Spinach (Spinacia oleracea) chloroplast sedoheptulose-1,7-bisphosphatase. Activation and deactivation, and immunological relationship to fructose-1,6-bisphosphatase.
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ABSTRACT: In this paper we study activation by dithiothreitol and reduced thioredoxins and deactivation by oxidized thioredoxins f of sedoheptulose-1,7-bisphosphatase. The behaviour of the enzyme when chromatographed on a thioredoxin-Sepharose column is also described. The enzyme is autoxidizable upon removal of reducing agents, and is activated when reduced by any of the thioredoxins. This mechanism may allow the regulation of the Calvin cycle upon light-dark and dark-light transitions. The formation of a stable complex between enzyme and thioredoxin could explain the inhibitory effect of high thioredoxin concentrations. The use of immunological techniques shows that sedoheptulose-1,7-bisphosphatase and fructose-1,6-bisphosphatase are poorly related immunologically.
Project description: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.
Project description:The aim of this paper is to study some steady-state kinetic properties of sedoheptulose-1,7-bisphosphatase, its pH-dependence and the effect of a substrate analogue, fructose 2,6-bisphosphate. Studies were carried out with sedoheptulose 1,7-bisphosphate and with fructose 1,6-bisphosphate, an alternative substrate. The pK values are identical for both substrates, and fructose 2,6-bisphosphate behaves like a competitive inhibitor. These results suggest that there exists a unique active site for either sedoheptulose 1,7-bisphosphate or fructose 1,6-bisphosphate on the enzyme molecule. Increasing Mg2+ concentrations shifted the optimum pH. As for fructose-1,6-bisphosphatase, we believe that this shift is due to the neutralization of negative charges near the active centre [Cadet, Meunier & Ferté (1987) Eur. J. Biochem. 162, 393-398]. The free species of sedoheptulose 1,7-bisphosphate and fructose 1,6-bisphosphate are not the usual substrates of enzyme, nor is Mg2+. But the kinetics relative to the (Mg2+-substrate4-)2- complex is not consistent with this complex being the substrate. An explanation of this discrepancy is proposed, involving both the negative charges near the active centre and the positive charges of Mg2+. The observed Vmax. of the reduced enzyme is 65% of the theoretical Vmax. for both substrates, but the observed Vmax. relative to sedoheptulose 1,7-bisphosphate is 3 times the one relative to fructose 1,6-bisphosphate. The specificity constant (kcat./Km), 1.62 x 10(6) M-1.s-1 with respect to sedoheptulose 1,7-bisphosphate compared with 5.5 x 10(4) M-1.s-1 with respect to fructose 1,6-bisphosphate, indicates that the enzyme specificity towards sedoheptulose 1,7-bisphosphate is high but not absolute.
Project description:Thiol-treated spinach (Spinacia oleracea) chloroplast fructose bisphosphatase (EC 3.1.3.11) is severely inhibited by H2O2, whereas the freshly purified enzyme is little affected. Dithiothreitol reverses inhibition by H2O2, indicating that essential thiol groups are oxidized during H2O2 inactivation. A new role for the dithiol and thioredoxin systems that are operative in illuminated chloroplasts is proposed.
Project description:Higher-plant sedoheptulose-1,7-bisphosphatase was isolated and purified over 200-fold from spinach (Spinacia oleracea) chloroplast stromal extracts to apparent electrophoretic homogeneity by DEAE-Fractogel, molecular sieving on Sephadex G-200 and Blue B dye-matrix affinity chromatography. It is a protein of Mr 66,000, made up of two apparently identical subunits (Mr 35,000). The enzyme is activated by reduced thioredoxin fb in the presence of dithiothreitol. Its specificity towards sedoheptulose 1,7-bisphosphate versus fructose 1,6-bisphosphate is high, but not absolute.
Project description:Thiol-treated spinach (Spinacia oleracea) chloroplast fructose bisphosphatase is powerfully inhibited by Ca2+ non-competitively with respect to its substrate, fructose 1,6-bisphosphate. 500 microM-Ca2+ causes virtually complete inhibition and the Ki is 40 microM. Severe inhibition of sedoheptulose bisphosphatase is also caused by Ca2+. A role for Ca2+ in regulation of the Calvin cycle in spinach chloroplasts is proposed.
Project description:The genome of the facultative ribulose monophosphate (RuMP) cycle methylotroph Bacillus methanolicus encodes two bisphosphatases (GlpX), one on the chromosome (GlpX(C)) and one on plasmid pBM19 (GlpX(P)), which is required for methylotrophy. Both enzymes were purified from recombinant Escherichia coli and were shown to be active as fructose 1,6-bisphosphatases (FBPases). The FBPase-negative Corynebacterium glutamicum ?fbp mutant could be phenotypically complemented with glpX(C) and glpX(P) from B. methanolicus. GlpX(P) and GlpX(C) share similar functional properties, as they were found here to be active as homotetramers in vitro, activated by Mn(2+) ions and inhibited by Li(+), but differed in terms of the kinetic parameters. GlpX(C) showed a much higher catalytic efficiency and a lower Km for fructose 1,6-bisphosphate (86.3 s(-1) mM(-1) and 14 ± 0.5 ?M, respectively) than GlpX(P) (8.8 s(-1) mM(-1) and 440 ± 7.6 ?M, respectively), indicating that GlpX(C) is the major FBPase of B. methanolicus. Both enzymes were tested for activity as sedoheptulose 1,7-bisphosphatase (SBPase), since a SBPase variant of the ribulose monophosphate cycle has been proposed for B. methanolicus. The substrate for the SBPase reaction, sedoheptulose 1,7-bisphosphate, could be synthesized in vitro by using both fructose 1,6-bisphosphate aldolase proteins from B. methanolicus. Evidence for activity as an SBPase could be obtained for GlpX(P) but not for GlpX(C). Based on these in vitro data, GlpX(P) is a promiscuous SBPase/FBPase and might function in the RuMP cycle of B. methanolicus.
Project description:The dual-function fructose-1,6/sedoheptulose-1,7-bisphosphatase (FBP/SBPase) in cyanobacteria carries out two activities in the Calvin cycle. Structures of this enzyme from the cyanobacterium Synechocystis sp. PCC 6803 exist, but only with adenosine monophosphate (AMP) or fructose-1,6-bisphosphate and AMP bound. The mechanisms which control both selectivity between the two sugars and the structural mechanisms for redox control are still unresolved. Here, the structure of the dual-function FBP/SBPase from the thermophilic cyanobacterium Thermosynechococcus elongatus is presented with sedoheptulose-7-phosphate bound and in the absence of AMP. The structure is globally very similar to the Synechocystis sp. PCC 6803 enzyme, but highlights features of selectivity at the active site and loop ordering at the AMP-binding site. Understanding the selectivity and control of this enzyme is critical for understanding the Calvin cycle in cyanobacteria and for possible biotechnological application in plants.
Project description:Clear cell renal cell carcinoma (ccRCC), the most common form of kidney cancer, is characterized by elevated glycogen levels and fat deposition. These consistent metabolic alterations are associated with normoxic stabilization of hypoxia-inducible factors (HIFs) secondary to von Hippel-Lindau (VHL) mutations that occur in over 90% of ccRCC tumours. However, kidney-specific VHL deletion in mice fails to elicit ccRCC-specific metabolic phenotypes and tumour formation, suggesting that additional mechanisms are essential. Recent large-scale sequencing analyses revealed the loss of several chromatin remodelling enzymes in a subset of ccRCC (these included polybromo-1, SET domain containing 2 and BRCA1-associated protein-1, among others), indicating that epigenetic perturbations are probably important contributors to the natural history of this disease. Here we used an integrative approach comprising pan-metabolomic profiling and metabolic gene set analysis and determined that the gluconeogenic enzyme fructose-1,6-bisphosphatase 1 (FBP1) is uniformly depleted in over six hundred ccRCC tumours examined. Notably, the human FBP1 locus resides on chromosome 9q22, the loss of which is associated with poor prognosis for ccRCC patients. Our data further indicate that FBP1 inhibits ccRCC progression through two distinct mechanisms. First, FBP1 antagonizes glycolytic flux in renal tubular epithelial cells, the presumptive ccRCC cell of origin, thereby inhibiting a potential Warburg effect. Second, in pVHL (the protein encoded by the VHL gene)-deficient ccRCC cells, FBP1 restrains cell proliferation, glycolysis and the pentose phosphate pathway in a catalytic-activity-independent manner, by inhibiting nuclear HIF function via direct interaction with the HIF inhibitory domain. This unique dual function of the FBP1 protein explains its ubiquitous loss in ccRCC, distinguishing FBP1 from previously identified tumour suppressors that are not consistently mutated in all tumours.
Project description:Fructose 1,6-bisphosphatase (FBPase) is a key enzyme in gluconeogenesis. It is a potential drug target in the treatment of type II diabetes. The protein is also associated with a rare inherited metabolic disease and some cancer cells lack FBPase activity which promotes glycolysis facilitating the Warburg effect. Thus, there is interest in both inhibiting the enzyme (for diabetes treatment) and restoring its activity (in relevant cancers). The mammalian enzyme is tetrameric, competitively inhibited by Fructose 2,6-bisphosphate and negatively allosterically regulated by AMP. This allosteric regulation requires information transmission between the AMP binding site and the active site of the enzyme. A recent paper by Topaz et al. (Bioscience Reports (2019) 39, pii:BSR20180960) has added additional detail to our understanding of this information transmission process. Two residues in the AMP binding site (Lys112 and Tyr113) were shown to be involved in initiating the message between the two sites. This tyrosine residue has recently be shown to be important with protein's interaction with the antidiabetic drug metformin. A variant designed to increase metal ion affinity (M248D) resulted in a five-fold increase in enzymatic activity. Interestingly alterations of two residues at the subunit interfaces (Tyr164 and Met177) resulted in increased responsiveness to AMP. Overall, these findings may have implications in the design of novel FBPase inhibitors or activators.
Project description:Freshly purified spinach chloroplast fructose bisphosphatase is powerfully inhibited by inorganic phosphate competitively with respect to its substrate fructose 1,6-bisphosphate. The concentrations of phosphate and substrate in the chloroplast stroma are such that the enzyme in this form could not operate at a significant rate in vivo. Incubation of the enzyme with dithiothreitol for 24 h decreases the Km for fructose 1,6-bisphosphate from 0.8 to 0.033 mM, decreases the Km for Mg2+ from 9 to 2 mM and substantially alleviates inhibition by inorganic phosphate. The physiological significance of thiol activation of the enzyme is discussed.