Activation of yeast enolase by Cd(II)

Activation of yeast enolase by Cd2+ exhibits properties similar to activation by the physiological cofactor Mg2+. The activity is weakly stimulated, then inhibited by increasing ionic strength. The activity increases, then falls with increasing Cd2+ concentration. The effect of pH on activity produced by Cd2+ is very similar to that produced by Mg2+, except that the Cd2+ profile is shifted one pH unit to more alkaline values, and the maximum activity of the Cd2+-enzyme is about 10% of that of the Mg2+-enzyme. The apparent kinetic parameters of Cd2+ activation show little effect of pH except for inhibition by high concentrations of Cd2+: the apparent Ki increases sharply with pH. This is interpreted as the result of Cd2+ being a less effective "catalytic" metal ion, and Cd2+ being more effective in stabilizing the enzyme at alkaline pH's. The similarity of effects of ionic strength, divalent cation, and pH may be due to interaction with the same six sites per mole of enzyme. We also characterized the dependence of what is believed to be the enzyme-catalyzed enolization of a substrate analog, D-tartronate semialdehyde-2-phosphate (TSP) on similar parameters of pH, ionic strength, etc. The putative enolization is dependent on catalytic metal ion, although the TSP binds to the conformational Cd2+-enzyme complex. The reaction is very slow and very pH dependent, increasing with pH with a midpoint of reaction velocity at pH 8.7. There is a strong qualitative correlation between pH dependencies of reaction velocity of substrate conversion and TSP enolization and absorbance of the enzyme-bound TSP enolate, whether with Mg2+ or Cd2+ as cofactor. The slowness of the Cd2+-TSP reaction is not limited by proton release or any reaction involving covalent bonds to hydrogen. The apparent reaction rate constant increases linearly with Cd2+ concentration. Addition of excess ethylenediaminetetraacetic acid reverses the TSP reaction, but again very slowly. The binding of Cd2+ to the catalytic sites is characterized by low association and dissociation rate constants.     

Cadmium(II)-113 NMR studies of the mechanism of metal ion activation of yeast enolase

Yeast enolase binds one mole of 113Cd2+ per subunit at a site that consists of all oxyligands in a distorted octahedral environment. This "conformational" metal ion's environment undergoes further distortion on addition of substrate/product or analogs. At pH's below the optimum value the shifted resonance tends to break up into several, suggesting the existence of several slowly exchanging intermediate forms. At acid pH's, on addition of one additional mole/subunit of 113Cd2+, which greatly increases catalysis, "conformational" resonance(s) further broadens, suggesting that the second, "catalytic" metal ion increases the rates of interconversion between "conformational" species. At more alkaline pH's, near the optimum pH, the "conformational" peak is sharpened, which suggests that very fast interconversion is occurring. The position of the "catalytic" metal ion resonance also suggests all oxyligands in a distorted octahedral geometry. The "catalytic" resonance is often broadened to the point where it cannot be seen, suggesting rapid changes in its geometry due to interconversion of substrate and product.     

Binding of terbium(III) to yeast enolase

Several independent criteria indicate 2 mol of terbium(III) bind to yeast enolase in the absence of substrate-fluorescence titrations of enzyme and metal, effects on thermal stability and published ultrafiltration and inhibition experiments. These measurements also suggest the terbium binding sites are the same as those normally occupied by “conformational” magnesium. Terbium binds much more strongly than magnesium, however, and measurements of the kinetics of the absorbance change in the terbium-enzyme on adding excess EDTA suggest the terbium-enzyme dissociation constant is about $\text{1}\text{500}$ that of the magnesium-enzyme. Measurements of enzyme activity as a function of substrate concentration show that terbium permits no enzymatic activity. However, magnesium competes more effectively with the lanthanide if the substrate analogue 3-aminoenolpyruvate 2-phosphate (AEP) is present.The fluorescence of the lanthanide is not readily observed on exciting the terbium-enzyme at 280 nm, indicating the absence of tyrosines or tryptophans in the coordination sphere of the metal. Excitation of terbium using 488 nm radiation from an argon ion laser shows the fluorescence of the metal is enhanced by binding to the enzyme. EDTA and carbonate have similar effects. This suggests carboxyl groups are involved in binding metal at the conformational sites of yeast enolase. Measurements of lifetimes of enzyme-bound terbium in the presence and absence of D₂O indicated three moles of water remained on each of the bound metals, independently of the buffer used. If enzyme-bound terbium is assumed to be nine-coordinate, the metal must bind to six groups from the enzyme. The presence of substrate does not markedly affect the emission spectrum of the bound terbium or the number of water molecules remaining on the metal, but calorimetric measurements show that substrate binds to the terbium enzyme.     

Studies of the role of catalytic and conformational metals in producing enzymatic activity in yeast enolase

Spectrophotometric titrations of yeast apoenolase with magnesium, the metal that produces the highest level of activity, nickel, which produces a very low level, and calcium, which produces no activity, suggest strong binding of 2 mol (1 per subunit) of all three metals at the same sites, called “conformational” sites. About two-thirds of the possible absorbance change in the chromophoric competitive inhibitor 3-aminoenolpyruvate-2-phosphate (AEP) that occurs when it binds to the enzyme in the presence of saturating levels of magnesium is produced when just 2 mol (1 per subunit) of magnesium is added. Since additional “catalytic” metal won't bind unless the AEP does, and the AEP won't bind unless the “conformational” sites are filled with metal, much of the absorbance change in the AEP must be produced by conformational metal.Metals that do not produce enzymatic activity do not produce the absorbance change in AEP whereas metals that permit any level of enzymatic activity produce the same absorbance change that magnesium does-the reaction is “all or none.” Studies of the effect of calcium, nickel, and magnesium on the CD spectrum of apoenzyme-AEP solutions suggest that activating metals produce an asymmetric chromophore in the AEP. This is interprested as indicating the chromophore in AEP bound to enzyme in the presence of an activating metal is a twisted carbon-carbon double bond.Calorimetric studies show the competitive inhibitor 3-phosphoglycerate binds to the calcium- and magnesium-enzyme with about the same change in enthalpy. The substrate or AEP reduces the rate of the apparent reaction of the calcium- or magnesium-enzyme with excess EDTA, suggesting that both substrate and AEP bind to the calcium-enzyme. The interpretation of these data is that the conformational metal plays a crucial role in activating the substrate while the catalytic metal controls the reaction rate. This interpretation is supported by experiments in which an enzyme with one type of conformational metal is reacted in the stopped-flow with catalytic metal and substrate. If an activating metal is the conformational metal, the initial activity is greater.     

A spectroscopic and electrochemical study of the interaction between copper (II) glycylglycyl-L-histidine-N-methylamide and iron (III) tetra-p-sulfophenylporphine

A binuclear complex has been produced by the reaction of an iron porphyrin (sodium tetra-p-sulfophenylporphine iron (III)-FeTPPS) with a copper metallo-tripeptide (copper (II) glycylglycyl-L-histidine-N-methylamide-CuGGH) in aqueous solution. The system has been characterized by electron spin resonance (ESR) spectroscopy, optical absorption spectroscopy, and electrochemical methods. Room-temperature ESR spectra of the copper complex and low-temperature ESR spectra of the iron porphine provide evidence for the formation of a binuclear complex. These findings are supported by absorption spectroscopy and electrochemical studies, and lead to a value of ca. 2 X 10(-3) M-1 (at room temperature) for the equilibrium constant for complex formation. The relevance of this system to the enzymic active site of mammalian cytochrome c oxidase is discussed.     

Preliminary studies on the inhibitory effect of novel Pd(II) complexes of vitamin B6 on cell divisions

Two novel complexes of Pd(II) involving vitamin B₆ compounds have been synthesized. They are compatible with the compositions Pd(P.H.)₂ C₂(P=pyridoxol) and Pd(PL.H)₂C₂ (PL = pyridoxal). The complexes inhibited the growth as well as the biosynthesis of RNA, DNA, and protein of E. coli B-766. Photoacoustic spectral (PAS) measurements showed that the complexes bound to DNA of the bacteria and were present only in the kidney of treated mice. The complexes inhibited the incorporation of ³H-thymidine as well as ¹⁴C-leucine in the DNA and protein, respectively, of liver cell cultures (BL8L). The inhibition of cell division of Walker-S-cells and human lymphocytes by the complexes was highly significant.     

Ultrastructure-function correlative studies for cardiac cryopreservation. V. Absence of a correlation between electrolyte toxicity and cryoinjury in the slowly frozen,cryoprotected rat heart

Hearts were frozen to ?17 °C in the initial presence of 2.1 m DMSO. Attempts were made to prevent or minimize the consequences of an osmotic shock based on Lovelock's classical hypothesis of freezing injury. Substitution of mannitol or potassium for NaCl before freezing did not improve the results, nor did perfusion of thawed hearts with hyperosmotic perfusate. It was found that freezing and thawing resulted in a significant attenuation of coronary flow and that, as a result of this, DMSO was apparently retained within the heart after thawing. DMSO was also difficult to remove at 30 °C in the absence of prior freezing and caused a significant drop in coronary flow upon institution of DMSO washout with balanced salt solution. The blanching of freezing and thawing was also seen, in milder form, in nonfrozen hearts. For both frozen-thawed and nonfrozen hearts, the blanching was associated with DMSO washout with balanced salt solution. Flow was improved by perfusion with hyperosmotic perfusate in both nonfrozen and in frozen-thawed hearts, but the improvement was largely temporary. Evidence from earlier studies indicates that electrolyte concentrations during freezing cannot be correlated with cardiac cryoinjury, in support of the present findings. It is suggested instead that cryoprotectant toxicity may be the chief agent of injury under the conditions studied.     

Oxidation of thymidylate synthase by inorganic compounds

Thymidylate synthase from methotrexate-resistant Lactobacillus casei was rapidly and completely inactivated by low concentrations of permanganate, periodate, or potassium triiodide at 0 degree C. The enzyme was not inactivated to any appreciable extent by iodate, iodide, ferricyanate, iodosobenzoate, or hydrogen peroxide. The inactivation by permanganate was retarded by the substrate 2'-deoxyuridylate and, to a lesser extent, by phosphate. Titration of enzyme activity with permanganate showed that two moles of permanganate were required to completely inactivate one mole of thymidylate synthase.     

Spectroscopic studies on the binding of iron, terbium, and zinc by apoferritin

Ultraviolet difference spectroscopy has been used to study Fe (III)-apoferritin complexes formed after addition of Fe (II) to apoferritin in air. At constant iron, the recorded spectra varied with time after Fe (II) addition and with the number of iron atoms/molecule (protein concentration). The results indicate that after production of an initial complex, rearrangement or migration of Fe (III) atoms occurs, with polynuclear species forming as end-product, probably by hydrolytic polymerization. The presence of Tb3+ or Zn2+ ions affected the Fe (III) spectra and their development in different ways. The combined data suggest that more than one site, or processes, are involved in ferritin iron-core formation and that some of the metal sites are clustered.     

Some studies on the metabolism of labelled molybdenum compounds in cattle

An initial experiment showed that [99Mo]di- and trithiomolybdates could be detected in bovine plasma after the introduction of [99Mo]molybdate into the rumen. It was felt that this justified the use of [99Mo]trithiomolybdate for the subsequent studies made of plasma thiomolybdate metabolism in vivo in cattle. Rapid intravenous injection of [99Mo]trithiomolybdate into cattle showed that doses of 50 mg Mo were subject to extensive hydrolysis over the first few minutes postinjection, but at lower dose rates this was reduced so that tracer doses (less than 1.5 mg Mo) were relatively stable. The plasma metabolism was unaffected by copper status within the limits of the experiments (that is, liver copper levels down to 9 mg/kg d.m.) The disappearance of [99Mo] and [35S]trithiomolybdate (1 mg Mo) from plasma was delayed for up to 10 hr by the immediate preinjection of copper, although no chemical modification of the thiomolybdate appeared to occur.     

Transesterification of oxo-osmium(VI) ligand complexes of thymine derivatives

We have measured the rate of transesterification reactions between glycols (L_O′) and oxo-osmium(VI) esters formed from thymine derivatives and a variety of nitrogen-containing ligands, L_OML_N, according to the equation: L_OML_N + L_O′

L_O′ML_N + L_O. We have also measured the rates of transesterification for this system with the addition of external nitrogen-containing ligands, L_N′. The order of effectiveness of the nitrogen-containing ligands in slowing the rate of transesterification is different depending on whether the ligand is present in the ester (L_N) or whether it is added externally (L_N′). Bathophenanthroline disulfonic acid is the most effective ligand when present in the ester. It forms exceptionally inert complexes. N,N,N′,N′-Tetramethylethylenediamine is the most effective ligand when added externally. The order of effectiveness of the nitrogen-containing ligand does not depend on the nature of either L_O or L_O′ but the nature of the glycol affects both the rate and the equilibria. We present mechanistic proposals, with limiting assumptions, to account for our findings.     

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.     

The binding of copper(II) and zinc(II) to oxidized glutathione

1H and 13C NMR studies of Zn(II) binding to oxidized glutathione (GSSG) in aqueous solution over the pH range 4-11 show that it forms a complex with a 1:1 Zn:GSSG stoichiometry. At pH values between 6 and 11 the metal ligands are the COO- and NH2 groups of the glutamate residues. Below pH 5 the glycine end of the molecule also binds to the metal ions. EPR and visible absorption spectra of Cu(II) GSSG solutions suggest that similar complexes are formed with Cu(II). The solid products obtained from these solutions are shown by analysis and EPR to be primarily binuclear with Cu2GSSG stoichiometry, although the structures depend on the pH and stoichiometry of the solution from which they were obtained.     

Purification of secretory granules on a urografin gradient

A procedure is described for the preparation of highly purified secretory granules from rat parotid glands. A 250g supernatant fraction of parotid homogenates is layered over a gradient composed of 20, 30, and 40% solution of Urografin. The secretory granules form a layer at the 30–40% interface. Chemical and enzymatic analysis show the purified granules contain 63 ± 8% of the amylase, 2 ± 0.8% of the succinic dehydrogenase, little or no RNA, and 10% of the protein present in the 250g supernatant fraction.     

Spectroscopic studies on the copper(II)—Inosine system

Interactions of inosine derivatives with copper(II) were studied in the pH range 1.4–13 in 50% H₂O-50% DMSO solution. The distinct pH dependence of the optical spectra observed in copper(II)-inosine complexes are correlated to their respective EPR changes as a function of pH. It was concluded that a simple 1:1 complex of copper(II)-inosine is formed in the pH range 1.4–5.0 and bis complexes are present in the pH 5.0–6.2 region solutions of inosine and Cu(II). From pH 6.2 to 7.8 a diamagnetic, hydroxybridged complex dominates. At pH 7.8–9.2 an insoluble, oxybridged species is formed in addition to the soluble paramagnetic Cu(NI)₄ complex. Starting from pH 9.1 the N-polymeric complex is formed which is stable up to pH 12.5, and above pH 12.5 the only species is the Cu(ribose)₂ complex.     

A multinuclear nmr relaxation study of the interaction of divalent metal ions with l-aspartic acid

Carbon-13 spin-lattice relaxation times, T₁, have been measured for aqueous solutions of L-aspartic acid, L-alanine, O-phospho-L-serine, and 2-mercapto-L-succinic acid in the presence of the paramagnetic metal ions, Cu²⁺ and Mn²⁺, and Mg²⁺ as a diamagnetic control, at ambient temperature and neutral pH. Nitrogen-15, oxygen-17 and proton relaxation times were also obtained for L-aspartic acid and phosphorus-31 relaxation times for O-phospho-L-serine under similar conditions. The structures of these complexes in solution were determined from the various metal ion-nuclei distances calculated from the paramagaetically-induced relaxation. These results indicate that the Cu²⁺ interaction with L-aspartic acid is through α-amino and β-carboxyl groups while Mn²⁺ coordinates most strongly through α-and β-carboxyl groups, with the possibility of a weak interaction through the amino group.An examination of the coordination of these divalent metal ions to an analog of L-aspartic acid in which the β-carboxyl group is replaced by a phosphate group (O-phospho-L-serine) indicated that Cu²⁺ coordination is now probably through the α-amino and phosphate groups, while this analog is a monodentate ligand for Mn²⁺ coordinating through the phosphate group. Removal of the β-carboxyl group (L-alanine) also results in Cu²⁺ coordination through the α-carboxyl and α-amino groups, and the same ligand interactions are observed with Mn²⁺. Replacement of the α-amino group of L-aspartic acid with an - SH group (2-mercapto-L-succinate) is sufficient to eliminate any specific coordination with either Cu²⁺ or Mn²⁺.     

Effects of glycol structure on the transesterification equilibria of oxo-osmium(VI) esters

We report equilibrium data on reactions between the N,N,N′,N′-tetramethylethylenediamine oxo-osmium(VI) ester of thymine with a variety of polyhydroxylated compounds including sugars, glycosides, sugar alcohols, and nucleosides. Some rate data are also included. The cis-furanosidic nucleosides showed the largest equilibrium constants; among the pyranosidic systems, the cis (ae) structure was preferred.     

Hydroxyl free radical production in iron-cysteine solutions and protection by zinc

Hydroxyl radicals (OH^?) can be formed on incubation of an oxygenated solution of ferrous sulphate and cysteine. This has been demonstrated by esr using the spin trap DMPO (5,5-dimethyl-1-pyrroline-1-oxide), catalase, and the radical scavengers ethanol and propan-2-ol. Hydroxyl radicals are not formed when excess zinc sulphate is present. These results provide support for the pro-oxidant action of iron and cysteine and a possible protective role for zinc.     

Regulation of peroxo-bridge formation between cobalt(II)-carnosine complexes by histidine: Possible involvement in polycythemia

The mechanisms by which histidine stabilizes the cobalt(II)-carnosine complex from oxidation to cobalt(III) in aqueous solution are investigated with ¹H-nmr, laser Raman, and Fourier transform-infrared spectroscopy. Histidine has at least three effects on the cobalt(II)-carnosine complex. First, over the concentration range of at least 5 to 250 mM, histidine stabilizes the cobalt(II)-carnosine complex from oxidation by excluding solvent molecules from the equatorial coordination positions. Second, at the upper end of this concentration range, histidine reduces the strained nonplanarity of the equatorial coordination positions around the cobalt(II) ion that results from tridentate chelation by carnosine. Bidentate ligation by histidine causes the carnosine to bind as a bidentate ligand also. Third, bidentate ligation of two carnosine molecules to the equatorial coordination positions of Co(II) ion places the β-alanyl residues inthe vicinity of the two axial coordination positions and thereby inhibits the binding of molecular oxygen. Substitution of a molecule of histidine for one of these two carnosine molecules makes an axial coordination position available for binding oxygen. The first two effects are expected to stabilize the cobalt(II) ion from rapid oxidation, whereas the third effect is expected to give long-term stability of the peroxo-bridged complex. Since bidentate ligation of histidine is favored over monodentate ligation only when the concentration of Co(II) ion is not limiting and is inhibited by high concentrations of carnosine in the same solution, the results presented provide a possible explanation for the observation that the stability of the Co(II) complexes toward oxidation and their ability to bind molecular oxygen depend on both the relative and absolute concentrations of Co(II) ion, carnosine, and histidine in solution. Furthermore, these results provide additional support to the suggestion that the high activity of carnosinase in kidney is involved in part in regulation of the oxygen sensor in this organ.