scholarly journals Phosphofructokinases A and B from Mycobacterium tuberculosis Display Different Catalytic Properties and Allosteric Regulation

2021 ◽  
Vol 22 (3) ◽  
pp. 1483
Author(s):  
Jan Snášel ◽  
Iva Machová ◽  
Veronika Šolínová ◽  
Václav Kašička ◽  
Marcela Krečmerová ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Biochemical Characterization ◽  
Metabolic Enzyme ◽  
Kinetic Properties ◽  
Reaction Products ◽  
Hypoxic Conditions ◽  
Glycolytic Activity ◽  
Gene And Protein Expression ◽  
Fructose 1,6 Bisphosphatase ◽  
Fructose 1,6 Bisphosphate

Tuberculosis (TB) remains one of the major health concerns worldwide. Mycobacterium tuberculosis (Mtb), the causative agent of TB, can flexibly change its metabolic processes during different life stages. Regulation of key metabolic enzyme activities by intracellular conditions, allosteric inhibition or feedback control can effectively contribute to Mtb survival under different conditions. Phosphofructokinase (Pfk) is one of the key enzymes regulating glycolysis. Mtb encodes two Pfk isoenzymes, Pfk A/Rv3010c and Pfk B/Rv2029c, which are differently expressed upon transition to the hypoxia-induced non-replicating state of the bacteria. While pfkB gene and protein expression are upregulated under hypoxic conditions, Pfk A levels decrease. Here, we present biochemical characterization of both Pfk isoenzymes, revealing that Pfk A and Pfk B display different kinetic properties. Although the glycolytic activity of Pfk A is higher than that of Pfk B, it is markedly inhibited by an excess of both substrates (fructose-6-phosphate and ATP), reaction products (fructose-1,6-bisphosphate and ADP) and common metabolic allosteric regulators. In contrast, synthesis of fructose-1,6-bisphosphatase catalyzed by Pfk B is not regulated by higher levels of substrates, and metabolites. Importantly, we found that only Pfk B can catalyze the reverse gluconeogenic reaction. Pfk B thus can support glycolysis under conditions inhibiting Pfk A function.

Regulation of Fructose 1,6-Bisphosphatase Activity of Chlorella by Mole Mass Change

1996 ◽  
Vol 51 (9-10) ◽  
pp. 639-645 ◽  
Author(s):  
N. Grotjohann
Keyword(s):  
Enzyme Regulation ◽  
Fold Increase ◽  
Substrate Affinity ◽  
Kinetic Properties ◽  
Enzyme Form ◽  
Phosphorylated Compounds ◽  
Fructose 1,6 Bisphosphatase ◽  
Chloroplast Stroma ◽  
Fructose 1,6 Bisphosphate

Fast protein liquid chromatography on Superose 6 of partially purified FBPase II from Chlorella reveals a 1350 kDa-form at pH 6.0 and a 67 kDa-form at pH 8.5. Treatment of the large enzyme form with 5mᴍ concentrations of Mg2+, F1,6P2, DTT or ATP leads to dissociation into smaller ones of 215 -470 kDa. Aggregation/dissoziation is a reversible process, as has been shown for the effect of F1,6P2 and of pH, by rechromatography. The change in mole mass results in alterations of the activitiy and of the kinetic properties of the enzyme forms, obtained. Dissociation results in a 4 - 6 fold increase in activity, as can be shown for F1,6P2-treated samples. Halfsaturation constants, as well as the degree of cooperativity of the 67- and the 1350- kDa form, are different for substrate affinity, activation by Mg2+ and DTT, and for inhibition by ATP. Both enzyme forms hydrolyse fructose 1,6 bisphosphate and seduheptulose 1,7 bisphosphate better than other phosphorylated compounds. The ratio of F1,6P2- to SDP-cleavage is 100:58 for the small enzyme form and 100: 84 for the large one. Activation of FBPase II in the light and inactivation in the dark is discussed on the basis of different oligomeric forms of the enzyme, generated by changes in the concentration of intermediates and effectors in the chloroplast stroma, leading to dissociation or aggregation. The conclusion is drawn that oligomerization of key enzymes, resulting in enzyme forms with different activities and different kinetic properties, might provide an effective mechanism for enzyme regulation in vivo

scholarly journals Properties of oxidized and reduced spinach (Spinacia oleracea) chloroplast fructose-1,6-bisphosphatase activated by various agents

1991 ◽  
Vol 278 (3) ◽  
pp. 787-791 ◽  
Author(s):  
T Chardot ◽  
J C Meunier
Keyword(s):  
Spinacia Oleracea ◽  
Acid Group ◽  
Lag Phase ◽  
Kinetic Properties ◽  
Amino Acid Group ◽  
Progress Curve ◽  
Fructose 1,6 Bisphosphatase ◽  
Specificity Constant ◽  
The Stability ◽  
Fructose 1,6 Bisphosphate

Fructose-1,6-bisphosphatase (FBPase) can be reduced and activated by either dithiothreitol or reduced thioredoxin. This activation is pH-dependent. An amino acid group with a pK value of 5.55 is involved in this process. Both enzyme forms can also be stimulated by agents such as fructose 1,6-bisphosphate, Mg2+, Ca2+ and Ca2+/fructose 1,6-bisphosphate. FBPase reduced by dithiothreitol is more strongly activated than the enzyme reduced by thioredoxin. The specificity constant (kcat./Km) is enhanced over 2.5-25-fold and 1.5-2-fold (depending on the agent used) for FBPase reduced by dithiothreitol and thioredoxin respectively. In both cases, no new kinetic properties appeared. The pH-activity profile of the stimulated enzyme is slightly shifted towards the acidic side with respect to the reduced enzyme. A lag phase is observed in the progress curve of both enzymic forms, treated or untreated. Each agent used to stimulate must induce a new conformation of the enzyme, more active than the initial one, characterized by a specificity constant and a relaxation time. This lag phase tends to disappear when the assay temperature is increased. Temperature has the same effect on the activity of oxidized, reduced and stimulated FBPase, but different effects on the stability of the different forms.

scholarly journals Effects of phosphorylation on the kinetic properties of rat liver fructose-1,6-bisphosphatase

1984 ◽  
Vol 222 (1) ◽  
pp. 125-130 ◽  
Author(s):  
D W Meek ◽  
H G Nimmo
Keyword(s):  
Protein Kinase ◽  
Cyclic Amp ◽  
Rat Liver ◽  
Kinetic Properties ◽  
Purification Procedure ◽  
Dependent Protein Kinase ◽  
Fructose 1,6 Bisphosphatase ◽  
Dependent Protein ◽  
A Subunit ◽  
Fructose 1,6 Bisphosphate

A new purification procedure for rat liver fructose-1,6-bisphosphatase that involves use of Procion Red-Sepharose is described. The purified enzyme was homogeneous, had a subunit Mr of 40 000-41 000 and seemed to be undegraded. The enzyme could be phosphorylated by cyclic AMP-dependent protein kinase with a stoicheiometry of one per subunit. Phosphorylation caused a 2-fold decrease in the Km of the enzyme for fructose 1,6-bisphosphate, but did not affect its allosteric responses to AMP, Mg2+ and fructose 2,6-bisphosphate.

scholarly journals Partial purification and characterisation of cytosolic Fructose-1, 6-bisphosphatase from Drymaria cordata

2018 ◽  
Vol 7 (4) ◽  
pp. 2126
Author(s):  
Niki Doma Sherpa ◽  
Raksha Mukhia ◽  
Dhani Raj Chhetri
Keyword(s):  
Enzyme Activity ◽  
Protein Concentration ◽  
Biochemical Characterization ◽  
Partial Purification ◽  
Streptomycin Sulphate ◽  
Fructose 1,6 Bisphosphatase ◽  
Ethnomedicinal Plant ◽  
Optimum Ph ◽  
Km Value ◽  
Fructose 1,6 Bisphosphate

Drymaria cordata is an important ethnomedicinal plant from which many important secondary metabolites have been reported. Partial purification of the enzyme, fructose 1,6-bisphosphatase was carried out following the methods of homogenization, streptomycin sulphate precipitation, ammonium sulphate cut and molecular sieve chromatography through Bio-Gel A-0.5m column. Biochemical characterization experiments were performed by standard methods with the enzyme preparation as purified from the column. Cytosolic fructose 1,6-bisphosphatase from the leaves of Drymaria cordata was purified to about 27-fold with 77% of recovery over homogenate fraction. The enzyme was highly specific to D-fructose-1,6-bisphosphate. With increase of protein concentration upto 300µg and incubation time upto120 minutes, the enzyme activity increased linearly. The metal ions Mg2+ or Mn2+ strongly stimulated the enzyme activity on the other hand Li+, Hg2+ and Zn2+ were potent inhibitors. The D. cordata enzyme showed temperature maxima at 40˚C while the optimum pH was at 8.0. The Km value of the enzyme for its substrate, Fructose 1,6-bisphosphate was 1.11µM proving its strong affinity.

scholarly journals Molecular and Biochemical Characterization of a Distinct Type of Fructose-1,6-Bisphosphatase from Pyrococcus furiosus

2002 ◽  
Vol 184 (12) ◽  
pp. 3401-3405 ◽  
Author(s):  
Corné H. Verhees ◽  
Jasper Akerboom ◽  
Emile Schiltz ◽  
Willem M. de Vos ◽  
John van der Oost
Keyword(s):  
Biochemical Characterization ◽  
Pyrococcus Furiosus ◽  
Distinct Type ◽  
Sequence Motifs ◽  
Conserved Sequence ◽  
New Family ◽  
Fructose 1,6 Bisphosphatase ◽  
Conserved Sequence Motifs ◽  
Fructose 1,6 Bisphosphate

ABSTRACT The Pyrococcus furiosus fbpA gene was cloned and expressed in Escherichia coli, and the fructose-1,6-bisphosphatase produced was subsequently purified and characterized. The dimeric enzyme showed a preference for fructose-1,6-bisphosphate, with a Km of 0.32 mM and a V max of 12.2 U/mg. The P. furiosus fructose-1,6-bisphosphatase was strongly inhibited by Li+ (50% inhibitory concentration, 1 mM). Based on the presence of conserved sequence motifs and the substrate specificity of the P. furiosus fructose-1,6-bisphosphatase, we propose that this enzyme belongs to a new family, class IV fructose-1,6-bisphosphatase.

Protection of astrocytes by fructose 1,6-bisphosphate and citrate ameliorates neuronal injury under hypoxic conditions

1996 ◽  
Vol 726 (1-2) ◽  
pp. 167-173 ◽  
Author(s):  
Judith A. Kelleher ◽  
Thelma Y.Y. Chan ◽  
Pak H. Chan ◽  
George A. Gregory
Keyword(s):  
Neuronal Injury ◽  
Hypoxic Conditions ◽  
Fructose 1,6 Bisphosphate

scholarly journals rhEPO (recombinant human eosinophil peroxidase): expression in Pichia pastoris and biochemical characterization

2006 ◽  
Vol 395 (2) ◽  
pp. 295-301 ◽  
Author(s):  
Chiara Ciaccio ◽  
Alessandra Gambacurta ◽  
Giampiero DE Sanctis ◽  
Domenico Spagnolo ◽  
Christina Sakarikou ◽  
...  
Keyword(s):  
Pichia Pastoris ◽  
Biochemical Characterization ◽  
Expression System ◽  
Gel Filtration ◽  
Proteolytic Processing ◽  
Kinetic Properties ◽  
Specific Substrate ◽  
Human Eosinophil ◽  
Eosinophil Peroxidase ◽  
Pichia Pastoris Expression System

A Pichia pastoris expression system has for the first time been successfully developed to produce rhEPO (recombinant human eosinophil peroxidase). The full-length rhEPO coding sequence was cloned into the pPIC9 vector in frame with the yeast α-Factor secretion signal under the transcriptional control of the AOX (acyl-CoA oxidase) promoter, and transformed into P. pastoris strain GS115. Evidence for the production of rhEPO by P. pastoris as a glycosylated dimer precursor of approx. 80 kDa was determined by SDS/PAGE and gel filtration chromatography. Recombinant hEPO undergoes proteolytic processing, similar to that in the native host, to generate two chains of approx. 50 and 20 kDa. A preliminary biochemical characterization of purified rhEPO demonstrated that the spectral and kinetic properties of the recombinant wild-type EPO are comparable with those of the native enzyme and are accompanied by oxidizing activity towards several physiological anionic substrates such as SCN−, Br− and Cl−. On the basis of the estimated Km and kcat values it is evident that the pseudohalide SCN− is the most specific substrate for rhEPO, consistent with the catalytic properties of other mammalian EPOs purified from blood.

scholarly journals Recombinant protein production provoked accumulation of ATP, fructose-1,6-bisphosphate and pyruvate in E. coli K12 strain TG1

2021 ◽  
Vol 20 (1) ◽  
Author(s):  
Jan Weber ◽  
Zhaopeng Li ◽  
Ursula Rinas
Keyword(s):  
Recombinant Protein ◽  
Recombinant Protein Production ◽  
Protein Production ◽  
Expression System ◽  
Limiting Factors ◽  
Expression Vectors ◽  
Metabolic Enzyme ◽  
E Coli ◽  
Metabolic Burden ◽  
Fructose 1,6 Bisphosphate

Abstract Background Recently it was shown that production of recombinant proteins in E. coli BL21(DE3) using pET based expression vectors leads to metabolic stress comparable to a carbon overfeeding response. Opposite to original expectations generation of energy as well as catabolic provision of precursor metabolites were excluded as limiting factors for growth and protein production. On the contrary, accumulation of ATP and precursor metabolites revealed their ample formation but insufficient withdrawal as a result of protein production mediated constraints in anabolic pathways. Thus, not limitation but excess of energy and precursor metabolites were identified as being connected to the protein production associated metabolic burden. Results Here we show that the protein production associated accumulation of energy and catabolic precursor metabolites is not unique to E. coli BL21(DE3) but also occurs in E. coli K12. Most notably, it was demonstrated that the IPTG-induced production of hFGF-2 using a tac-promoter based expression vector in the E. coli K12 strain TG1 was leading to persistent accumulation of key regulatory molecules such as ATP, fructose-1,6-bisphosphate and pyruvate. Conclusions Excessive energy generation, respectively, accumulation of ATP during recombinant protein production is not unique to the BL21(DE3)/T7 promoter based expression system but also observed in the E. coli K12 strain TG1 using another promoter/vector combination. These findings confirm that energy is not a limiting factor for recombinant protein production. Moreover, the data also show that an accelerated glycolytic pathway flux aggravates the protein production associated “metabolic burden”. Under conditions of compromised anabolic capacities cells are not able to reorganize their metabolic enzyme repertoire as required for reduced carbon processing.

Biochemical Characterization of VapC46 Toxin from Mycobacterium tuberculosis

2020 ◽  
Vol 62 (6-7) ◽  
pp. 335-343
Author(s):  
Madhurima Roy ◽  
Madhuparna Bose ◽  
Kamakshi Bankoti ◽  
Anirban Kundu ◽  
Santanu Dhara ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Biochemical Characterization
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