Biomedical subjects

B Gontero

Publications and source records attributed to B Gontero.

At least 19 recordsLinked to original sources

PubMed2000

Thioredoxin activation of phosphoribulokinase in a bi-enzyme complex from Chlamydomonas reinhardtii chloroplasts.

The activation of oxidized phosphoribulokinase either "free" or as part of a bi-enzyme complex by reduced thioredoxins during the enzyme reaction was studied. In the presence of reduced thioredoxin, the product of the reaction catalyzed by phosphoribulokinase within the bi-enzyme complex does not appear in a linear fashion. It follows a mono-exponential pattern that suggests a slow dissociation process of the bi-enzyme complex in the assay cuvette. A plot of the steady state of product appearance against thioredoxin concentration gave a sigmoid curve. On the basis of our experimental results, we propose a minimum model of the activation of phosphoribulokinase by reduced thioredoxin. Reduced thioredoxin may act on the phosphoribulokinase, either within the complex or in the dissociated metastable form. However, the time required to activate the enzyme as part of the complex is shorter (about 20 s) than that required to activate the dissociated form (about 10 min). This might be of physiological relevance, and we discuss the role of the interactions between phosphoribulokinase and glyceraldehyde-3-phosphate dehydrogenase in the regulation of the Calvin cycle.

Animals↗

PubMed2000

Angiotensin II induces nuclear factor- kappa B activation in cultured neonatal rat cardiomyocytes through protein kinase C signaling pathway.

Rat neonatal ventricular cardiomyocytes (RNVM) possess G protein-coupled AT(1)receptors for angiotensin II (AngII) that activate multiple intracellular pathways. To elucidate potential signaling mechanisms involved, we focussed on the nuclear transcription factor-kappa B (NF- kappa B) in RNVM culture. Using specific antibody to NF- kappa Bp65, immunolocalization of NF- kappa B was cytoplasmic in unstimulated cardiomyocytes, whereas NF- kappa B was translocated into the RNVM nucleus in response to AngII. This translocation was inhibited in the presence of calphostin C, a specific inhibitor of protein kinase C (PKC). Western blot analysis showed an increase of NF- kappa B in AngII-stimulated cardiomyocyte nuclear extracts as compared to controls. Biomolecular interaction analysis (BIA analysis) of NF- kappa B activation showed that only AngII-nuclear extracts bound to NF- kappa B consensus sequence with a high degree of affinity. This DNA-binding capacity was completely lost in calphostin C-treated cells. At transcriptional level in RNVM, AngII mediates the upregulation of matrix gelatinase (MMP-9), which is totally inhibited by calphostin C treatment. In conclusion, cardiomyocyte nuclear NF- kappa B translocation in response to Ang II via PKC pathway activates cardiomyocyte-specific transcription of MMP-9 and may activate transcription from responsive genes which are involved in cardiac hypertrophy process and/or cardiac remodeling.

Angiotensin II↗

PubMed1999

Memory and imprinting in multienzyme complexes. Evidence for information transfer from glyceraldehyde-3-phosphate dehydrogenase to phosphoribulokinase under reduced state in Chlamydomonas reinhardtii.

The phosphoribulokinase, when it is in a reduced state in a bi-enzyme complex, is more active than when it is oxidized. This complex dissociates upon dilution to give a metastable reduced form of phosphoribulokinase, which differs from the stable form isolated beside the complex. The kinetic parameters of the reduced stable phosphoribulokinase and those of the complex are very similar, unlike those of the metastable form. Although the kinetic mechanism of the reduced stable form is ordered, with ribulose-5-phosphate binding first, ATP binds first to the phosphoribulokinase in the complex and to the metastable form. Therefore, phosphoribulokinase bears an imprint from glyceraldehyde-3-phosphate dehydrogenase after disruption of the complex. Dissociation of phosphoribulokinase from the complex also enhances its flexibility. The imprinting and greater flexibility result in the catalytic constant of dissociated phosphoribulokinase being 10-fold higher than that of the enzyme in the complex. Imprinting corresponds to stabilization-destabilization energies resulting from conformation changes generated by protein-protein interactions. The energy stored within the metastable phosphoribulokinase is mainly used to decrease the energy barrier to catalysis.

Animals↗

PubMed1997

Information transfer in multienzyme complexes--1. Thermodynamics of conformational constraints and memory effects in the bienzyme glyceraldehyde-3-phosphate-dehydrogenase-phosphoribulokinase complex of Chlamydomonas reinhardtii chloroplasts.

Oxidized phosphoribulokinase is almost inactive in its isolated state but becomes active when associated with glyceraldehyde-3-phosphate dehydrogenase. There is therefore an information transfer that takes place between these two enzymes. However, when the complex dissociates, free oxidized phosphoribulokinase is even more active than when it is associated with glyceraldehyde-3-phosphate dehydrogenase. This means that glyceraldehyde-3-phosphate dehydrogenase exerts an imprinting effect upon phosphoribulokinase which persists for a while after the parting of the two proteins. Various methods derived from statistical thermodynamics can be used to estimate the fraction of energy transferred from glyceraldehyde-3-phosphate dehydrogenase to phosphoribulokinase and which alters the kinetic parameters of the latter enzyme. In the complex, the decrease of the free energy associated with the binding of ribulose 5-phosphate is larger than that of ATP. This implies that the mutual association of the two enzymes facilitates the binding of the former substrate but is without effect on that of the latter. The main effect exerted by the association of the two enzymes is to decrease by about 10 kJ/mol the height of the energy barrier of the catalytic process. Phosphoribulokinase keeps an imprinting effect exerted by glyceraldehyde-3-phosphate dehydrogenase after the parting of the two enzymes. Part of the energy transferred from one protein to the other is used to decrease slightly the apparent binding free energy of the two substrates of phosphoribulokinase by about 1.5 kJ/mol. Whereas the previous association of the two enzymes does not significantly alter substrate binding to phosphoribulokinase, it greatly affects catalysis and decreases by about 16 kJ/mol the height of the energy barrier pertaining to this step. Therefore, within multienzyme complexes, information and energy can be transferred between proteins. Statistical thermodynamics offers the possibility of estimating how this energy is used to alter the various kinetic parameters of the reaction.

Animals↗

PubMed1997

Information transfer in multienzyme complexes--2. The role of Arg64 of Chlamydomonas reinhardtii phosphoribulokinase in the information transfer between glyceraldehyde-3-phosphate dehydrogenase and phosphoribulokinase.

A mutant phosphoribulokinase has been isolated from the 12-2B mutant of Chlamydomonas reinhardtii. In this mutant, Arg64 has been replaced by Cys. The enzyme, which may exist in the dimeric and tetrameric states, is almost devoid of activity. Neither of these enzymes is able to form a complex with glyceraldehyde-3-phosphate dehydrogenase. The phosphoribulokinase gene has been expressed in Escherichia coli. The resulting recombinant protein, after isolation and purification, is apparently identical to the native enzyme extracted from the chloroplast. Three mutants have been generated by site directed mutagenesis. Arg64 has been replaced by Ala, Lys or Glu. With the exception of the latter, the two other mutants, [A64]phosphoribulokinase and [K64]phosphoribulokinase, are active when they are reduced, and nearly totally inactive in their oxidized state. Their activity, however, is decreased relative to that of the native, or to that of the wild-type recombinant phosphoribulokinase. Both the catalytic constant and the apparent affinity of ribulose 5-phosphate are decreased relative to the corresponding values obtained for the wild-type, the native or the recombinant enzyme. Whereas the [A64]phosphoribulokinase is unable to form a complex with glyceraldehyde-3-phosphate dehydrogenase, [K64]phosphoribulokinase does, but the stability of the resulting complex is much decreased relative to that of the wild-type complex. The oxidized mutant [K64]phosphoribulokinase becomes active in the presence of glyceraldehyde-3-phosphate dehydrogenase but this activity is smaller than that of the corresponding wild-type enzyme. Taken together, these results show that Arg64 plays a major role in the association of the two enzymes and in the information transfer that takes place between glyceraldehyde-3-phosphate dehydrogenase and phosphoribulokinase. As this residue also appears to be important for catalytic activity, it may be tempting to consider that it stabilizes a conformation that is required for both the catalytic activity and the formation of the bienzyme complex.

Animals↗

PubMed1997

Memory and imprinting effects in multienzyme complexes--I. Isolation, dissociation, and reassociation of a phosphoribulokinase-glyceraldehyde-3-phosphate dehydrogenase complex from Chlamydomonas reinhardtii chloroplasts.

A bienzyme complex made up of phosphoribulokinase and glyceraldehyde-3-phosphate dehydrogenase has been isolated and purified from chloroplasts of Chlamydomonas reinhardtii. The complex contains four phosphoribulokinase and eight glyceraldehyde-3-phosphate dehydrogenase polypeptide chains. As phosphoribulokinase is dimeric and glyceraldehyde-3-phosphate dehydrogenase tetrameric, it is concluded that the complex comprises two phosphoribulokinase and two glyceraldehyde-3-phosphate dehydrogenase molecules. Its overall molecular mass is 460 kDa, which is in excellent agreement with its stoichiometry. Moreover, owing to the nature of the two enzymes, this complex must catalyse two nonconsecutive reactions. The bienzyme complex tended to spontaneously dissociate into the free enzymes upon dilution. This dissociation process was considerably promoted by reducing agents such as dithiothreitol or reduced thioredoxin. The kinetics of the dissociation process induced by dithiothreitol or reduced thioredoxin were paralleled by an increase of activity of phosphoribulokinase. The dissociation of the complex was reversible. If oxidized phosphoribulokinase and glyceraldehyde-3-phosphate dehydrogenase were mixed, a certain amount of the complex was formed. The reconstituted complex displayed properties that were indistinguishable from those of the native complex extracted from chloroplasts of Chlamydomonas reinhardtii. These results suggest that the concentration of the complex in vivo must vary depending on the light intensity.

Animals↗

PubMed1997

Memory and imprinting effects in multienzyme complexes--II. Kinetics of the bienzyme complex from Chlamydomonas reinhardtii and hysteretic activation of chloroplast oxidized phosphoribulokinase.

Oxidized, free, stable phosphoribulokinase from Chlamydomonas reinhardtii was almost completely devoid of catalytic activity (0.06 s(-1)/site). However, when it was bound to glyceraldehyde-3-phosphate dehydrogenase from the same organism, it displayed significant activity (3.25 s(-1)/site). Moreover, this complex tended to spontaneously dissociate upon dilution; the isolated phosphoribulokinase activity increased up to 56 s(-1)/site, subsequently decreased, and finally became almost completely inactive. Its intrinsic kinetic properties (Km and k(cat)) changed with the variation of the overall activity. These effects were paralleled by changes of conformation of the enzyme as revealed by fluorescence analysis. A model is proposed that allows quantitative expression of the dynamics of the dissociation of the oxidized bienzyme complex and the effects of either of the two substrates, ATP and ribulose 5-phosphate, on this dissociation process. Whereas ATP destabilized the complex and promoted its dissociation, ribulose 5-phosphate tended to stabilize this complex. Inactive, stable, oxidized phosphoribulokinase may form a complex with glyceraldehyde-3-phosphate dehydrogenase regaining its catalytic activity. In this case, glyceraldehyde-3-phosphate dehydrogenase acts in a manner similar, but not identical to a chaperonin. The information content of the phosphoribulokinase gene, as defined by the sequence of its base pairs, was therefore not sufficient to specify full enzyme activity. It needed the presence of glyceraldehyde-3-phosphate dehydrogenase to give the oxidized phosphoribulokinase a conformation competent for its activity. The potential biological significance of these effects remains to be discovered.

Adenosine Triphosphate↗

PubMed1994

The modulation of enzyme reaction rates within multi-enzyme complexes. 1. Statistical thermodynamics of information transfer through multi-enzyme complexes.

There is now experimental evidence that association of different enzymes as a multi-enzyme complex may result in an alteration of the catalytic properties of the enzymes present in this complex. This effect is not related to the channelling of reaction intermediates between different active sites. It appears as a consequence of an information transfer that occurs within the multi-enzyme complex. A theory, based on statistical thermodynamics, has been developed which provides an understanding, on a physical basis, for how isologous as well as heterologous interactions between identical, or different, enzymes of the complex may modulate the catalytic properties of an oligomeric enzyme of that complex. The theory predicts three possible types of effects: an alteration, through heterologous interactions, of an already existing co-operativity of the oligomeric enzyme within the complex; a co-operativity, generated by heterologous interactions in the complex that could not occur if the oligomeric enzyme were isolated from the rest of the complex; a Michaelis-Menten character of the oligomeric enzyme within the complex, but with altered values of Vm and Km relative to what would have been observed with the naked enzyme. All these effects appear as a consequence of a transfer of information between different enzymes of the same multi-protein complex. The following paper in this journal shows how one can demonstrate and characterize experimentally these effects in a multi-enzyme complex containing ribulose bisphosphate carboxylase-oxygenase.

Catalysis↗

PubMed1994

The modulation of enzyme reaction rates within multi-enzyme complexes. 2. Information transfer within a chloroplast multi-enzyme complex containing ribulose bisphosphate carboxylase-oxygenase.

Octameric ribulose bisphosphate carboxylase-oxygenase binds in an independent manner its substrate (ribulose bisphosphate) and a substrate analog (6-phosphogluconate). The eight active sites of the free enzyme are thus independent. The kinetic behaviour of the active site becomes different if ribulose bisphosphate carboxylase-oxygenase is inserted in the five-enzyme complex previously isolated from chloroplasts. Ribulose bisphosphate carboxylase-oxygenase then becomes more active than the corresponding free enzyme form. By comparing the behaviour of the same enzyme in the free state and in the associated state it then becomes possible to study how the thermodynamics of protein-protein interactions alters the kinetic behaviour of ribulose bisphosphate carboxylase-oxygenase. This alteration may be expressed in terms of stabilization-destabilization energies exerted upon the various intermediate states of the enzyme reaction, within the multi-protein complex. Heterologous interactions within this complex exert a constant stabilization energy on the enzyme ground states along the reaction co-ordinate of -1.68 kJ/mol and a constant stabilization energy of -3.79 kJ/mol on the enzyme transition states. These stabilization energies express how information propagates within the multi-enzyme complex as to increase the apparent affinity of the substrate for the active sites of ribulose bisphosphate carboxylase-oxygenase, as well as to increase the catalytic rate constant. The binding of the substrate analog 6-phosphogluconate to free ribulose bisphosphate carboxylase-oxygenase is non-co-operative. It becomes positively co-operative if this enzyme is inserted in the multi-protein complex. Under these conditions, only one type of enzyme-inhibitor complex is detected experimentally. Here again heterologous interactions stabilize this enzyme-inhibitor complex relative to that expected if ribulose bisphosphate carboxylase oxygenase is free. The extent of stabilization is -1.03 kJ/mol. Neither free nor associated ribulose bisphosphate carboxylase-oxygenase display any co-operativity relative to substrate binding. However, in the presence of the substrate analog 6-phosphogluconate, this enzyme displays positive co-operativity relative to substrate, although not if it is naked. These results can be explained theoretically and show that the maximum value of the Hill coefficient is < or = 2. As 6-phosphogluconate and other substrate analogs are present in chloroplasts under normal conditions, this co-operativity might be of functional importance in vivo.

Binding Sites↗

PubMed1993

Structural and functional properties of a multi-enzyme complex from spinach chloroplasts. 1. Stoichiometry of the polypeptide chains.

Antibodies have been raised specifically against chloroplast phosphoribulokinase, glyceraldehyde-3-phosphate dehydrogenase and ribulose 1,5-bisphosphate carboxylase-oxygenase. Each of these antibodies recognizes the same macromolecular entity isolated and purified from chloroplasts. This entity is a multi-enzyme complex, previously isolated and made up of ribose-phosphate isomerase, phosphoribulokinase, ribulose 1,5-bisphosphate carboxylase-oxygenase, phosphoglycerate kinase and glyceraldehyde-3-phosphate dehydrogenase. Under denaturing conditions the multi-enzyme complex contains two polypeptides of 54 kDa and 15 kDa corresponding to the large and the small subunits of ribulose 1,5-bisphosphate carboxylase-oxygenase, the two polypeptides of the glyceraldehyde-3-phosphate dehydrogenase of 39 kDa and 37 kDa, one polypeptide of 40 kDa pertaining to phosphoribulokinase and one polypeptide of 30 kDa very likely pertaining to ribose-phosphate isomerase. The combined use of immunochemical and densitometric techniques allows one to determine the number and the stoichiometry of the various types of polypeptide chains and to compare them with the quaternary structure of the corresponding isolated enzymes. Ribulose 1,5-bisphosphate carboxylase-oxygenase of higher plants consists of eight large and eight small subunits. Glyceraldehyde-3-phosphate dehydrogenase is made up of two types of polypeptide chains called A and B and its simplest quaternary structure is A2B2. Finally, phosphoribulokinase is a dimer made up of two identical subunits. Therefore, for the three isolated enzymes, the stoichiometry of the polypeptide chains is always 1:1. Within this multi-enzyme complex, there are two subunits of phosphoribulokinase, two A and B subunits of glyceraldehyde-3-phosphate dehydrogenase and two large and four small subunits of ribulose 1,5-bisphosphate carboxylase-oxygenase. Therefore the number and the stoichiometry of the polypeptide chains of phosphoribulokinase and glyceraldehyde-3-phosphate dehydrogenase are the same in the multi-enzyme complex and in the free enzymes, but those of ribulose 1,5-bisphosphate carboxylase-oxygenase are completely different. This conclusion that the multi-enzyme complex contains two active sites for ribulose 1,5-bisphosphate may be confirmed independently by kinetic inhibition studies using 6-phosphogluconate.

Binding Sites↗

PubMed1993

Structural and functional properties of a multi-enzyme complex from spinach chloroplasts. 2. Modulation of the kinetic properties of enzymes in the aggregated state.

The carboxylase activity of free ribulose 1,5-bisphosphate carboxylase-oxygenase has been compared to that of the five-enzyme complex present in chloroplasts. Kinetic results have shown that the V/active site is lower for the free enzyme than for the complex. Conversely the Km is smaller for the complex than for the free enzyme. This implies that the catalytic activity of the enzyme is enhanced when it is embedded in the complex. Under reducing conditions and in the presence of reduced thioredoxin, inactive oxidized phosphoribulokinase, free in solution or inserted in the multi-enzyme complex, becomes active. The kinetics of this activation process has been studied and shown to be exponential. The time constant of this exponential decreases, for the free enzyme, as thioredoxin concentration is increased. Alternatively, for the enzyme embedded in the complex, this time constant increases with thioredoxin concentration almost in a linear fashion. This implies that the complex is much more rapidly activated by reduced thioredoxin than is the free phosphoribulokinase. The variation of the amplitude of this activation process as a function of thioredoxin concentration is a hyperbola. The concentration of thioredoxin which results in half the asymptotic value of this hyperbola is smaller for the complex than for the free enzyme. A kinetic model has been proposed and the dynamic equations resulting from this model have been derived. They fit the experimental results exactly. From the variation of the amplitude of the activation process one may derive the binding constants of thioredoxin on either the oxidized enzyme or on a partly dithiothreitol-reduced enzyme (both of them free or inserted in the complex). In either case, the affinity of reduced thioredoxin is larger for the complex than for the free enzyme. The individual values of some of the rate constants have also been estimated from the variation of the time constants as a function of thioredoxin concentration. Taken together, these results show that at least two enzymes, ribulose 1,5-bisphosphate carboxylase-oxygenase and phosphoribulokinase, have quite different kinetic properties depending on whether they are in free solution or embedded in the multi-enzyme complex.

Chloroplasts↗

PubMed1991

Thioredoxin activation of phosphoribulokinase in a chloroplast multi-enzyme complex.

The activation process of spinach phosphoribulokinase by thioredoxin f has been studied with the enzyme in a free, isolated state, or integrated in a multi-enzyme complex. The time periods required for enzyme activation are always smaller and the maximal enzyme velocities are always greater when chloroplast phosphoribulokinase is included in the multi-enzyme complex than when it is in the isolated state. Comparative kinetic studies show that phosphoribulokinase extracted from the complex behaves exactly as in the isolated state. The reduced form of the kinase, whatever it has been included in the complex or isolated from the chloroplasts, are deactivated by oxidized thioredoxins. In the absence of thioredoxin f however, the reduced form of the isolated enzyme undergoes spontaneous oxidation whereas the reduced kinase included in the multi-enzyme complex is stable. 'Unspecific' proteins such as bovine serum albumin do not provide any protection of the kinase against autooxidation, whereas 'homologous' specific proteins such as ribulose-1,5-bisphosphate carboxylase/oxygenase dramatically decrease the rate of this autooxidation process. These results therefore support the view that interactions between phosphoribulokinase and the other components of the multi-enzyme complex play an important role in the modulation of the activity of this enzyme. The possible part of these interactions in the control of the Calvin cycle is discussed.

Adenosine Triphosphate↗

PubMed1988

Reactivity of the thiol groups of Escherichia coli phosphofructo-1-kinase.

Modification of Escherichia coli phosphofructo-1-kinase (6-phosphofructokinase; EC 2.7.1.11) with several thiol modifying reagents led to a pseudo-first-order loss of activity that was associated with the modification of a single cysteine residue, identified as the cysteine at position 119 in the protein sequence. This cysteine was protected from reaction with vinyl pyridine, bromopyruvate, and dithionitrobenzoic acid by the substrate, fructose-6-P. In the crystal structure of the highly homologous phosphofructokinase from Bacillus stearothermophilus, cysteine 119 is sufficiently distant from the catalytic site to exclude a direct steric inhibition of the binding of substrate as a mechanism of inactivation for the modification. Thus, the inhibition is unlikely to be a direct one but to be the result of interference with the conformational change that is associated with fructose-6-P binding. A second thiol, position 283, was shown to be protected from reaction when the enzyme was in its native conformation. In contrast to the previously published sequence for the E. coli enzyme six cysteines as opposed to seven have been found both in enzyme from strain LE392 and in enzyme produced by a plasmid that was derived from pLC 16-4. The position in question, 75, was identified as phenylalanine.

Chemical Phenomena↗

PubMed1988

A functional five-enzyme complex of chloroplasts involved in the Calvin cycle.

A complex of five different enzymes: ribose-phosphate isomerase, phosphoribulokinase, ribulose-bisphosphate carboxylase/oxygenase, phosphoglycerate kinase and glyceraldehyde-phosphate dehydrogenase, has been purified from spinach chloroplasts. These enzymes catalyse five consecutive reactions of the Calvin cycle, of which the two reactions catalysed by phosphoribulokinase and ribulose-bisphosphate carboxylase/oxygenase are unique to this cycle. The five-enzyme complex has been purified by successive chromatographies on DEAE-Trisacryl, Sephadex G-200 and hydroxyapatite. The homogeneity of the complex has been tested by analytical centrifugation and electrophoresis. Depending on the technique used to estimate its molecular mass, the value obtained varies between 520 kDa and 536 kDa. In addition to the five enzymes mentioned above, the complex contains a 65-kDa polypeptide. The quaternary structure of these enzymes in the complex appears to be different from what has been described for the individual enzymes in the 'noncomplexed state'. The five-enzyme complex is functional as glyceraldehyde 3-phosphate is formed from ribose 5-phosphate. Preliminary experiments suggest that channelling of reaction intermediates occurs within the complex.

Aldose-Ketose Isomerases↗

PubMed1987

Kinetics of the modulation of chloroplastic fructose-1,6-bisphosphatase activity by thioredoxin fb.

The inactivation of reduced chloroplast fructose-bisphosphatase by oxidized thioredoxin fb has been studied during the enzyme reaction along the principle of Tian and Tsou [Biochemistry (1982) 21, 1028-1032]. A minimum model for this process is presented and its kinetic and equilibrium parameters have been determined. Thioredoxin fb binding to the enzyme is fast relative to catalysis and product desorption. Under quasi-equilibrium conditions oxidized thioredoxin is a non-competitive inhibitor of the enzyme reaction and must bind to a regulatory 'thioredoxin site'. The slow deactivation is thermodynamically favoured, and as expected from binding data, slowed down by the presence of substrate, fructose bisphosphate. The desorption of thioredoxin fb from the enzyme is extremely slow and this small protein may be regarded as a 'regulatory' subunit of fructose-bisphosphatase.

Chloroplast Thioredoxins↗

PubMed1987

Function, structure and evolution of fructose-1,6-bisphosphatase.

The hydrolysis of fructose-1,6-bisphosphate to fructose-6-phosphate is a key reaction of carbohydrate metabolism. The enzyme that catalyzes this reaction, fructose-1,6-bisphosphatase, appears to be present in all forms of living organisms. Regulation of the enzyme activity, however, occurs by a variety of distinct mechanisms. These include AMP inhibition (most sources), cyclic AMP-dependent phosphorylation (yeast), and light-dependent activation (chloroplast). In this short review, we have analyzed the function of several fructose-1,6-bisphosphatases and we have made a comparison of partial amino acid sequences obtained from the enzymes of the yeast Saccharomyces cerevisiae, Escherichia coli, and spinach chloroplasts with the known entire amino acid sequence of a mammalian gluconeogenic fructose-1,6-bisphosphatase. These results demonstrate a very high degree of sequence conservation, suggesting a common evolutionary origin for all fructose-1,6-bisphosphatases.

Amino Acid Sequence↗

PubMed1986

Amino acid sequence homology among fructose-1,6-bisphosphatases.

The hydrolysis of fructose 1,6-bisphosphate to fructose 6-phosphate is a key reaction of carbohydrate metabolism. The enzyme that catalyzes this reaction, fructose-1,6-bisphosphatase, appears to be present in all forms of living organisms. Regulation of the enzyme activity, however, occurs by a variety of distinct mechanisms. These include AMP inhibition (most sources), cyclic AMP-dependent phosphorylation (yeast), and light-dependent activation (chloroplast). In the present studies, we have made a comparison of the primary structure of mammalian fructose-1,6-bisphosphatase with the sequence of peptides isolated from the yeast Saccharomyces cerevisiae, Escherichia coli, and spinach chloroplast enzymes. Our results demonstrate a high degree of sequence homology, suggesting a common evolutionary origin for all fructose-1,6-bisphosphatases.

Amino Acid Sequence↗

PubMed1984

The 'slow' pH-induced conformational transition of chloroplast fructose 1,6-bisphosphatase and the control of the Calvin cycle.

A slow conformation change of chloroplastic reduced fructose bisphosphatase is detected upon raising the pH from 7 to 8 or upon lowering the pH from 8 to 7. These conformation changes are fully reversible. In the time scale investigated these processes occur in one step. Their time constants and their amplitudes have been determined and analyzed as a function of proton concentration. The results obtained are consistent with the view that upon ionization or protonation of a strategic ionizable group the protein undergoes a 'slow' conformational transition that may be followed by conventional fluorescence techniques. Since illumination brings about a pH rise of chloroplastic stroma from 7 to 8, the above results suggest that light activation of fructose bisphosphatase is at least in part due to a slow conformation change of this enzyme.

Chloroplasts↗