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PubMed1995

Characterization of the glycolysis in lactate dehydrogenase-A deficiency.

Recurrent rhabdomyolysis due to decreased glycolysis occurred during strenuous exercise by patients with lactate dehydrogenase-A subunit (LDH-A; muscle) deficiency. We report the glycolytic features of 4 patients from 2 families in whom the severity of the disease differed. There was no difference in the gene abnormality. The enzyme activity of LDH in the muscle was less than 5% that of the control value. Glycolysis in the muscle showed that the respective sums of the pyruvate and lactate levels in the patients with mild and severe symptoms were reduced to approximately 65% and 35% that of the control value. Comparable amounts of glycerol 3-phosphate were produced. Glycerol 3-phosphate dehydrogenase activity in the muscles of patients with mild symptoms was three times the control value. These findings suggest that the disease severity in our patients may be related to the degree of NADH reoxidation by glycerol 3-phosphate dehydrogenase substituting for LDH.

Adult↗
PubMed1995

Glycolytic defects in muscle: aspects of collaboration between basic science and clinical medicine.

The molecular heterogeneities of enzyme abnormality have been identified successfully since 1990 for major clinical entities of glycogenolytic and glycolytic defects in skeletal muscle. The interchange between clinical medicine and basic science, which enabled these achievements, has a long history. This review introduces several important examples of this interchange, which has borne much fruit in the comprehensive understanding of glycogenolysis-glycolysis in skeletal muscle and the related defects that cause various metabolic diseases. For instance, the presence of "glycogen synthase" was mainly suggested by the pathophysiology of McArdle's disease. Clinical manifestations of muscle phosphofructokinase (PFK) deficiency have indicated that there could be PFK isozymes under separate genetic control. Although glycolysis is a unidirectional pathway, enzyme defects at each step do not necessarily cause similar manifestations. Glycogen accumulation is mostly associated with enzyme defects in glycogenolysis and in the first stage of glycolysis. Since the original report of phosphoglycerate mutase deficiency in 1981, no newly recognized glycolytic defects have been presented. Glycolytic steps for which no enzyme deficiency has been identified seem to provide another important impetus for further study of "fail-safe" mechanisms in regard to monogenic disorders.

Biochemical Phenomena↗
PubMed1992

pH dependence of the reverse reaction catalyzed by phosphofructokinase I from Escherichia coli: implications for the role of Asp 127.

The kinetics of the reverse reaction catalyzed by Escherichia coli phosphofructokinase, i.e., the synthesis of ATP and fructose-6-phosphate from ADP and fructose-1,6-bisphosphate, have been studied at different pH values, from pH 6 to pH 9.2. Hyperbolic saturations of the enzyme are observed for both substrates. The affinity for fructose-1,6-bisphosphate decreases with pH following the ionization of a group with a pK of 6.6, whereas the catalytic rate constant and perhaps the affinity for ADP are controlled by the ionization of a group with a pK of 6. Several arguments show that the pK of 6.6 is probably that of the carboxyl group of Asp 127, whereas the pK of 6 is tentatively attributed to the carboxyl group of Asp 103. The pK of 6.6 is assigned to the carboxyl group of Asp 127 in the free enzyme, and a simple model suggests that the same group would have an abnormally high pK, above 9.6, in the complex between phosphofructokinase and fructose-1,6-bisphosphate. It is proposed that the large pK shift of more than 3 pH units upon binding of fructose-1,6-bisphosphate is due to an electrostatic repulsion that could exist between the 1-phosphate group and the carboxyl group of Asp 127, which are close to each other in the crystal structure of phosphofructokinase (Shirakihara, Y. & Evans, P.R., 1988, J. Mol. Biol. 204, 973-994). The same interpretation would also explain the much higher affinity of the enzyme for fructose-1,6-bisphosphate when Asp 127 is protonated.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Diphosphate↗
PubMed1995

Covalent control of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase: insights into autoregulation of a bifunctional enzyme.

The hepatic bifunctional enzyme, 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (6PF-2-K/Fru-2,6-P2ase), E.C. 2.7-1-105/E.C. 3-1-3-46, is one member of a family of unique bifunctional proteins that catalyze the synthesis and degradation of the regulatory metabolite fructose-2,6-bisphosphate (Fru-2,6-P2). Fru-2,6-P2 is a potent activator of the glycolytic enzyme 6-phosphofructo-1-kinase and an inhibitor of the gluconeogenic enzyme fructose-1,6-bisphosphatase, and provides a switching mechanism between these two opposing pathways of hepatic carbohydrate metabolism. The activities of the hepatic 6PF-2-K/Fru-2,6-P2ase isoform are reciprocally regulated by a cyclic AMP-dependent protein kinase (cAPK)-catalyzed phosphorylation at a single NH2-terminal residue, Ser-32. Phosphorylation at Ser-32 inhibits the kinase and activates the bisphosphatase, in part through an electrostatic mechanism. Substitution of Asp for Ser-32 mimics the effects of cAPK-catalyzed phosphorylation. In the dephosphorylated homodimer, the NH2- and COOH-terminal tail regions also have an interaction with their respective active sites on the same subunit to produce an autoregulatory inhibition of the bisphosphatase and activation of the kinase. In support of this hypothesis, deletion of either the NH2- or COOH-terminal tail region, or both regions, leads to a disruption of these interactions with a maximal activation of the bisphosphatase. Inhibition of the kinase is observed with the NH2-truncated forms, in which there is also a diminution of cAPK phosphorylation to decrease the Km for Fru-6-P. Phosphorylation of the bifunctional enzyme by cAPK disrupts these autoregulatory interactions, resulting in inhibition of the kinase and activation of the bisphosphatase. Therefore, effects of cyclic AMP-dependent phosphorylation are mediated by a combination of electrostatic and autoregulatory control mechanisms.

Amino Acid Sequence↗
PubMed1993

Different signals control the activation of glycolysis in the yeast Saccharomyces cerevisiae.

The glycolytic pathway in Saccharomyces cerevisiae is activated by fermentable sugars at several steps. Mutants with deletions of genes coding for enzymes of the upper part of glycolysis were used to characterize the triggering mechanisms. Synthesis of fructose-2,6-bisphophate is catalysed by two 6-phosphofructo-2-kinase isoenzymes, one of which is activated by fermentable sugars while synthesis of the second enzyme is induced (Kretschmer and Fraenkel, 1991). Increase in the level of fructose-2,6-bisphosphate is demonstrated to depend on an internal metabolite upstream of the phosphoglucose isomerase reaction. The signalling process correlates with distinct temporal changes in the concentration of glucose-6-phosphate but not with its absolute level, indicating an adaptational mechanism. It is independent of the uptake and phosphorylation systems used by different sugars. Interestingly, this increase, although delayed, could also be observed in strains lacking the rapid cAMP increase after sugar addition which is thought to be responsible for the activating process. Synthesis of glucose-6-P and fructose-6-P is needed for the complete induction of pyruvate kinase and inactivation of fructose-1,6-bisphosphatase. On the other hand, induction of pyruvate decarboxylase depends mainly on a signal in the lower part of glycolysis.

Carbohydrate Metabolism↗
PubMed1993

Stimulation of glucose utilization by fructose in isolated rat hepatocytes.

In rat hepatocytes fructose at low concentrations (below 1 mM) stimulated the glycolytic flux as measured by the release of 3H2O from [3-3H]glucose and increased fructose 2,6-bisphosphate [Fru(2,6)P2] levels, without modifying the activity of 6-phosphofructo-2-kinase. Maximal stimulation of the glycolytic pathway by 0.1 mM fructose was observed when hepatocytes were incubated in the presence of physiological concentrations of glucose (8 mM). The rise in Fru(2,6)P2 levels was probably due to an increase in glucose 6-phosphate, which in turn resulted from the stimulation of glucose phosphorylation as measured by the formation of 3H2O from [2-3H]glucose. Furthermore, no effects of low doses of fructose on the glycolytic flux or on glucose phosphorylation were observed in hepatocytes from streptozocin-diabetic rats in which glucokinase is almost absent, or in hepatocytes incubated in the presence of mannoheptulose where glucokinase is inhibited. These results suggest that fructose at low concentrations increases the glycolytic flux by raising Fru(2,6)P2 levels solely as a consequence of the stimulation of glucose phosphorylation.

Animals↗
PubMed1993

Pyrophosphate: fructose-6-phosphate 1-phosphotransferase and fructose 2,6-bisphosphate in the bundle sheath of maize leaves.

The aim of this work was to discover whether the cells of the bundle sheath of the leaves of maize (Zea mays) contained pyrophosphate:fructose-6-phosphate 1-phosphotransferase (PFP) and fructose 2,6-bisphosphate (Fru-2,6-P2). Physiologically active preparations of bundle sheath cells from leaves of 4- to 6-week-old plants showed activities of PFP, 6-phosphofructo-2-kinase (6-PF-2-K), and fructose-2,6-bisphosphatase (Fru-2,6-Pase) of 38, 1.8, and 15 nmol min-1 mg-1 chlorophyll, respectively, and contained 75 pmol mg-1 chlorophyll Fru-2,6-P2. For the above enzymes, and marker enzymes for the bundle sheath and for mesophyll cells, the ratios of the activities in leaf extracts to those in bundle sheath extracts were determined. The ratios for PFP, 6-PF-2-K, Fru-2,6-Pase, and Fru-2,6-P2 were intermediate between those found for the mesophyll markers and bundle sheath markers. The distribution of PFP activity after nonaqueous fractionation of leaves differed from that of the bundle sheath and mesophyll marker enzymes. It is argued that maize bundle sheaths can contain significant activity of PFP and amounts of Fru-2,6-P2.

Carbon Dioxide↗
PubMed1993

Phosphofructokinase from liver of the rainbow trout, Oncorhynchus mykiss.

Phosphofructokinase (PFK) from liver of the rainbow trout Oncorhynchus mykiss was purified to homogeneity with a recovery of 35% of total activity. The purified enzyme was a homotetramer with a native molecular weight of 297,000 +/- 16,000 and a subunit M(r) of 76,000 +/- 3000. Arrhenius plots of enzyme activity were linear over 5-27 degrees C with an activation energy of 52.3 +/- 2.1 kJ/mol. The binding of fructose 6-phosphate was cooperative. High ATP increased the Hill coefficient and produced a marked allotropic inhibition of the enzyme activity. The affinity of the enzyme for fructose 6-phosphate was increased by the addition of the enzyme activators such as inorganic phosphate, ammonium ions, AMP, and fructose 2,6-bisphosphate; the activators also reduced the inhibitory effect of ATP. Trout liver PFK was activated by phosphoenolpyruvate at physiological concentrations but was not affected by citrate.

Adenosine Monophosphate↗
PubMed1993

Cloning, expression, and sequence of an allosteric mutant ADPglucose pyrophosphorylase from Escherichia coli B.

Escherichia coli B mutant strain SG14 accumulates glycogen at 28% of the rate observed for the parent strain. This is due to the presence of an ADPglucose pyrophosphorylase with altered allosteric properties including lower apparent affinities for substrates, the activator, fructose-1,6-bisphosphate, and the inhibitor, AMP. The mutant enzyme also is completely insensitive to activation by NADPH. To clone this mutant, an SG14 library was constructed by insertion of the chromosomal DNA into the PstI site of pBR322. Screening of the library via colony hybridization with a wild-type gene (glgC) probe resulted in the successful isolation of a recombinant plasmid, designated pPJ2, which contained the mutant glgC gene. The enzyme expressed from pPJ2 was partially purified and found to be very similar in kinetic and allosteric properties to the enzyme isolated from the SG14 strain. The mutant glgC gene, a HincII fragment from pPJ2, was then subcloned into pUC118/119 for dideoxy sequencing of both strands. One amino acid change was found in a region that is highly conserved in all known sequences: a single point mutation at the deduced amino acid residue 44 resulted in a change of alanine to threonine. The properties of this mutant are discussed in comparison to other known allosteric mutants.

Adenosine Monophosphate↗
PubMed1994

Importance of the modulation of glycolysis in the control of lactate metabolism by fatty acids in isolated hepatocytes from fed rats.

In liver cells from fed rats, lactate utilization depends on its extracellular concentration and the threshold concentration at which lactate uptake equilibrates release is about 3 mM. Even-chain fatty acids (butyrate, octanoate, or oleate) played a crucial role (i) to depress the lactate release, from 40% (butyrate or oleate) to 72% (octanoate), and (ii) to lower the threshold concentration for lactate utilization (down to 1 mM with octanoate). The effects of fatty acids were connected to their inhibition of hepatic glycolysis, estimated by the detritiation of [6-3H]glucose (about -30% with butyrate or oleate and -45% with octanoate). Fatty acids depressed the cellular concentration of pyruvate which, at physiological concentration of lactate, favors its utilization. The rise in ketone bodies concentration in response to fatty acids reflected an enhanced acetyl CoA production, resulting in an accumulation of citrate. In parallel there was a drop of the cellular concentration of fructose 2,6-biphosphate. As a result, there was an inhibition of the flux through 6-phosphofructo-1 kinase (50, 75, or 40% inhibition with butyrate, octanoate, or oleate, respectively). The other regulatory glycolysis steps, catalyzed by glucokinase and pyruvate kinase, were not affected by fatty acids. Inhibition of hepatic glycolysis by fatty acids seems connected to acetyl-CoA generation since octanoate, readily metabolized to acetyl-CoA and ketone bodies by hepatocytes, had a more potent stimulatory effect on the hepatic uptake of lactate than butyrate or oleate. Propionate, which yields practically no acetyl CoA, slightly stimulated lactate release and elevated the threshold of lactate utilization. The present data suggest thus that, in hepatocytes from fed rats, fatty acids effectively inhibit glycolysis and switch liver cell metabolism toward gluconeogenic conditions, which promotes lactate utilization.

Acetyl Coenzyme A↗
PubMed1995

Mutagenesis of an amino acid residue in the activator-binding site of cyanobacterial ADP-glucose pyrophosphorylase causes alteration in activator specificity.

The specificity for activator of ADP-glucose pyrophosphorylase is closely related to the corresponding major carbon-assimilation pathway. The enzyme from Escherichia coli is mainly activated by fructose-1,6-P2, while the cyanobacterial, algal, and higher-plant enzymes are activated by 3-P-glycerate. Previous results have shown that Lys39 of the E. coli enzyme is involved in the binding of fructose-1,6-P2 while for the Anabaena enzyme, lysine residues 382 and 419 have been shown to be involved in the binding of 3-phosphoglycerate. This report shows that if Lys419 of the Anabaena enzyme is changed to glutamine, activation of the cyanobacterial enzyme by fructose-1,6-P2 becomes more effective than that of 3-P-glycerate at lower concentrations. Kinetic studies show that fructose-1,6-P2 competitively inhibits 3-P-glycerate activation of the Anabaena wild-type enzyme, suggesting that these two compounds bind to the same site. Thus a change of one amino acid at the activator binding domain can affect the specificity of activation of the Anabaena ADP-glucose pyrophosphorylase.

Allosteric Regulation↗
PubMed1995

Site-directed mutagenesis of the substrate binding site of porcine fructose-1,6-bisphosphatase.

Asn 212, Arg 243, Tyr 244, Tyr 264, and Lys 274, which are conserved in all known primary sequences of fructose-1, 6-bisphosphatase, are located in the substrate binding domain on the basis of the crystal structure of the enzyme. Mutations of the five residues of porcine liver fructose-1,6-bisphosphatase (Asn212Ala, Arg243-Met, Tyr244Phe, Tyr264Phe, and Lys274Leu) were carried out by site-directed mutagenesis. The wild-type and mutant forms of the enzyme were purified to homogeneity and characterized by initial rate kinetics and circular dichroism spectrometry. The mutants exhibited kcat values that are similar to those of the wild-type enzyme. The Km values for fructose 1,6-bisphosphate of the mutants are 6- to 44-fold higher than that of the wild-type enzyme. The Ki values for fructose 2,6-bisphosphate and AMP of the mutants increased from 56- to 1950-fold and 12- to 27-fold, respectively, relative to the wild-type enzyme. The alteration of inhibition constants for both inhibitors suggest that these five active site residues are involved in the inhibition by fructose 2,6-bisphosphate and AMP. No apparent differences in secondary structure of the wild-type and mutant forms of fructose-1,6-bisphosphatase were observed as measured by circular dichroism spectrometry. This report demonstrates that Asn 212, Arg 243, Tyr 244, Tyr 264, and Lys 274 not only are the sites for substrate binding, but also play an important role in the binding affinity of inhibitors fructose 2,6-bisphosphate and AMP.

Adenosine Monophosphate↗
PubMed1995

Anomeric specificity of rat hepatic 6-phosphofructo-2-kinase: an NMR study.

The anomeric specificity of 6-phosphofructo-2-kinase for D-fructose-6-phosphate was determined by nuclear magnetic spectroscopy. A mutant 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (His258-Ala) was used to minimize degradation of fructose-2,6-bisphosphate by the bisphosphatase activity. The 1H NMR spectrum of the fructose-2,6-bisphosphate formed from the reaction was identical in the spectral region (3.5 to 4.0 ppm) to that reported for D-fructose-2,6-bisphosphate by Voll et al. (7). The integration of this region accounted for the 7 nonexchangeable protons of the furanose form of fructose. The measured coupling constants and the chemical shifts were identical to those of commercially prepared D-fructose-2,6-bisphosphate. The long range (through 4-bond: P-2, O-2, C-2, C-3, and H-3) coupling between P-2 and H-3, 4JH-3, P-2, was found to be 1.06 Hz and provides strong evidence for the beta-anomer. Additionally, failure to find a similar coupling to the H-la peak ruled out the possibility of existence of the alpha-anomer. These results indicate that only beta-D-fructose-2,6-bisphosphate was synthesized via the 6-phosphofructo-2-kinase reaction. It was concluded that 6-phosphofructo-2-kinase has an absolute stereo specificity for the beta-anomer of D-fructose-6-phosphate.

Animals↗
PubMed1995

A pH-dependent allosteric transition in Ascaris suum phosphofructokinase distinct from that observed with fructose 2,6-bisphosphate.

Ascaris suum phosphofructokinase exhibits dramatic shifts in its circular dichroic spectra in the pH range 6 to 8. These shifts are quite distinct from those induced by the activators AMP and fructose 2,6-bisphosphate. Concomitant with these pH-induced spectral shifts, the enzyme also displays changes in its allosteric behavior. Inorganic ions such as K+, NH+4, SO4(2-), and PO4(3-) also cause CD-spectral shifts similar to those produced by a change in pH. Based on the evidence derived from gel filtration and sedimentation equilibrium studies, the observed CD-spectral shifts are interpreted as due to conformational changes in the enzyme tetramer rather than due to a change in its aggregation state. Further, since the pK value of 6.4 obtained from pH dependence of increase in ellipticity at 210 nm agrees very well with the pK value of 6.8 for the loss of ATP inhibition due to modification of a histidine residue (G. S. J. Rao, B. A. Wariso, P. F. Cook, and B. G. Harris (1987) J. Biol. Chem. 262, 14068-14073), it is concluded that a single histidine residue in the ATP-inhibitory site acts as a trigger for the structural changes accompanying ATP inhibition of the enzyme. This view is strongly supported by the observation that the enzyme desensitized to ATP inhibition by chemical modification of a histidine residue in the ATP-inhibitory site shows absolutely no change in its CD spectrum in the pH range 6 to 8. This study demonstrates that the mechanism of activation of phosphofructokinase at higher pH and by inorganic ions involves conformational transitions that are quite distinct from those induced by AMP and fructose 2,6-bisphosphate. A scheme is presented that incorporates all of the different states of the enzyme dependent upon effectors and pH.

Adenosine Monophosphate↗
PubMed1997

Regulatory role of fructose-2,6-bisP on glucose metabolism in frog oocytes: in vivo inhibition of glycogen synthesis.

Glycogen synthesis following glucose microinjection in frog oocytes proceeds preferentially by an indirect pathway involving gluconeogenesis from triose compounds. Because of the known regulatory role of fructose-2,6-bisP on glucose utilization in most vertebrate tissues we coinjected [U-14C]glucose and fructose-2,6-bisP into oocytes and observed a marked inhibition of label incorporation into glycogen, with an I50 value of 2 microM, which is similar to the value measured for the in vitro inhibition of oocyte fructose-1,6-bisphosphatase. Other hexoses-bisP were tested: 2,5-anhydromannitol-1,6-bisP was as effective as inhibitor as fructose-2,6-bisP; glucose-1,6-bisP showed some effect although 50% inhibition was obtained at a concentration 10 times higher than with fructose-2,6-bisP; fructose-1,6-bisP had no effect at all. The inhibition pattern for the in vivo glycogen synthesis by these analogs closely matched the one obtained with partially purified oocyte fructose-1,6-bisphosphatase. The intracellular concentration of fructose-2,6-bisP in unperturbed oocytes was found to be between 0.1 and 0.2 microM. Fructose-6-phosphate,2-kinase levels measured in oocyte homogenates were between 0.02 and 0.06 mU per gram of ovary. After 60 min incubation, fructose-2,6-bisP microinjected into the oocytes was almost completely degraded, suggesting that fructose-2,6-bisphosphatase is active in vivo. The results presented in this paper indicate that fructose-2,6-bisP plays an important role in the in vivo regulation of glucose utilization in frog-grown oocytes.

Animals↗
PubMed1998

Conserved active site aspartates and domain-domain interactions in regulatory properties of the sugar kinase superfamily.

The structures of the sugar kinase/heat shock 70/actin superfamily of enzymes show that the active site is located in a deep cleft between two domains whose relative movement defines a domain closure conformational change thought to be involved in the catalytic and regulatory properties of members of the superfamily. To investigate the role of the domain closure in the regulatory behavior, site-directed mutagenesis is used to alter specific domain-domain interactions in Escherichia coli glycerol kinase (EC 2.7.1.30; ATP:glycerol 3-phosphotransferase), a member of this superfamily. Two active site aspartate residues are conserved throughout the superfamily, one (Asp245 in glycerol kinase) which is proposed to act as a general base during catalysis and one (Asp10 in glycerol kinase) which interacts with the Mg(II) ion of the bound Mg(II)-nucleotide complex. Each of these residues participates in domain-domain interactions that are mediated by the bound substrates. The enzymes containing the substitutions Asp245 to Asn (D245N) or Asp10 to Asn (D10N) were purified by affinity chromatography, and the effects of the substitutions on the catalytic properties and regulation by the allosteric effectors, fructose 1,6-bisphosphate (FBP), and the glucose-specific phosphocarrier protein, IIIGlc (also known as IIAGlc), were determined. Each of the residues participates in catalysis; kcat/Katp is decreased 300-fold by the D245N substitution and 100-fold by the D10N substitution. Affinity labeling with the glycerol analog 1,3-dichloroacetone shows that the level of activity seen for the D245N mutant enzyme is not due to deamidation of the substituted asparagine. Each of the substitutions has little effect on regulation by FBP and the apparent affinity for IIIGlc, and the D245N substitution does not affect the extent of inhibition by IIIGlc. However, the D10N substitution decreases the maximum extent of inhibition by IIIGlc from 100 to 60%, thus changing the action of IIIGlc to that of a partial inhibitor. The different sensitivities of the extents of FBP and IIIGlc inhibition to perturbation of a domain-domain interaction mediated by Asp10 suggest that the relations of the actions of these allosteric effectors to the domain closure conformational change are different.

Allosteric Regulation↗
PubMed1998

A kinetic study of site-directed mutants of Escherichia coli ADP-glucose pyrophosphorylase: the role of residue 295 in allosteric regulation.

The effects of amino acid substitutions at residue 295 on the regulatory properties of Escherichia coli ADP-glucose pyrophosphorylase were studied. In previous studies, this residue, altered from proline to serine (P295S) in the gene of a mutant strain of E. coli, resulted in a high-activity form of enzyme [higher activity in absence of activator fructose 1,6-bisphosphate (FBP), higher apparent affinity for FBP and substrates, and lower apparent affinity for the inhibitor, AMP]. The effects of size and charge on this site were explored by replacing Pro with Gly, Asp, Asn, Gln, or Glu. All mutant enzymes were expressed and purified for kinetic analysis. All mutant enzymes, to varying extents, were in more active form than the wild-type enzyme. Enzymes with a substituted negative charge (P295D, P295E) had the highest activity in the absence of FBP, while the P295G enzyme was most similar to the wild type. The P295D and P295E enzymes had the lowest apparent affinities for AMP; this effect was partially abolished by the neutral substitutions P295N and P295Q. Another mutation, G336D, had previously been found to produce an even higher activity enzyme form. In order to examine interactions between substitutions at the 295 and 336 positions, the double mutant P295D-G336D was constructed and characterized. The double mutant enzyme was more active in the absence of FBP, with a higher affinity for FBP and a lower apparent affinity for AMP than either single mutated enzyme. The significance of residue 295 in regulation is discussed.

Adenosine Monophosphate↗
PubMed1999

cDNA sequence and kinetic properties of human lung fructose(1, 6)bisphosphatase.

A cDNA encoding fructose(1,6)bisphosphatase was isolated from total human lung RNA. The cDNA contained an open reading frame encoding 337 amino acids. The determined nucleotide sequence of the lung cDNA was significantly different from muscle cDNA and slightly differed from human liver cDNA in a single mutation (Gly-336 for Ala-336) and a T for C substitution in position 648. The human lung fructose(1, 6)bisphosphatase [Fru(1,6)Pase] was isolated and its kinetic parameters were compared with liver and muscle isoenzymes. Values of kcat for the lung Fru(1,6)Pase were lower than for the liver and muscle enzyme. Like the liver isoenzyme, lung Fru(1,6)Pase is significantly less inhibited by AMP than the muscle enzyme. The values of I0.5 were 9.5, 9.8, and 0.3 microM for the liver, lung, and muscle enzyme, respectively. The lung enzyme was slightly more sensitive to fructose(2,6)bisphosphate [Fru(2,6)P2] inhibition than the liver enzyme. Ki was 75 microM for the lung and 96 microM for the liver enzyme. The synergistic effect of AMP and Fru(2,6)P2 on the lung and liver Fru(1,6)Pase was also observed. In the presence of AMP the corresponding values of Ki for Fru(2,6)P2 were 16 microM for the lung and 10 microM for the liver enzyme.

Adenosine Monophosphate↗