Value Of Mn2 Research Articles

Kinetic and structural analysis of Escherichia coli phosphoenolpyruvate carboxykinase mutants

BackgroundPhosphoenolpyruvate carboxykinase (PEPCK) is a metabolic enzyme in the gluconeogenesis pathway, where it catalyzes the reversible conversion of oxaloacetate (OAA) to phosphoenolpyruvate (PEP) and CO2. The substrates for Escherichia coli PEPCK are OAA and MgATP, with Mn2+ acting as a cofactor. Analysis of PEPCK structures have revealed amino acid residues involved in substrate/cofactor coordination during catalysis. MethodsKey residues involved in coordinating the different substrates and cofactor bound to E. coli PEPCK were mutated. Purified mutant enzymes were used for kinetic assays. The structure of some mutant enzymes were determined using X-ray crystallography. ResultsMutation of residues D269 and H232, which comprise part of the coordination sphere of Mn2+, reduced kcat by 14-fold, and significantly increased the Km values for Mn2+ and OAA. Mutation of K254 a key residue in the P-loop motif that interacts with MgATP, significantly elevated the Km value for MgATP and reduced kcat. R65 and R333 are key residues that interacts with OAA. The R65Q and R333Q mutations significantly increased the Km value for OAA and reduced kcat respectively. ConclusionsOur results show that mutation of residues involved in coordinating OAA, MgATP and Mn2+ significantly reduce PEPCK activity. K254 plays an important role in phosphoryl transfer, while R333 is involved in both OAA decarboxylation and phosphoryl transfer by E. coli PEPCK. General significanceIn higher organisms including humans, PEPCK helps to regulate blood glucose levels, hence PEPCK is a potential drug target for patients with non-insulin dependent diabetes mellitus.

Photoacoustic and Photoluminescence Spectra of ZnS:Mn Nanocrystals

The optical absorption spectra of ZnS:Mn nanocrystals can be obtained by applying photoacoustic (PA) spectroscopy, which is a powerful technique for detecting small amounts of highly scattered materials. The peak position in PA difference spectra increases linearly within experimental accuracy with the decrease of Mn2+ ion concentration. These results correspond to the following possibilities, \\ding172 the increase of the higher excited energy states relative to the ground state of 6A1, \\ding173 the increase of state density for the higher excited states and \\ding174 the increase of transition probability from the 6A1 state to the higher excited states. These possibilities suggest the hybridization of the s-p states of the ZnS nanocrystal host and the d state of the Mn2+ ion impurity. The peak position of the PL spectrum (the transition from 4T1 to 6A1 states) is ∼ 2.08 eV with a half width of 0.24 eV, similar to that of the bulk ZnS:Mn, and it is independent of the Mn2+ ion concentration. This result might be attributed to the fact that the Mn2+ emission is due to phonon assisted transition. Hence, there might be a slight shift of the peak position with increase of Mn2+ ion concentration due to hybridization of the s-p states of the ZnS nanocrystal host and the d state of the Mn2+ ion impurity. The peak intensity of the PL spectrum shows a maximum at a certain value of Mn2+ ion concentration and it decreases with the increase of concentration of Mn2+ ion impurities (concentration quenching).

Phytoene synthase from tomato (Lycopersicon esculentum) chloroplasts--partial purification and biochemical properties.

Phytoene synthase activity in tomato chloroplasts is membrane-associated, requiring treatment with high ionic strength buffer or mild non-ionic detergent for solubilisation. Using a combination of ammonium sulphate precipitation, cation and anion exchange, dye-ligand and hydrophobic interaction chromatography, phytoene synthase has been purified 600-fold from tomato (Lycopersion esculentum Mill.) chloroplasts. The native molecular mass of the enzyme was 43 kDa. with an isoelectric point of 4.6. Although phytoene synthase was functional in a monomeric state, under optimal native conditions it was associated with a large (at least 200 kDa) protein complex which contained other terpenoid enzymes such as isopentenyl diphosphate isomerase and geranylgeranyl diphosphate (GGPP) synthase. Both Mn2+ and ATP, in combination, were essential for catalytic activity; their effect was stochiometric from 0.5 to 2 mM, with Km values for Mn2+, ATP and the substrate GGPP of 0.4 mM, 2.0 mM and 5 microM, respectively. The detergents Tween 60 and Triton X-100 (0.1 w/v) stimulated (5-fold) enzyme activity, but lipids (crude chloroplast lipids and phospholipids) had no such effect and could not compensate for the absence of detergent. A number of metabolites with possible regulatory effects were investigated, including beta-carotene, which reduced enzyme activity in vitro some 2-fold. A comparison of phytoene synthase activity from partially purified chloroplast and chromoplast preparations indicated biochemical differences.

Lysine Residues 162 and 340 Are Involved in the Catalysis and Coenzyme Binding of NADP+-Dependent Malic Enzyme from Pigeon

Alanine-scanning site-directed mutagenesis was carried out on all conserved lysine residues of pigeon cytosolic NADP+-dependent malic enzyme. Only two mutant enzymes, K162A and K340A, showed significant effect on their kinetic parameters. Both mutant enzymes have Km values for Mn2+ and l-malate similar to those of wild-type. The Km value for NADP+ of K162A is identical to that of wild-type. However, K162A demonstrated a 235-fold decrease in the kcat value (0.17 ± 0.01 vs 40.0 ± 1.3 s−1). These data suggested that the side chain of K162 is important for the enzyme catalytic reaction. We propose that the ϵ-amino group of K162 may serve as a general acid to protonate the 3-carbon of enolpyruvate after decarboxylation. The K340A mutant demonstrated no effect on the kcat value. However, its Km value for NADP+ was increased by a factor of 65 (225.7 ± 5.07 vs 3.49 ± 0.05 μM). We propose that the NADP+ specificity is determined by the electrostatic interaction between the ϵ-amino group of K340 and 2′-phosphate of NADP+.

Metal requirements of a diadenosine pyrophosphatase from Bartonella bacilliformis: magnetic resonance and kinetic studies of the role of Mn2+.

Recombinant IalA protein from Bartonella bacilliformis is a monomeric adenosine 5'-tetraphospho-5'-adenosine (Ap4A) pyrophosphatase of 170 amino acids that catalyzes the hydrolysis of Ap4A, Ap5A, and Ap6A by attack at the delta-phosphorus, with the departure of ATP as the leaving group [Cartwright et al. (1999) Biochem. Biophys. Res. Commun. 256, 474-479]. When various divalent cations were tested over a 300-fold concentration range, Mg2+, Mn2+, and Zn2+ ions were found to activate the enzyme, while Ca2+ did not. Sigmoidal activation curves were observed with Mn2+ and Mg2+ with Hill coefficients of 3.0 and 1.6 and K0.5 values of 0.9 and 5.3 mM, respectively. The substrate M2+ x Ap4A showed hyperbolic kinetics with Km values of 0.34 mM for both Mn2+ x Ap4A and Mg2+ x Ap4A. Direct Mn2+ binding studies by electron paramagnetic resonance (EPR) and by the enhancement of the longitudinal relaxation rate of water protons revealed two Mn2+ binding sites per molecule of Ap4A pyrophosphatase with dissociation constants of 1.1 mM, comparable to the kinetically determined K0.5 value of Mn2+. The enhancement factor of the longitudinal relaxation rate of water protons due to bound Mn2+ (epsilon b) decreased with increasing site occupancy from a value of 12.9 with one site occupied to 3.3 when both are occupied, indicating site-site interaction between the two enzyme-bound Mn2+ ions. Assuming the decrease in epsilon(b) to result from cross-relaxation between the two bound Mn2+ ions yields an estimated distance of 5.9 +/- 0.4 A between them. The substrate Ap4A binds one Mn2+ (Kd = 0.43 mM) with an epsilon b value of 2.6, consistent with the molecular weight of the Mn2+ x Ap4A complex. Mg2+ binding studies, in competition with Mn2+, reveal two Mg2+ binding sites on the enzyme with Kd values of 8.6 mM and one Mg2+ binding site on Ap4A with a Kd of 3.9 mM, values that are comparable to the K0.5 for Mg2+. Hence, with both Mn2+ and Mg2+, a total of three metal binding sites were found-two on the enzyme and one on the substrate-with dissociation constants comparable to the kinetically determined K0.5 values, suggesting a role in catalysis for three bound divalent cations. Ca2+ does not activate Ap4A pyrophosphatase but inhibits the Mn2+-activated enzyme competitively with a Ki = 1.9 +/- 1.3 mM. Ca2+ binding studies, in competition with Mn2+, revealed two sites on the enzyme with dissociation constants (4.3 +/- 1.3 mM) and one on Ap4A with a dissociation constant of 2.1 mM. These values are similar to its Ki suggesting that inhibition by Ca2+ results from the complete displacement of Mn2+ from the active site. Unlike the homologous MutT pyrophosphohydrolase, which requires only one enzyme-bound divalent cation in an E x M2+ x NTP x M2+ complex for catalytic activity, Ap4A pyrophosphatase requires two enzyme-bound divalent cations that function in an active E x (M2+)2 x Ap4A x M2+ complex.

Different manganese binding sites in photosystem II probed by selective chemical modification of histidyl and carboxylic acid residues.

The binding of Mn2+ to manganese-depleted photosystem II was investigated after chemical modification of histidyl and carboxylic acid residues in the presence or absence of the native manganese cluster. K(M) values for Mn2+ were determined from steady-state electron transfer between Mn2+ and 2,6-dichlorophenolindophenol, the dissociation constant for Mn2+ was measured by observing the effect of added Mn2+ on the reduction of the primary donor P680+ after a saturating flash, and single-turnover electron donation from Mn2+ was followed by monitoring the decay kinetics of the EPR signal from the flash-induced tyrosine Zox radical. K(M) values for Mn2+ were found to be highly pH-dependent in both modified and unmodified photosystem II membranes. Treatment with histidine modifiers after removal of the manganese complex increased the K(M) values between 2.5 and 10 times and increased the dissociation constant for Mn2+ 8-fold, compared to membranes that were modified in the presence of the manganese cluster. Modification of carboxylic acid residues after removal of the manganese cluster increased the K(M) about 5-fold compared to membranes that were modified in the presence of the manganese cluster. The reduction rate of tyrosine Zox by Mn2+ was diminished after modification of either histidine or carboxylic acid residues. The apparent second-order rate constant decreased from 2.6 x 10(6) M(-1) s(-1) to 0.05 x 10(6) M(-1) s(-1) after histidine modification in the presence or absence of manganese, to 0.77 x 10(6) M(-1) s(-1) after carboxylic acid residue modification in the presence of manganese, and to 0.18 x 10(6) M(-1) s(-1) after carboxylic acid modification in the absence of manganese. Our results indicate the existence of two different manganese binding sites containing histidine, and at least two manganese sites with carboxylic acid residues, which are differently shielded against modifying agents by the native manganese cluster.

A comparative study of malolactic enzyme and malic enzyme of different lactic acid bacteria

Malolactic enzyme of lactic acid bacteria catalyzes the decarboxylation of L-malate to L-lactate. The appropriate enzyme of Lactobacillus casei, Leuconostoc oenos, and Leuconostoc mesenteroides, as well as the malic enzyme of Lactobacillus casei, were purified to electrophoretic homogeneity by salmine sulphate precipitation, ion-exchange chromatography, hydrophobic chromatography, and gel filtration. The malolactic enzymes investigated were similar and showed only minor variations in the isoelectric point and the temperature optimum. The molecular weight of the subunit of all malolactic enzymes was about 65 000. Aggregates were formed, depending on the pH. The optimum activity of malolactic enzyme was observed at pH 5.8–6.0, and at this pH the dimer was stable. In addition to Mn2+ and NAD, the malolactic enzyme required K+, which was replaceable by NH4+, for maximum activity. The Km values for L-malate were 10.9 mM (Leuconostoc mesenteroides B116) and 3 mM (Leuconostoc oenos). The Km values for Mn2+ were 0.1 mM (Leuconostoc mesenteroides B116) and 0.017 mM (Leoconostoc oenos). Malic enzyme oxidatively decarboxylates L-malate to pyruvate. This enzyme consists of a 37 000 subunit that forms dimers and tetramers. The NAD-dependent malic enzyme of Lactobacillus casei decarboxylates oxalacetate and is therefore regarded as a L-malate:NAD+ oxidoreductase (oxalacetate decarboxylating), EC 1.1.1.38. Key words: malolactic enzyme, malic enzyme, Lactobacillus, Leuconostoc.

Efficiency of manganese and copper uptake by excised roots of maize genotypes

Abstract In this study three maize (Zea mays L.) genotypes, a single‐cross hybrid and its parents were evaluated for their efficiency to absorb Mn2+ and Cu2+. All experiments were run using excised roots and uptake efficiency was expressed as Vmax and Km. Also, the genotypes were investigated for possible mutual effects between Mn2+ and Cu2+ during their absorption by roots. Vmax values for both Mn2+ and Cu2+ showed that an appreciable heterotic effect occurred, higher for Mn2+ than for Cu2+ At the same time the hybrid displayed with respect to the inbreds higher Km values for Mn2+ and only slightly lower Km values for Cu2+. Hence a clear positive level of heterosis was evidenced with reference to the maximum absorption capacity (Vmax) but not to the affinity (1/Km) for substrates. Cu2+ inhibited uptake of Mn2+ in a non‐competitive fashion (Vmax for Mn2+ fell in the presence of Cu2+ while Km remained constant) and this effect appeared to a lesser extent in the hybrid than in its parentals. At the same tim...

Carbon dioxide metabolism by Capnocytophaga ochracea: identification, characterization, and regulation of a phosphoenolpyruvate carboxykinase.

Cell suspensions of Capnocytophaga ochracea incorporated [14C]NaHCO3 into a major four-carbon fermentation product, succinate, and cell-free extracts from this organism contained high levels of phosphoenolpyruvate carboxykinase (PEPCK). PEPCK is the major, if not the only, CO2(HCO-3)-fixing enzyme in C. ochracea since cell-free extracts were devoid of pyruvate-dependent and other phosphoenolpyruvate (PEP)-dependent CO2(HCO-3)-fixing enzymes. The reaction products of the enzyme, which was partially purified by diethylaminoethylcellulose column chromatography, were identified as adenosine 5'-triphosphate (ATP) and oxalacetate. The enzyme showed maximum activity when manganese (Mn2+) was the divalent cation in the incubation mixture, and it had an absolute requirement for the nucleoside 5-'diphosphate adenosine 5'-diphosphate (ADP). PEPCK showed a sigmoidal kinetic response to the Mn2+ concentration and homotropic interactions in its kinetic responses to each of its three substrates PEP, ADP, and CO2(HCO-3). The (S)0.5v values for Mn2+, PEP, ADP, and CO2(CHO-3) were approximately 0.08, 0.3, 0.1, and 10 mM, respectively, and Hill coefficients for these same ligands were 2.60, 1.7, 1.9, and 3.0, respectively. In addition, C. ochracea PEPCK is under metabolic control by the nucleoside -5'-triphosphate ATP, and it also showed a sigmoidal kinetic response to this allosteric effector. The Hill coefficient for ATP was 2.70.