were studied by means of a micro-centrifugation technique using radioactive 36Cl- as a ligand. Chloride did not significantly alter the binding of activating Zn2+, or binding of Co2+ to the essential metal sites. Both the native Zn2+ enzyme and Co2+-substituted aminopeptidase I bind stoichiometric amounts of C1- (1 Cl-/subunit) with apparent dissociation constants of 0.1-0.2 mM. Additional Cl- was bound at higher concentrations. In contrast to the metal-containing enzyme forms the apoenzyme did not express the high-affinity chloride binding site.

Protease susceptibility of zinc - and APO-carboxypeptidase A

Proteases in preparations of carboxypeptidase A progressively inactivate solutions of the apoenzyme but not the metal-containing enzyme. Free amino acids generated by proteolysis interfere with spectral studies after reconstituting the apoenzyme with cobalt. Purification by affinity chromatography eliminates this effect. Affinity-purified apoenzyme is susceptible to digestion with chymotrypsin but the metalloenzyme is not.

Thio-analogues of inorganic pyrophosphate inhibit the replication of influenza virus a in vitro

Mono- and bisthiopyrophosphate can inhibit the replication of influenza virus A/X49 in Madin-Darby canine kidney (MDCK) cells at concentrations at which no cytotoxic effect is observed after 3 days. The thiopyrophosphate analogues inhibit the RNA transcriptase activity of this virus possibly by chelating with an essential metal ion in the transcriptase complex. [ 31P]NMR spectroscopy indicates that bisthiopyrophosphate coordinates to zinc through sulphur and magnesium through oxygen which may influence the inhibitory properties of this compound with metal-containing enzymes.

Complex formation of picloram and related chemicals with metal lons

The complex formation of metal ions with pyridine carboxylic acids was estimated with polarography and spectrophotometry. Picloram (4-amino-3,5,6-trichloropicolinic acid), α-picolinic acid, fusaric acid, dipicolinic acid, and quinolinic acid formed complexes with Fe(III) or Cu(II) whose coordination involves, most probably, a lone pair of electrons of pyridine nitrogen and a carboxylic group. Picloram-metal complexes were, however, estimated to be relatively unstable compared to other pyridine-α-carboxylic acids tested. Effects of pyridine carboxylic acids on oxidation of indole-3-acetic acid (IAA) were tested in vitro in a horseradish protoheme peroxidase system. No significant effect of the pyridine carboxylic acids was observed at 2 × 10 −4 M. Also, no concentration effect of picloram (10 −5 to 3 × 10 −3 M) was obtained. These results suggest that the phytotoxic action of picloram may not result from the depletion of free metal ions in plants nor inhibition of activity of metal-containing enzymes through strong chelation as hypothesized. Thus, auxin activity of picloram should be explained in other wasy.

Microbial Oxidation of Gaseous Hydrocarbons: Production of Secondary Alcohols from Corresponding n -Alkanes by Methane-Utilizing Bacteria

Over 20 new strains of methane-utilizing bacteria were isolated from lake water and soil samples. Cell suspensions of these and of other known strains of methane-utilizing bacteria oxidized n-alkanes (propane, butane, pentane, hexane) to their corresponding secondary alcohols (2-propanol, 2-butanol, 2-pentanol, 2-hexanol). The product secondary alcohols accumulated extracellularly. The rate of production of secondary alcohols varied with the organism used for oxidation. The average rate of 2-propanol, 2-butanol, 2-pentanol, and 2-hexanol production was 1.5, 1.0, 0.15, and 0.08 mumol/h per 5.0 mg of protein in cell suspensions, respectively. Secondary alcohols were slowly oxidized further to the corresponding methylketones. Primary alcohols and aldehydes were also detected in low amounts (rate of production were 0.05 to 0.08 mumol/h per 5.0 mg of protein in cell suspensions) as products of n-alkane (propane and butane) oxidation. However, primary alcohols and aldehydes were rapidly metabolized further by cell suspensions. Methanol-grown cells of methane-utilizing bacteria did not oxidize n-alkanes to their corresponding secondary alcohols, indicating that the enzymatic system required for oxidation of n-alkanes was induced only during growth on methane. The optimal conditions for in vivo secondary alcohol formation from n-alkanes were investigated in Methylosinus sp. (CRL-15). The rate of 2-propanol and 2-butanol production was linear for the 40-min incubation period and increased directly with cell protein concentration up to 12 mg/ml. The optimal temperature and pH for the production of 2-propanol and 2-butanol were 40 degrees C and pH 7.0. Metalchelating agents inhibited the production of secondary alcohols. The activities for the hydroxylation of n-alkanes in various methylotrophic bacteria were localized in the cell-free particulate fractions precipitated by centrifugation between 10,000 and 40,000 x g. Both oxygen and reduced nicotinamide adenine dinucleotide were required for hydroxylation activity. The metal-chelating agents inhibited hydroxylation of n-alkanes by the particulate fraction, indicating the involvement of a metal-containing enzyme system in the oxidation of n-alkanes. The production of 2-propanol from the corresponding n-alkane by the particulate fraction was inhibited in the presence of methane, suggesting that the subterminal hydroxylation of n-alkanes may be catalyzed by methane monooxygenase.

Competitive Inhibition of Enzyme Activity by Urea

SUMMARY Urea Inhibition of fifetallo-enzymes--From the data here presented, it appears that, in general, urea is relatively ineffective as an inhibitor of those metallo-enzymes whose metal component is involved in the formation of the enzyme-substrate complex. Thus, hexokinase and alkaline phosphatase which show an ab- solute dependence upon the presence of Mg++, and the ferri- porphyrin proteins, peroxidase and cytochrome oxidase, are com- pletely unaffected by urea even at 2 M concentration. Uricase, a copper-protein (45), carboxypeptidase, a zinc protein (46), catalase, a hemoprotein, and inorganic pyrophosphatase which is dependent upon Mg++ in the medium are inhibited only at very high concentrations of urea. Much evidence suggests that the metal of many metal-requiring or metal-containing enzymes serves as a bridge between enzyme and substrate in the formation of the enzyme-substrate complex (47). It is extremely unlikely that urea can prevent chelation or coordination of the enzyme or substrate by a metal. Similarly, it is unlikely that urea could competitively inhibit the action of enzymes on inorganic sub- strates such as pyrophosphate, sulfite or peroxide since, in these instances, electrostatic forces rather than hydrogen-bonding or van der Waals’ forces must be involved in formation of the en- zyme-substrate complex. Xanthine oxidase has been found to be competitively inhibited by low levels of urea. Analysis of urea inhibition of a series of diverse enzymes indicates that urea is, in general, a competitive inhibitor of enzymes which act upon organic substrates. Guani- dine is much more effective than urea as a competitive inhibitor of the same group of enzymes. Relatively higher concentrations of urea are required to effect inhibition of enzymes which act upon inorganic substrates and such inhibitions are either “non- competitive” or “uncompetitive.” In general, enzymes which require metal ions as cofactors or which contain a metal as an integral part of the enzyme molecule were found to be resistant to inhibition by urea.

Azione del 2–4 Dinitrofenolo, del Cianuro, Dell'azide e di Alcuni Inibitori Dell'attività Deidrogenasica Sulla Riduzione “In Vivo“ del Cloruro di Trifeniltetrazolio

Summary The effects of some dehydrogenase inhibitors (iodoacetate, fluoride, malonate), of some inhibitors of metal containing-enzymes (diethyldithiocarbamate, azide, cyanide) and of a compound uncoupling oxidations from phosphorylations, (2–4 dinitrophenol), on triphenyltetrazolium chloride (TTC), reduction in disks from Helianthus tubers was investigated. Fluoride and iodacetate, at concentrations resp. 2.5 × 10−2 and 3 × 10−3 M, inhibited of more than 75% the TTC reduction. Malonate at cone. 5 × 10−2 inhibited the TTC reduction of about 33%. Diethyldithiocarbamate, at a conc, clearly active on catecol oxidase (2 × 10−3 M), was practically ineffective on TTC reduction. On the countrary, azide and cyanide at concentrazions 10−2 M and even lower, distinctly inhibited the reaction. The effect of these inhibitors appeared by far more evident in tissues incubated in air then in vacuum. As both compounds are known to inhibit catalase, and thus the breakdown of oxigen peroxide, a strong poison of sulphydryl gr...

THE CYTOCHROME-C CONTENT OF BOVINE CRYSTALLINE LENS

THE CRYSTALLINE lens is rich in both pyruvic acid1and its reduction product, lactic acid.2The citric-acid cycle has been identified as probably playing a role in the utilization of these substrates.3The final hydrogen mediator in the lens is unknown. Previous experiments indicate that a large portion of the respiration of the crystalline lens is inhibited by cyanide.4This fact indicates that the normal oxygen consumption of bovine crystalline lens is dependent on the presence of a heavy metal-containing enzyme, most probably iron. The commonest enzyme system which is dependent on the presence of iron is the cytochromecytochrome oxidase system. A previous attempt to isolate and identify cytochrome c in the lens met with failure.4The present report details another attempt to isolate and to identify cytochrome c in the crystalline lens. METHODS A quantitative spectrophotometric determination was made to see whether cytochrome c