Effects of metal ions on sphingomyelinase activity of Bacillus cereus

Some divalent metal ions were examined for their effects on sphingomyelinase activity of Bacillus cereus. The enzyme activity toward mixed micelles of sphingomyelin and Triton X-100 proved to be stimulated by Co2+ and Mn2+, as well as by Mg2+. Km's for Co2+ and Mn2+ were 7.4 and 1.7 microM, respectively, being smaller than the Km for Mg2+ (38 microM). Sr2+ proved to be a competitive inhibitor against Mg2+, with a Ki value of 1 mM. Zn2+ completely abolished the enzyme activity at concentrations above 0.5 mM. The concentration of Zn2+ causing 50% inhibition of the enzyme activity was 2.5 microM. Inhibition by Zn2+ was not restored by increasing concentrations of Mg2+ when the concentration of Zn2+ was above 10 microM. Ba2+ was without effect. When sphingomyelinase was incubated with unsealed ghosts of bovine erythrocytes at 37 degrees C, the enzyme was significantly adsorbed onto the membrane in the presence of Mn2+, Co2+, Sr2+ or Ba2+. Incubation with intact or Pronase-treated erythrocytes caused enzyme adsorption only in the presence of Mn2+. In the course of incubation, the enzyme was first adsorbed on the membranes of intact bovine erythrocytes in the presence of Mn2+; then sphingomyelin breakdown proceeded with ensuing desorption of adsorbed enzyme. Hot-cold hemolysis occurred in parallel with sphingomyelin breakdown. In this case, the hydrolysis of membranous sphingomyelin as well as the initial enzyme adsorption took place in the following order: unsealed ghosts greater than Pronase-treated erythrocytes greater than intact erythrocytes.     

Purification and Mn2+ activation of Rhodospirillum rubrum nitrogenase activating enzyme.

The Fe protein activating enzyme for Rhodospirillum rubrum nitrogenase was purified to approximately 90% homogeneity, using DE52-cellulose chromatography and sucrose density gradient centrifugation. Activating enzyme consists of a single polypeptide of molecular weight approximately 24,000. ATP was required for catalytic activity, but was relatively ineffective in the absence of Mg2+. When the concentration of MgATP2- was held in excess, there was an additional requirement for a free divalent metal ion (Mn2+) for enzyme activity. Kinetic experiments showed that the presence of Mg2+ influenced the apparent binding of Mn2+ by the enzyme, resulting in a lowering of the concentration of Mn2+ required to give half-maximum activity (K alpha) as the free Mg2+ concentration was increased. A low concentration of Mn2+ had a sparing effect on the requirement for free Mg2+. There is apparently a single metal-binding site on activating enzyme which preferentially binds Mn2+ as a positive effector, and free Mg2+ can compete for this site.     

Effect of manganese(ous) and sulfate on activity of human placental glucose 6-phosphate-dependent form of glycogen synthase.

The human placental glucose-6-P-dependent form of glycogen synthase, in the absence of glucose-6-P, can be activated by MnSO4. Separately, Mn2+ and SO4(2-) have no significant effect. In the presence of glucose-6-P, Mn2+ activates the enzyme, but SO4(2-) inhibits; MnSO4 synergetically increases the enzyme activity. Mn2+ reduces the Ka for glucose-6-P to one-tenth of the control value; SO4(2-) increases the Ka 5-fold; however, MnSO4 has no effect on Ka. MnSO4, like glucose-6-P, increases the Vmax of the enzyme in the presence of its substrate, UDP-glucose; it slightly increases the Km for UDP-glucose. In the presence of glucose-6-P, Mn2+ increases and SO4(2-) decreases the Vmax of the enzyme, but neither has an effect on the Km for UDP-glucose. At physiological concentrations of UDP-glucose and glucose-6-P, either Mn2+ or MnSO4 at concentrations less than 1 mM increases the enzyme activity as much as 8 mM glucose-6-P does. At physiological concentrations of UDP-glucose and glucose-6-P, Mn2+ or MnSO4 reverses the inhibition of the enzyme by ATP.     

L-threonine dehydrogenase from Escherichia coli K-12: thiol-dependent activation by Mn2+

Addition of 1 mM Mn2+ to all solutions in the final chromatographic step used to purify L-threonine dehydrogenase (L-threonine:NAD+ oxidoreductase, EC 1.1.1.103) from extracts of Escherichia coli K-12 routinely provides 30-40 mg of pure enzyme per 100 g wet weight of cells with specific activity = 20-30 units/mg. Enzyme dialyzed exhaustively against buffers containing Chelex-100 resin has a specific activity = 8 units/mg and contains 0.003 or 0.02 mol of Mn2+/mol of enzyme as determined by radiolabeling studies with 54Mn2+ or by atomic absorption spectroscopy, respectively. Dehydrogenase activity is completely abolished by low concentrations of either Hg2+ or Ag+; of a large spectrum of other metal ions tested, only Mn2+ and Cd2+ have an activating effect. Activation of threonine dehydrogenase by Mn2+ is thiol-dependent and is saturable with an activation Kd = 9.0 microM and a Vmax = 105 units/mg. Stoichiometry of Mn2+ binding was found to be 0.86 mol of Mn2+/mol of enzyme subunit with a dissociation constant (Kd) = 8.5 microM. Mn2+ appears to interact directly with threonine dehydrogenase; gel filtration studies with the dehydrogenase plus 54Mn2+ in the presence of either NAD+, NADH, L-threonine, or combinations thereof show that only Mn2+ coelutes with the enzyme whereas all other ligands elute in the salt front and the stoichiometry of the dehydrogenase-Mn2+ interaction is not affected in any instance. A theoretical curve fit to data for the pH-activity profile of Mn2+-saturated enzyme has a pKa = 7.95 for one proton ionization. The data establish L-threonine dehydrogenase of E. coli to be a metal ion activated enzyme.     

Guanylate cyclase: assay and properties of the particulate and supernatant enzymes in mouse parotid.

A new, very sensitive, rapid and reliable assay for guanylate cyclase has been established based on conversion of [32P]GTP to [32P]guanosine 3':5'-monophosphate and its separation on Dowex 50 and aluminium oxide columns. The optimum conditions for the assay of mouse parotid guanylate cyclase have been established and using this procedure the properties of the enzyme have been investigated. The enzyme was found in both the particulate and supernatant fractions. The particulate enzyme was activated 12-fold by Triton X-100 and the supernatant enzyme activity increased 2-fold. In the presence of detergent guanylate cyclase activity was distributed 85% in the particulate and 15% in the supernatant fractions, respectively. The particulate activity was localised in a plasma membrane fraction. Guanylate cyclase activity was also assayed in a wide variety of other tissues. In all cases enzymatic activity was found in both the particulate and supernatant fractions. The distribution varied with the tissue but only the intestinal mucosa had a greater proportion of total guanylate cyclase activity in the particulate fraction than the parotid. The two enzymes showed some similar properties. Their pH optima were pH 7.4, both enzymes were inhibited by ATP, dATP, dGTP and ITP, required Mn2+ for activity and plots of activity versus Mn2+ concentration were sigmoidal. However, in many properties the enzymes were dissimilar. The ratios of Mn2+ to GTP for optimum activity were 4 and 1.5 for the supernatant and plasma-bound enzymes, respectively. The slope of Hill plots for the supernatant enzyme with varying Mn2+ was 2. The particulate enzyme plots also had a slope of 2 at low Mn2+ concentration but at higher concentrations (above 0.7 mM) the Hill coefficient shifted abruptly to 4. Calcium ions reduced sigmoidicity of the kinetics lowering the Hill coefficient, activated the enzyme at all Mn2+ concentrations but had no effect on the Mn2+:GTP ratio with the supernatant enzyme while with the plasma membrane enzyme Ca2+ had no effect on the sigmoid form of the kinetics at low Mn2+ but prevented the shift to a greater Hill coefficient at higher Mn2+, inhibited the activity at low Mn2+ and shifted the Mn2+:GTP optimum ratio to 4. For the particulate enzyme plots of activity versus GTP concentration were sigmoid (n = 1.3), while the supernatant enzyme exhibited hyperbolic kinetics.     

Extended X-ray absorption fine structure of Mn2+ and Mn2+ X ATP complex bound to coupling factor 1 of the H+-ATPase from chloroplasts

The spinach chloroplast ATPase, coupling factor 1, contains three tight Mn2+-binding sites which interact cooperatively. The bound manganese coordinations were studied by x-ray absorption fine structure analysis. Mn2+ was found to be bound to the enzyme with an average Mn-O bond length of 2.15 +/- 0.15 A, significantly shorter than the 2.15 +/- 0.15 A of the Mn-O bond of the average first hydration shell for Mn2+ in aqueous solution. On adding ATP to the manganese-enzyme mixture, a tertiary complex of Mn2+ X ATP X enzyme was formed as indicated by the appearance of a second shell. Mn-P bond distances were estimated at 4.95 +/- 0.15 A in the tertiary Mn2+ X ATP X enzyme complex, which was considerably longer than the Mn-P bond distance of 3.36 +/- 0.15 A for the Mn2+ X ATP complex in aqueous solution. The Mn-P bond distance in the tertiary Mn2+ X ATP X enzyme complex decreased to 4.32 +/- 0.15 A when selenite, a potent effector of ATPase activity, was added. Based on these results, it is suggested that the tertiary complex is required for catalysis. The stimulation of ATP hydrolysis by anions such as selenite may be the result of shortening the distance between Mn2+ and the ATP phosphates in the enzyme active site.     

Association-dissociation of mammalian brain glutamine synthetase: effects of metal ions and other ligands

Glutamine synthetase from ovine brain has been found to exist in vivo and in vitro as a Mn4E complex, where E is octameric enzyme [F. C. Wedler, R. B. Denman, and W. G. Roby (1982) Biochemistry 24, 6389-6396]. Previously observed anomolous effects of added metal ions and protein concentration on the observed specific activity in vitro can now be explained in terms of association-dissociation of the native octamer. In the absence of glycerol, added to stabilize the enzyme for long-term storage, activity decreases sharply below 4 micrograms/ml (20 nM octamer) in assay mixtures due to dissociation of octamer to tetramer and thence to inactive monomer. No dimeric species were detectable under any conditions. The octameric species Mn4EMn4 could be activated further by Mn(II) to form a species Mn4EMn4Mn8 that has a specific activity of ca. 900 U/mg in the transferase assay. Enzyme with one Mn(II)/subunit, Mn4EMn4, associated to octamers more extensively than Mn4E. At the low concentrations of enzyme at which the tetramer predominates, addition of substrates alone or in pairs caused partial reassociation to octamers, the most effective combinations being ATP and glutamate, ADP and L-glutamine, or ATP and L-methionine sulfoximine. Analysis of the data by the methods of Kurganov or Thomes and co-workers indicate that the tetramer/octamer equilibrium has a Kd value of ca. 2.5 X 10(-6) M, comparable to values calculated for other enzyme systems. The specific activities for octamer and monomer in the Mg(II)-dependent transferase assay were calculated to be 200 +/- 20 and 0 U/mg, respectively. Direct determination of the specific activity of pure tetramer is hampered by its substrate-promoted reassociation to octamer under assay conditions. Tetramers, produced by 2 M urea and then immobilized on CNBr-activated Sepharose 4B, exhibited a specific activity that was 86% of that of the identically treated octamers. This indicates a specific activity of ca. 172 (+/- 20) for tetramers in solution. Light-scattering experiments showed that, with 1.7-2.0 Mn(II) bound per subunit, the octameric enzyme octamers can associate further to an oligomeric species (Mn4EMn4Mn8)n, where n greater than or equal to 5. This oligomerization also was promoted strongly by lanthanide ions. Mg(II), however, caused only the association of tetramer to octamer.(ABSTRACT TRUNCATED AT 400 WORDS)     

Properties of digitonin-solubilized calmodulin-dependent guanylate cyclase from the plasma membranes of Tetrahymena pyriformis NT-1 cells

Calmodulin-dependent guanylate cyclase from Tetrahymena plasma membranes was solubilized in about a 22% yield by using digitonin in the presence of 0.2 mM CaCl2 and 20% glycerol. The detergent, when present in the assay at concentrations above 0.05%, diminished the basal and calmodulin-stimulated activity of the enzyme. Guanylate cyclase solubilized with digitonin was eluted from DEAE-cellulose with 200 mM KCl in a yield of 50%. Properties of the solubilized enzyme were similar to those of the native membrane-bound enzyme. The Kms for Mg-GTP and Mn-GTP were 140 and 30 microM, respectively. The enzyme required Mn2+ for maximum activity, the relative activity in the presence of Mg2+ being 30% of the activity with Mn2+. The solubilized enzyme retained the ability to be activated by calmodulin, with its extent being reduced as compared to the membrane-bound enzyme. The presence of a Ca2+-dependent calmodulin-binding site on the solubilized enzyme was shown by the Ca2+-dependent retention of the enzyme on a calmodulin-Sepharose-4B column.     

High activity of NADP-dependent malic enzyme in mitochondria from abdomen muscle of the crayfish Orconectes limosus.

1. Mitochondria isolated from abdomen muscle of crayfish Orconectes limosus exhibit malic enzyme activity in the presence of L-malate, NADP and Mn2+ ions after addition of Triton X-100. Under optimal conditions about 230 nmole of reduced NADP and an equivalent amount of pyruvate are produced per min per mg of mitochondrial protein. 2. The pH optimum for decarboxylation of L-malate is about 7.5. 3. The apparent Km for L-malate, NADP and Mn2+ ions was found to be 0.66, 0.012, and 0.0025 mM, respectively. 4. The requirement for Mn2+ can be replaced by Mg2+, Co2+ and Ni2+ ions; however, higher concentrations of these ions than Mn2+ are required for a full stimulation of malic enzyme activity. 5. Oxaloacetate and pyruvate inhibited the enzyme activity in a competitive manner with apparent Ki values of 0.05 mM and 5.4 mM, respectively.     

桉树人工林地土壤酶活性与微量元素含量的关系

运用典范相关分析研究了桉树人工林地土壤酶活性和土壤微量元素含量关系。结果表明,Zn和Mn对土壤蛋白酶活性的促进作用最大。Zn在一定程度上对脲酶和过氧化氢酶有抑制作用;而Mn对脲酶和过氧化氢酶有促进作用。结合林地生物的生长特征等因子,"综合土壤酶因子"可作为土壤肥力评价的一个指标,对桉树林地土种的划分有一定意义。     

Characterization of an adenylyl cyclase activity in particulate preparations from epimastigote forms of Trypanosoma cruzi.

Particulate preparations from epimastigote forms of Trypanosoma cruzi contain an adenylyl cyclase (ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1) which could be stored at --20 degree C and resisted 5 cycles of freezing and thawing over 10 days without significant loss of activity. The enzyme reaction strictly required Mn2+, had a pH optimum of 7.7 and was not inhibited or stimulated by NaF. Particles prepared in the presence of 10 mM Mn2+ or Mg2+ were 3--4 times more active than particles prepared in the absence of these cations. However, Mg2+ could not substitute for Mn2+ during enzyme assay nor did it enhance activity in the presence of saturating concentrations of Mn2+. The binary complex Mn - ATP2- was shown to be the true substrate for the adenylyl cyclase and free ATP was highly inhibitory. Plots of enzyme activity against equimolar concentrations of ATP - Mn gave sigmoid curves with n values in Hill plots ranging between 1.5 and 2.0. Excess Mn2+ activated the cyclase catalyzed reaction at low but not at high concentrations of ATP - Mn. In the presence of an excess of 1 mM Mn2+, which transforms 97% of the added ATP to productive Mn - ATP2- complex, the substrate saturation curve assumed a Michaelian pattern with an apparent Km =0.2 mM.     

ADP, chloride ion, and metal ion binding to bovine brain glutamine synthetase

The binding of divalent cations and nucleotide to bovine brain glutamine synthetase and their effects on the activity of the enzyme were investigated. In ADP-supported gamma-glutamyl transfer at pH 7.2, kinetic analyses of saturation functions gave [S]0.5 values of approximately 1 microM for Mn2+, approximately 2 mM for Mg2+, 19 nM for ADP.Mn, and 7.2 microM for ADP.Mg. The method of continuous variation applied to the Mn2+-supported reaction indicated that all subunits of the purified enzyme express activity when 1.0 equiv of ADP is bound per subunit. Measurements of equilibrium binding of Mn2+ to the enzyme in the absence and presence of ADP were consistent with each subunit binding free Mn2+ (KA approximately equal to 1.5 X 10(5) M-1) before binding the Mn.ADP complex (KA' approximately equal to 1.1 X 10(6) M-1). The binding of the first Mn2+ or Mg2+ to each subunit produces structural perturbations in the octameric enzyme, as evidenced by UV spectral and tryptophanyl residue fluorescence changes. The enzyme, therefore, has one structural site per subunit for Mn2+ or Mg2+ and a second site per subunit for the metal ion-nucleotide complex, both of which must be filled for activity expression. Chloride binding (KA' approximately equal to 10(4) M-1) to the enzyme was found to have a specific effect on the protein conformation, producing a substantial (30%) quench of tryptophanyl fluorescence and increasing the affinity of the enzyme 2-4-fold for Mg2+ or Mn2+. Arsenate, which activates the gamma-glutamyl transfer activity by binding to an allosteric site, and L-glutamate also cause conformational changes similar to those produced by Cl- binding. Anion binding to allosteric sites and divalent metal ion binding at active sites both produce tryptophanyl residue exposure and tyrosyl residue burial without changing the quaternary enzyme structure.     

Study of properties of NADP malate dehydrogenase from corn leaves]

Kinetic properties of purified chloroplast isoenzyme of the "malic" enzyme from corn leaves were studied. The enzyme had optimum activity at pH 8.0 and 36 degrees C. Under standart conditions the Michaelis constants for the "malic" enzyme with Mn2+ as cofactor are 0.091 mM for malate and 0.04 mM for NADP. In case of Mg2+ as cofactor they are 0.66 and 0.02 mM respectively. Respective Km values for the cofactors Mn2+ and Mg2+ are 0.018 and 0.091 mM. The activity of the "malic" enzyme was inhibited by reduced NADP and NAD, ATP, ADP, fructose-1,6-diphosphate, oxaloacetic, oxalic, glyoxylic, glycolic and alpha-ketoglutaric acids, as well as by phosphate anions and pyrophosphate. The inhibitory effect of all metabolites and ions is more pronounced in case of Mn, rather than Mg, used as cofactors for the reaction. A possibility of metabolic regulation of NADP-"malic" enzyme activity in the leaves of C4-plants, is discussed.     

Purification and properties of mitochondrial phosphoenolpyruvate carboxykinase from liver of Squalus acanthias

Liver from Squalus acanthias (spiny dogfish), a representative elasmobranch, contains approximately 1.4 units (mumol/min) of phosphoenolpyruvate carboxykinase activity per gram and approximately 90% of the total units of activity are localized in the mitochondria. The mitochondrial phosphoenolpyruvate carboxykinase was isolated and characterized. The purified enzyme has properties generally similar to those found in mammalian and avian species. The enzyme has a molecular weight of approximately 70,000 and exists in a functional state as a monomer. The isolated enzyme displays a dual cation requirement (e.g., 6 mM Mg2+ and 10 microM Mn2+) for maximal activity; very little activity is observed when Mg2+ is present alone, and the maximal activity attained with Mn2+ alone (millimolar concentrations required) is significantly less than that observed under optimal conditions with both cations present. When assayed in the direction of oxalacetate formation there is a lag in product formation with time; the lag can be eliminated by the presence of 50 microM GTP (product). The Km for substrates is not affected by Mn2+ concentration, suggesting that the role of Mn2+ may not be related to substrate binding. The apparent Km for phosphoenolpyruvate (approximately 1 mM) is substantially higher than that reported for phosphoenolpyruvate carboxykinase from other species. The activity of phosphoenolpyruvate carboxykinase is increased 70% by physiological concentrations of urea. Maximal velocity of the reaction in the direction of oxalacetate formation is approximately half that of the reverse reaction.     

Ureidoglycolate amidohydrolase from developing French bean fruits (Phaseolus vulgaris [L.].)

Ureidoglycolate is an intermediate of allantoin catabolism in ureide-transporting legumes. This report describes the first purification of ureidoglycolate degrading activity (UGDA) from plant tissue in which the enzyme has been separated from urease. The enzyme from developing fruits of Phaseolus vulgaris has been purified 48-fold to give a preparation free of allantoinase and urease activity. UGDA was inhibited by EDTA while the Vmax was increased in the presence of Mn2+. The Km values for ureidoglycolate in the presence and the absence of Mn2+ were 2.0 and 5.4 mM, respectively. In the absence of Mn2+ UGDA was heat labile at 40 degrees C, but in the presence of Mn2+ the activity was stable up to temperatures of 60 degrees C. The Mr of UGDA was determined to be 300,000 by gel filtration chromatography and the pH optimum ranged from pH 7.0 to 8.5. Ammonia was determined to be the nitrogen-containing product of UGDA by a microdiffusion assay. This enzyme should therefore be described as ureidoglycolate amidohydrolase. The activity was shown to be associated with peroxisomes by fractionation of a crude extract on a sucrose density gradient. The products of ureidoglycolate degradation are glyoxylate, ammonia, and presumably carbon dioxide, which can be readily utilized by pathways of metabolism that are known to be present in this organelle.     

Manganese-dependent inhibition of human liver arginase by borate

Full activation of human liver arginase (EC 3.5.3.1), by incubation with 5 mM Mn2+ for 10 min at 60 degrees C, resulted in increased Vmax and a higher sensitivity of the enzyme to borate inhibition, with no change in the K(m) for arginine. Borate behaved as an S-hyperbolic I-hyperbolic non-competitive inhibitor and had no effect on the interaction of the enzyme with the competitive inhibitors L-ornithine (Ki = 2 +/- 0.5 mM), L-lysine (Ki = 2.5 +/- 0.4 mM), and guanidinium chloride (Ki = 100 +/- 10 mM). The pH dependence of the inhibition was consistent with tetrahedral B(OH)4- being the inhibitor, rather than trigonal B(OH)3. We suggest that arginase activity is associated with a tightly bound Mn2+ whose catalytic action may be stimulated by addition of a more loosely bound Mn2+, to generate a fully activated enzyme form. The Mn2+ dependence and partial character of borate inhibition are explained by assuming that borate binds in close proximity to the loosely bound Mn2+ and interferes with its stimulatory action. Although borate protects against inactivation of the enzyme by diethyl pyrocarbonate (DEPC), the DEPC-sensitive residue is not considered as a ligand for borate binding, since chemically modified species, which retain about 10% of enzymatic activity, were also sensitive to the inhibitor.     

A histidine residue at the active site of avian liver phosphoenolpyruvate carboxykinase

The histidine-selective reagents diethylpyrocarbonate (DEPC) and dimethylpyrocarbonate were used to study active site residues of phosphoenolpyruvate carboxykinase. Both reagents show pseudo first-order inhibition of enzyme activity at 22 +/- 1 degree C with calculated second-order rate constants of 2.8 and 4.6 M-1 s-1, respectively. The inhibition appears partially reversible. Substrates affect the rate of inhibition: KHCO3 enhances the rate, Mn2+ has little effect, and phosphoenolpyruvate decreases the rate. The best protection is obtained by IDP or IDP and Mn2+. The kinetic studies show that modification of histidine is specific and leads to loss of enzymatic activity. Two histidines per enzyme are modified by DEPC, as measured by an absorption change at 240 nm, in the absence of substrate, leading to loss in activity. One histidine per molecule is modified in the presence of KHCO3, giving inactivation. Cysteine and lysine residues are not affected. A study of the inhibition rate constant as a function of pH gives a pKa of 6.7. Enzyme modified by DEPC in the absence of substrate (1% remaining activity) shows no binding of ITP or of phosphoenolpyruvate to the enzyme.Mn2+ complex as studied by proton relaxation rates. When enzyme is modified in the presence of KHCO3 (44% remaining activity), ITP and KHCO3 bind to the enzyme.Mn2+ complex similarly to the binding to native enzyme. Phosphoenolpyruvate binding to modified enzyme.Mn results in an enhancement of proton relaxation rates rather than the decrease observed with native enzyme.Mn. The CD spectra of histidine-modified enzyme show a decrease in alpha-helical and random structure with an increase in anti-parallel beta-sheet structure compared to native enzyme. These results show that avian phosphoenolpyruvate carboxykinase has 2 histidine residues which are reactive with DEPC and dimethylpyrocarbonate, and one of the 15 histidine residues in the protein is at or near the phosphoenolpyruvate binding site and is involved in catalysis.     

Kinetic and thermodynamic characterization of the functional properties of a hybrid versatile peroxidase using isothermal titration calorimetry: Insight into manganese peroxidase activation and lignin peroxidase inhibition

Isothermal titration calorimetry (ITC) was developed for measuring lignin peroxidase (LiP) and manganese peroxidase (MnP) activities of versatile peroxidase (VP) from Bjerkandera adusta. Developing an ITC approach provided an alternative to colorimetric methods that enabled reaction kinetics to be accurately determined. Although VP from Bjerkandera adjusta is a hybrid enzyme, specific conditions of [Mn+2] and pH were defined that limited activity to either LiP or MnP activities, or enabled both to be active simultaneously. MnP activity was found to be more efficient than LiP activity, with activity increasing with increasing concentrations of Mn+2. These properties of MnP were explained by a second metal binding site involved in homotropic substrate (Mn+2) activation. The activation of MnP was also accompanied by a decrease in both activation energy and substrate (Mn) affinity, reflecting a flexible enzyme structure. In contrast to MnP activity, LiP activity was inhibited by high dye (substrate) concentrations arising from uncompetitive substrate inhibition caused by substrate binding to a site distinct from the catalytic site. Our study provides a new level of understanding about the mechanism of substrate regulation of catalysis in VP from B. adjusta, providing insight into a class of enzyme, hybrid class II peroxidases, for which little experimental data is available.     

Characterization of guanylate cyclase of rod outer segments of the bovine retina.

Guanylate cyclase (GTP pyrophosphate-lyse (cyclizing), EC 4.6.1.2.) of bovine retinal rod outer segments is almost completely particulate, i.e. associated with rod outer segment membranes. In contrast to particulate guanylate cyclase in other tissues, treatment of rod outer segments with Triton X-100 does not solublize the enzyme but inhibits it. Enzyme activity is dependent on the presence of divalent cation, especially Mn2+ with only poor activation by Mg2+ (10-fold lower) and no activation seen with other cation. Ezpression of maximal activity required Nm2+ and GTP in equimolar concentrations with an apparent Km of 8 . 10(-4) M and V of 10 nmol/min per mg protein. Excess of Mn2+ over that required for the formation of the Mn . GTP complex was inhibitory. Ca2+, Ba2+ and Co2+ inhibited enzyme activity when assayed with the Mn . GTP substrate complex. In the presence of a fixed concentration of 1mM Mn2+, the enzyme exhibited strong negative cooperative interactions with GTP, characterized by an intermediary plateau region in the substrate vs. enzyme activity curve, a curve of downward concavity in the double reciprocal plot and a Hill coefficient of 0.5. Nucleotides such as ITP, ATP and UTP at higher concentrations (1 mM) stimulates activity by 40%. NaN3 has no effect on the guanylate cyclase. It is thus possible that the guanylate cyclase may be regulated in vivo by both the metal : GTP substrate ratio and the free divalent cation concentration as well as by the ATP concentration and thus play an important but yet undefined role in the visual process.     

Spatial proximity of two divalent metal ions at the active site of S-adenosylmethionine synthetase

S-Adenosylmethionine synthetase from Escherichia coli is shown to require 2 divalent metal ions/enzyme subunit for maximal enzymatic activity. In the absence of substrate, the tetrameric enzyme binds 1 Mn(II) ion/subunit, whereas in the presence of a nucleotide substrate, adenylylimidodiphosphate, or the product pyrophosphate, there are two Mn(II)-binding sites/subunit. Electron paramagnetic resonance spectra of Mn(II) bound to the enzyme reveal a spin exchange interaction between 2 Mn(II) ions in complexes of enzyme and Mn(II) which also contain adenosylmethionine, K+, and either pyrophosphate or imidotriphosphate. Since a spin exchange interaction requires orbital overlap between the 2 ions, the metal ions must be bound close to one another, and they may share a common ligand.