Activation of liver cytosol phosphoenolpyruvate carboxykinase by Ca2+ through intracellular redistribution of Mn2+

Calcium has no known direct effect on phosphoenolpyruvate carboxykinase from rat liver cytosol. However, addition of calcium salts to liver postnuclear supernatant led to an increase in assayable enzyme activity in cytosols. This indicates that mitochondria and microsomes present in postnuclear supernatant can participate in observed enzyme activation. The stimulation of phosphoenolpyruvate carboxykinase was prevented by the manganese complexion 1-(2-pyridylazo)-2-naphthol, was not additive with activation by MnCl2 and was inhibited by La3+, Sr2+ and ruthenium red. These data indicate that manganese and mitochondrial or microsomal calcium carriers participate in the mechanism of indirect calcium effect. Measuring of manganese content in cytosols directly, by atomic absorption spectrometry, has provided evidence that there is a pool of manganese associated with mitochondrial and microsomal fraction of rat liver that can be mobilized to the cytosol by calcium ions. The direct addition of this pool of manganese to the cytosol caused the stimulation of phosphoenolpyruvate carboxykinase activity to the same levels as did calcium ions in the postnuclear supernatant. It is postulated that calcium can effect enzyme activity indirectly by releasing manganese from specific cellular compartments into the cytosol.     

Mn(II) binding sites of calf liver arginase

Commercial calf liver arginase was further purified through gel filtration column chromatography. The enzyme is nearly homogeneous in SDS-PAGE; it contains 4 manganese atoms per molecule of enzyme. By dialysis against 1,10-o-phenanthroline at 4°C it is possible to obtain an arginase apo-form containing 2 manganese atoms per molecule of enzyme (apo-2 form) while a treatment of the pur enzyme with o-phenanthroline at 37°C followed by dialysis against 0.1 M NaCl is capable of producing an apoenzyme with only 1 manganese atom per molecule (apo-1 form). The apo-2 and apo-1 arginases retain respectively about 50% and 25% of the full enzymatic activity. NMR titrations of both apo-arginases with increasing concentrations of manganese allowed us to determine the affinity constants for the binding of Mn²⁺ to the protein. It was shown that in this enzyme two manganese atoms are weakly bound, one is more strongly bound and the fourth one is bound so tightly that it is not removed under the experimental conditions used.     

Nitrate reductase from Chlorella vulgaris. Reaction with manganese (III) pyrophosphate and with ferric o-phenanthroline.

Millimolar concentrations of tervalent manganese pyrophosphate can partially activate nitrate reductase which has been inactivated with NADH and HCN. The tervalent manganese complex is nevertheless not reduced by NADH in the presence of the enzyme, that is, it is not a substrate for the diaphorase moiety of the nitrate reductase. Ferric o-phenanthroline, on the other hand, is a good diaphorase substrate, but fails to activate the inactive enzyme.     

Structural explanation for the role of Mn2+ in the activity of phi6 RNA-dependent RNA polymerase

The biological role of manganese (Mn(2+)) has been a long-standing puzzle, since at low concentrations it activates several polymerases whilst at higher concentrations it inhibits. Viral RNA polymerases possess a common architecture, reminiscent of a closed right hand. The RNA-dependent RNA polymerase (RdRp) of bacteriophage 6 is one of the best understood examples of this important class of polymerases. We have probed the role of Mn(2+) by biochemical, biophysical and structural analyses of the wild-type enzyme and of a mutant form with an altered Mn(2+)-binding site (E491 to Q). The E491Q mutant has much reduced affinity for Mn(2+), reduced RNA binding and a compromised elongation rate. Loss of Mn(2+) binding structurally stabilizes the enzyme. These data and a re-examination of the structures of other viral RNA polymerases clarify the role of manganese in the activation of polymerization: Mn(2+) coordination of a catalytic aspartate is necessary to allow the active site to properly engage with the triphosphates of the incoming NTPs. The structural flexibility caused by Mn(2+) is also important for the enzyme dynamics, explaining the requirement for manganese throughout RNA polymerization.     

Purification and characterization of an aminoacyl proline hydrolase from guinea-pig intestinal mucosa.

The purification of an aminoacylproline hydrolase from guinea-pig intestinal mucosa is described. The enzyme, which is an aminopeptidase has a molecular weight of 112 000 and is activated by manganese and inhibited by zinc. Unlike other aminoacylproline hydrolases this enzyme displayed a broad substrate specificity. However, it was preferentially active against dipeptides containing proline in the C-terminal position.     

The insulin-mimetic action of Mn2+: involvement of cyclic nucleotides and insulin in the regulation of hepatic hexokinase and glucokinase

Manganese causes a significant rise in hepatic glucokinase and hexokinase in 16-day-old suckling rats, and has an insulinomimetic effect in producing a precocious emergence of glucokinase (EC 2.7.1.2) and a rise in the low Km, hexokinases (EC 2.7.1.1) activities. These enzyme changes occur within 6 hr of manganese administration and there are accompanying increases in plasma insulin and hepatic cyclic GMP. That the effect of manganese is at a site other than, or in addition to, insulin secretion is suggested by the significant increases in glucokinase and hexokinase in 16-day-old streptozotocin-diabetic rats; in this group there is also an increase in hepatic cGMP similar in time scale to that of the normal-manganese-treated group. The effects of manganese and insulin were not additive. It is proposed that one site of action of manganese may be at the level of cyclic GMP systems. The results are also discussed in relation to the known action of manganese at the level of the protein phosphatases.     

Regulation of Escherichia coli glutamine synthetase. Evidence for the action of some feedback modifiers at the active site of the unadenylylated enzyme.

The interaction of unadenylylated form of Escherichia coli glutamine synthetase with several substrates and effectors has been examined by magnetic resonance techniques. These studies show that two manganese ions bind per enzyme subunit. From the dramatic line broadening observed in the alanine spectra in the presence of manganese and enzyme, it is concluded that the binding of alanine occurs at a site nearer one of the two manganese sites. Electron spin resonance (ESR) titration experiments suggest apparent dissociation constants of 20 and 120 muM for manganese to these sites in the presence of 1.0 mM magnesium ion. The manganese concentration dependence of the broadening of alanine suggests an affinity of 30 muM for the manganese closest to the alanine binding site. This suggests that alanine binds closer to the more tightly bound manganese ion. Glutamate appears to displace the alanine and also appears to bind close to the strongly bound manganese ion. It is proposed that alanine and glutamine bind competitively and in the same site. The binding of alanine and ATP is shown to thermodynamically interact such that the presence of one ligand increases the affinity of the enzyme for the other ligand. The presence of ATP dramatically sharpens the alanine line width when manganese and glutamine synthetase are present. Addition of ADP or phosphate alone has little effect on the alanine line width but the addition of both ADP and phosphate shows the same dramatic sharpening as the addition of ATP alone, suggesting an induced fit conformational change in the enzyme induced by ATP or by both ADP and phosphate. A binding scheme is proposed in which all feedback inhibitors of the enzyme bind in a competitive fashion with substrates.     

Influence of manganese on enzyme synthesis and citric acid accumulation inAspergillus niger

Summary A comparison of citric acid fermentations in manganese-deficient and manganese-containing media showed that manganese strongly influences idiophase metabolism. In the presence of manganese, cell growth increases, sugar consumption is diminished and acidogenesis decreases drastically. An investigation of the key enzymes of glycolysis, the pentosephosphate pathway, TCA-cycle, nitrogen metabolism, and gluconeogenesis indicated that manganese deficiency was accompanied by a repression of anabolic and TCA-cycle-enzymes with the exception of citrate synthase. The activity of this enzyme and the enzymes of glycolysis paralleled the sugar consumption rate. In the presence of manganese, no repression of enzyme synthesis was observed. Activities of 2-oxoglutarate dehydrogenase and isocitrate lyase could not be detected in either case. The results support the hypothesis that manganese deficiency mainly affects the operation of biosynthetic reactions inAspergillus niger, thus leading to an overflow of citric acid as an end product of glycolysis.     

Ribonucleotide reductase of Brevibacterium ammoniagenes is a manganese enzyme

Ribonucleotide reduction and not DNA replication is the site for the specific manganese requirement of DNA synthesis and cell growth in the coryneform bacterium Brevibacterium ammoniagenes. To characterize the metal effect we have isolated and purified ribonucleoside-diphosphate reductase from overproducing bacteria that were first deprived of and then reactivated by manganese ions. Purification on columns of Sephacryl S400, DEAE-cellulose and hydroxyapatite provided an apparently homogeneous enzyme consisting of two protein subunits. These were characterized by affinity chromatography on 2',5'-ADP-Sepharose as nucleotide-binding protein B1 (Mr = 80,000) and catalytic protein B2 (Mr = 100,000, composed of two Mr = 50,000 polypeptides), which were both necessary for activity. In vitro the purified enzyme does not require added metal ions except for an unspecific, twofold activity increase observed in the presence of Mg2+ and other divalent cations. Enzyme activity is inhibited by hydroxyurea (I50 = 2.5 mM). The electronic spectrum with maxima around 455 nm and 485 nm closely resembles that of manganese(III)-containing pseudocatalase and of oxo-bridged binuclear Mn(III) model complexes. Denaturation of the enzyme in trichloroacetic acid liberated an equimolar amount of Mn(II) which was detected by EPR spectroscopy. It was not possible to remove and reintroduce metal ions without loss of enzyme activity. Manganese-deficient cell cultures were also grown in the presence of 54MnCl2. Ribonucleotide reductase activity and radioactivity cochromatographed in several systems. Non-denaturing polyacrylamide gel electrophoresis showed that protein subunit B2 was specifically 54Mn-labeled. All these properties suggest that the ribonucleotide reductase of B. ammoniagenes is a manganese-containing analog of the non-heme-iron-containing reductases of Escherichia coli and eukaryotes.     

Biosynthetic Ca2+/Sr2+ exchange in the photosystem II oxygen-evolving enzyme of Thermosynechococcus elongatus

The thermophilic cyanobacterium, Thermosynechococcus elongatus, has been grown in the presence of Sr2+ instead of Ca2+ with the aim of biosynthetically replacing the Ca2+ of the oxygen-evolving enzyme with Sr2+. Not only were the cells able to grow normally with Sr2+, they actively accumulated the ion to levels higher than those of Ca2+ in the normal cultures. A protocol was developed to purify a fully active Sr(2+)-containing photosystem II (PSII). The modified enzyme contained a normal polypeptide profile and 1 strontium/4 manganese, indicating that the normal enzyme contains 1 calcium/4 manganese. The Sr(2+)- and Ca(2+)-containing enzymes were compared using EPR spectroscopy, UV-visible absorption spectroscopy, and O2 polarography. The Ca2+/Sr2+ exchange resulted in the modification of the EPR spectrum of the manganese cluster and a slower turnover of the redox cycle (the so-called S-state cycle), resulting in diminished O2 evolution activity under continuous saturating light: all features reported previously by biochemical Ca2+/Sr2+ exchange in plant PSII. This allays doubts that these changes could be because of secondary effects induced by the biochemical treatments themselves. In addition, the Sr(2+)-containing PSII has other kinetics modifications: 1) it has an increased stability of the S3 redox state; 2) it shows an increase in the rate of electron donation from TyrD, the redox-active tyrosine of the D2 protein, to the oxygen-evolving complex in the S3-state forming S2; 3) the rate of oxidation of the S0-state to the S1-state by TyrD* is increased; and 4) the release of O2 is slowed down to an extent similar to that seen for the slowdown of the S3TyrZ* to S0TyrZ transition, consistent with the latter constituting the limiting step of the water oxidation mechanism in Sr(2+)-substituted enzyme as well as in the normal enzyme. The replacement of Ca2+ by Sr2+ appears to have multiple effects on kinetics properties of the enzyme that may be explained by S-state-dependent shifts in the redox properties of both the manganese complex and TyrZ as well as structural effects.     

Evidence for a new extracellular peroxidase. Manganese-inhibited peroxidase from the white-rot fungus Bjerkandera sp. BOS 55.

A novel enzyme activity was detected in the extracellular fluid of Bjerkandera sp. BOS 55. The purified enzyme could oxidize several compounds, such as Phenol red, 2,6-dimethoxyphenol (DMP), Poly R-478, ABTS and guaiacol, with H2O2 as an electron acceptor. In contrast, veratryl alcohol was not a substrate. This enzyme also had the capacity to oxidize DMP in the absence of H2O2. With some substrates, a strong inhibition of the peroxidative activity by Mn2+ was observed. Phenol red oxidation was inhibited by 84% with only 1 mM of this metal ion. Because DMP oxidation by this enzyme is only slightly inhibited by Mn2+, this substrate should not be used in assays to detect manganese peroxidase. The enzyme is tentatively named 'Manganese-Inhibited Peroxidase'.     

Reduction of CuA induces a conformational change in cytochrome c oxidase from Paracoccus denitrificans.

Cytochrome c oxidase (cytochrome aa3) from Paracoccus denitrificans contains a tightly bound manganese(II) ion, which responds to reduction of the enzyme by a change in its EPR signal (Seelig et al. (1981) Biochim. Biophys. Acta 636, 162-167). In this paper, the nature of this phenomenon is studied and the bound manganese is used as a reporter group to monitor a redox-linked conformational change in the protein. A reductive titration of the cyanide-inhibited enzyme shows that the change in the manganese EPR signal is associated with reduction of CuA. The change appears to reflect a rearrangement in the rhombic octahedral coordination environment of the central Mn2+ atom and is indicative of a redox-linked conformational transition in the enzyme. The manganese is likely to reside at the interface of subunits I and II, near the periplasmic side of the membrane. One of its ligands may be provided by the transmembrane segment X of subunit I, which has been suggested to contribute ligands to cytochrome a and CuB as well. Another manganese ligand is a water oxygen, as indicated by broadening of the manganese EPR signal in the presence of H2(17)O.     

Structural identity between the iron- and manganese-containing superoxide dismutases

We have recently reported the first complete amino acid sequence of an iron-containing superoxide dismutase. The iron enzyme is thought to be closely homologous to the manganese-containing superoxide dismutases. The availability of complete amino acid sequence information for four manganese superoxide dismutases and the crystal structures for two iron and two manganese superoxide dismutases prompted us to investigate the degree of homology between the two proteins at various levels. We report that it is not possible to clearly distinguish the two proteins on the basis of their secondary or tertiary structures. It would appear that a small number of single site substitutions are responsible for conferring distinguishing properties between the two proteins. Substitution of glycine 77 and glutamine 154 by a glutamine and an alanine respectively in Photobacterium leiognathi iron superoxide dismutase may distinguish the kinetic and other particular properties of this protein from the manganese protein (and other iron superoxide dismutases). Furthermore the primary structure of both the iron and manganese proteins does not appear to have any homology with any other known amino acid sequence.     

Manganese(II) oxidation by manganese peroxidase from the basidiomycete Phanerochaete chrysosporium. Kinetic mechanism and role of chelators.

Manganese oxidation by manganese peroxidase (MnP) was investigated. Stoichiometric, kinetic, and MnII binding studies demonstrated that MnP has a single manganese binding site near the heme, and two MnIII equivalents are formed at the expense of one H2O2 equivalent. Since each catalytic cycle step is irreversible, the data fit a peroxidase ping-pong mechanism rather than an ordered bi-bi ping-pong mechanism. MnIII-organic acid complexes oxidize terminal phenolic substrates in a second-order reaction. MnIII-lactate and -tartrate also react slowly with H2O2, with third-order kinetics. The latter slow reaction does not interfere with the rapid MnP oxidation of phenols. Oxalate and malonate are the only organic acid chelators secreted by the fungus in significant amounts. No relationship between stimulation of enzyme activity and chelator size was found, suggesting that the substrate is free MnII rather than a MnII-chelator complex. The enzyme competes with chelators for free MnII. Optimal chelators, such as malonate, facilitate MnIII dissociation from the enzyme, stabilize MnIII in aqueous solution, and have a relatively low MnII binding constant.     

Crystal structure of the di-iron/radical protein of ribonucleotide reductase from Corynebacterium ammoniagenes.

Ribonucleotide reductase (RNR) is the enzyme performing de novo production of the four deoxyribonucleotides needed for DNA synthesis. All mammals as well as some prokaryotes express the class I enzyme which is an alpha(2)beta(2) protein. The smaller of the homodimers, denoted R2, contains a di-iron carboxylate site which, upon reaction with molecular oxygen, generates a stable tyrosyl radical needed for catalysis. The three-dimensional structure of the oxidized class Ib RNR R2 from Corynebacterium ammoniagenes has been determined at 1.85 A resolution and refined to an R-value of 15.8% (R(free) = 21.3%). In addition, structures of both the reduced iron-containing, and manganese-substituted protein have been solved. The C. ammoniagenes R2 has been proposed to be manganese-dependent. The present structure provides evidence that manganese is not oxidized by the protein, in agreement with recent biochemical data, and that no obvious structural abnormalities are seen in the oxidized and reduced iron-containing forms, giving further support that the protein is indeed an iron-dependent RNR R2. The di-manganese structure also provides an explanation for the magnetic properties of this site. The structure of the oxidized C. ammoniagenes R2 also reveals an additional water molecule bridging the radical and the iron site, which has not previously been seen in any other R2 structure and which might have important mechanistic implications.     

Engineering and characterization of human manganese superoxide dismutase mutants with high activity and low product inhibition

Human manganese superoxide dismutase is a mitochondrial metalloenzyme that is involved in protecting aerobic organisms against superoxide toxicity, and has been implicated in slowing tumor growth. Unfortunately, this enzyme exhibits strong product inhibition, which limits its potential biomedical applications. Previous efforts to alleviate human manganese superoxide dismutase product inhibition utilized rational protein design and site-directed mutagenesis. These efforts led to variants of human manganese superoxide dismutase at residue 143 with dramatically reduced product inhibition, but also reduced catalytic activity and efficiency. Here, we report the use of a directed evolution approach to engineer two variants of the Q143A human manganese superoxide dismutase mutant enzyme with improved catalytic activity and efficiency. Two separate activity-restoring mutations were found--C140S and N73S--that increase the catalytic efficiency of the parent Q143A human manganese superoxide dismutase enzyme by up to five-fold while maintaining low product inhibition. Interestingly, C140S is a context-dependent mutation, and the C140S-Q143A human manganese superoxide dismutase did not follow Michaelis-Menten kinetics. The re-engineered human manganese superoxide dismutase mutants should be useful for biomedical applications, and our kinetic and structural studies also provide new insights into the structure-function relationships of human manganese superoxide dismutase.     

Aspartokinase from wheat germ: isolation, characterization, and regulation

Aspartokinase has been isolated from wheat germ and a preliminary survey made of its properties in a partially purified extract. The enzyme has an absolute requirement for ATP and a divalent metal ion. The phosphate donor can be either ATP or GTP, but other nucleotides are ineffective. Both magnesium and manganese will activate the enzyme, whereas calcium shows a trace amount of activity. The enzyme has a Km of 16.7 mm for aspartate, 1.2 mm for ATP, and 3.3 mm for MgCl(2). Lysine inhibits the reaction at fairly low concentrations, and threonine inhibits at high concentrations. Other amino acids which are derived from aspartate (methionine, homoserine, threonine, and isoleucine) have little effect. When lysine and threonine are added together, they show a concerted inhibition of the reaction. The enzyme is also stabilized against heat inactivation by lysine and threonine together but not by either when added separately. It is suggested that aspartokinase from plants is a regulatory enzyme and exhibits a concerted feedback mechanism.     

Metallation state of human manganese superoxide dismutase expressed in Saccharomyces cerevisiae

Human manganese superoxide dismutase (Sod2p) has been expressed in yeast and the protein purified from isolated yeast mitochondria, yielding both the metallated protein and the less stable apoprotein in a single chromatographic step. At 30 °C growth temperature, more than half of the purified enzyme is apoprotein that can be fully activated following reconstitution, while the remainder contains a mixture of manganese and iron. In contrast, only fully metallated enzyme was isolated from a similarly constructed yeast strain expressing the homologous yeast manganese superoxide dismutase. Both the manganese content and superoxide dismutase activity of the recombinant human enzyme increased with increasing growth temperatures. The dependence of in vivo metallation state on growth temperature resembles the in vitro thermal activation behavior of human manganese superoxide dismutase observed in previous studies. Partially metallated human superoxide dismutase is fully active in protecting yeast against superoxide stress produced by addition of paraquat to the growth medium. However, a splice variant of human manganese superoxide dismutase (isoform B) is expressed as insoluble protein in both Escherichia coli and yeast mitochondria and did not protect yeast against superoxide stress.     

Manganese requirement of phosphoglycerate phosphomutase and its consequences for growth and sporulation of Bacillus subtilis.

In the absence of manganese, rapidly metabolizable carbohydrates such as glucose or glycerol are not completely metabolized by Bacillus subtilis growing in a nutrient sporulation medium: 3-phosphoglyceric acid (3PGA) accumulates inside the cells, growth stops at a low cell titer, and normal sporulation remains suppressed (no prespore septa). Upon the addition of manganese, 3PGA disappears, growth resumes, and normal sporulation takes place. These effects results from a specific manganese requirement of phosphoglycerate phosphomutase which catalyzes the interconversion of 3PGA and 2-phosphoglyceric acid (2PGA). Other metal ions cannot replace manganese, for which the enzyme has an apparent Km of 0.22 mM.     

Prostaglandin endoperoxide synthetase from bovine vesicular gland microsomes. Inactivation and activation by heme and other metalloporphyrins.

Prostaglandin endoperoxide synthetase purified to apparent homogeneity from bovine vesicular gland microsomes contained iron far below the equimolar amount and essentially no heme. However, the enzyme required various metalloporphyrins including hematin or several hemoproteins such as hemoglobin. Preincubation of the enzyme with hematin or hemoglobin resulted in the loss of enzyme activity. The enzyme inactivation was protected by tryptophan or various other aromatic compounds. Furthermore, the simultaneous presence of tryptophan brought about activation of enzyme; namely, the enzyme preincubated with heme and tryptophan showed an almost full activity with a heme concentration in the reaction mixture far below the saturating level. Such inactivation and activation of the enzyme were also observed with manganese protoporphyrin. An identical heme requirement, heme-induced inactivation, and activation of the enzyme were observed in three types of reactions catalyzed by the enzyme: 1) bis-dioxygenation of 8,11,14-eicosatrienoic acid to produce prostaglandin G1, 2) 15-hydroperoxide cleavage of prostaglandin G1 to produce prostaglandin H1, and 3) guaiacol peroxidation. When heme was replaced by manganese protoporphyrin, the enzyme catalyzed only the bis-dioxygenation producing prostaglandin G1 and the activities of the latter two reactions were not detectable.