Biomedical subjects

K Uyeda

Publications and source records attributed to K Uyeda.

At least 19 recordsLinked to original sources

PubMed1999

Reaction mechanism of fructose-2,6-bisphosphatase. A mutation of nucleophilic catalyst, histidine 256, induces an alteration in the reaction pathway.

A bifunctional enzyme, fructose-6-phosphate,2-kinase/fructose 2, 6-bisphosphatase (Fru-6-P,2-kinase/Fru-2,6-Pase), catalyzes synthesis and degradation of fructose 2,6-bisphosphate (Fru-2,6-P2). Previously, the rat liver Fru-2,6-Pase reaction (Fru-2,6-P2 --> Fru-6-P + Pi) has been shown to proceed via a phosphoenzyme intermediate with His258 phosphorylated, and mutation of the histidine to alanine resulted in complete loss of activity (Tauler, A., Lin, K., and Pilkis, S. J. (1990) J. Biol. Chem. 265, 15617-15622). In the present study, it is shown that mutation of the corresponding histidine (His256) of the rat testis enzyme decreases activity by less than a factor of 10 with a kcat of 17% compared with the wild type enzyme. Mutation of His390 (in close proximity to His256) to Ala results in a kcat of 12.5% compared with the wild type enzyme. Attempts to detect a phosphohistidine intermediate with the H256A mutant enzyme were unsuccessful, but the phosphoenzyme is detected in the wild type, H390A, R255A, R305S, and E325A mutant enzymes. Data demonstrate that the mutation of His256 induces a change in the phosphatase hydrolytic reaction mechanism. Elimination of the nucleophilic catalyst, H256A, results in a change in mechanism. In the H256A mutant enzyme, His390 likely acts as a general base to activate water for direct hydrolysis of the 2-phosphate of Fru-2,6-P2. Mutation of Arg255 and Arg305 suggests that the arginines probably have a role in neutralizing excess charge on the 2-phosphate and polarizing the phosphoryl for subsequent transfer to either His256 or water. The role of Glu325 is less certain, but it may serve as a general acid, protonating the leaving 2-hydroxyl of Fru-2,6-P2.

Animals↗

PubMed1999

Crystal structure of the H256A mutant of rat testis fructose-6-phosphate,2-kinase/fructose-2,6-bisphosphatase. Fructose 6-phosphate in the active site leads to mechanisms for both mutant and wild type bisphosphatase activities.

Fructose-6-phosphate,2-kinase/fructose-2,6-bisphosphatase (Fru-6-P, 2-kinase/Fru-2,6-Pase) is a bifunctional enzyme, catalyzing the interconversion of beta-D-fructose- 6-phosphate (Fru-6-P) and fructose-2,6-bisphosphate (Fru-2,6-P2) at distinct active sites. A mutant rat testis isozyme with an alanine replacement for the catalytic histidine (H256A) in the Fru-2,6-Pase domain retains 17% of the wild type activity (Mizuguchi, H., Cook, P. F., Tai, C-H., Hasemann, C. A., and Uyeda, K. (1998) J. Biol. Chem. 274, 2166-2175). We have solved the crystal structure of H256A to a resolution of 2. 4 A by molecular replacement. Clear electron density for Fru-6-P is found at the Fru-2,6-Pase active site, revealing the important interactions in substrate/product binding. A superposition of the H256A structure with the RT2K-Wo structure reveals no significant reorganization of the active site resulting from the binding of Fru-6-P or the H256A mutation. Using this superposition, we have built a view of the Fru-2,6-P2-bound enzyme and identify the residues responsible for catalysis. This analysis yields distinct catalytic mechanisms for the wild type and mutant proteins. The wild type mechanism would lead to an inefficient transfer of a proton to the leaving group Fru-6-P, which is consistent with a view of this event being rate-limiting, explaining the extremely slow turnover (0. 032 s-1) of the Fru-2,6-Pase in all Fru-6-P,2-kinase/Fru-2,6-Pase isozymes.

Amino Acid Sequence↗

PubMed1999

A novel factor binding to the glucose response elements of liver pyruvate kinase and fatty acid synthase genes.

Transcription of the liver type pyruvate kinase and lipogenesis enzyme genes is induced by high carbohydrate in liver. We have found a novel protein factor in rat liver nuclei that binds to the glucose response element (CACGTG motifs) of the pyruvate kinase gene (Liu, Z. , Thompson, K. S., and Towle, H. C. (1993) J. Biol. Chem. 268, 12787-12795) and the "insulin response element" of fatty acid synthase gene. The amounts of this DNA-binding protein, termed "glucose response element binding protein" (GRBP) in the nuclear extract, were increased in liver by a high carbohydrate diet and decreased by starvation, high fat, and high protein diet. GRBP also occurs in cytosols of liver and is dependent on carbohydrate. Both the nuclear and the cytosolic GRBP showed similar properties, except the former was more resistant to thermal inactivation than the latter. Kinetics of glucose activation of the cytosolic GRBP in a primary culture of hepatocytes indicated that a half-maximum activation was achieved after 6 h, and glucose concentration required for the maximum activation of the GRBP was approximately 12 mM. Dibutyryl-cAMP, okadaic acid, and forskolin inhibited glucose activation of both GRBP and liver pyruvate kinase transcription. These results suggested that GRBP may be a factor that recognizes the glucose response motif site and may be involved in mediating carbohydrate response of the pyruvate kinase gene.

Animals↗

PubMed1998

Site-directed mutants of rat testis fructose 6-phosphate, 2-kinase/fructose 2,6-bisphosphatase: localization of conformational alterations induced by ligand binding.

Site-directed mutagenesis was utilized to construct mutants, containing one or two tryptophan residues, of the bifunctional enzyme fructose 6-phosphate,2-kinase-fructose 2,6-bisphosphatase. Two of the single-tryptophan mutants (W15 and W64) had the tryptophan residue located in the kinase domain, which is in the N-terminal half, and two (W299 and W320) had the tryptophan residue located in the phosphatase domain, which is in the C-terminal half. The double-tryptophan mutants were W15/W64, W15/W299, W64/W299, and W299/W320. Dynamic polarization data indicated that these tryptophan residues had varying degrees of local mobility. Steady-state polarization data revealed energy transfer between the tryptophan residues in the double mutant W299/W320 but not in the W15/W64, W15/W299, or W64/W299 mutants, indicating the proximity of the W299 and W320 residues. The binding of fructose-6-phosphate resulted in a significant increase in the anisotropy of the W15 mutants, but did not affect the anisotropies of any of the other single-tryptophan mutants. Binding of fructose-2,6-bisphosphate also significantly increased the anisotropy of W15. In the case of fructose-6-phosphate binding, the increased anisotropy was shown to be due to a restriction of the tryptophan residue's local mobility in the presence of bound ligand, which suggests that the N-terminus is located near the kinase active site. These increases in anisotropies were used to estimate the dissociation constants of fructose-6-phosphate and fructose-2,6-bisphosphate, which were 29 +/- 3 and 2.1 +/- 0.3 microM, respectively. These observations are considered in light of the recently published crystal structure for this bifunctional enzyme.

Amino Acid Substitution↗

PubMed1997

Effect of replacement of the amino and the carboxyl termini of rat testis fructose 6-phosphate, 2-kinase:fructose 2,6-bisphosphatase with those of the liver and heart isozymes.

Fru 6-P,2-kinase:Fru 2,6-Pase is a bifunctional enzyme, consisting of highly conserved catalytic domains and variable regulatory domains. The regulatory domains reside in either the N- or the C-terminus, depending upon the isozyme. The rat testis enzyme (RT2K) lacks the regulatory domain, but the rat liver and the bovine heart enzymes contain phosphorylation site(s) in the N- and the C-termini, respectively. In order to determine whether the regulatory domains can be swapped, we have constructed mutant enzymes in which the N- or the C-terminal tail of the testis enzyme was replaced with that of either the liver or the heart enzyme. The substitution with the N-terminus of the liver enzyme (RLN-RT2K) resulted in a small change in the kinetic properties of Fru 6-P,2-kinase, but that with the heart enzyme increased the KFru 6-P 18-fold without affecting the Vmax. The substitution with the C-terminus of the heart enzyme had little effect. The phosphorylation of RLN-RT2K increased KFru 6-P fivefold as in the liver enzyme but did not affect the Fru 2,6-Pase, unlike the liver enzyme. All these mutant enzymes were more thermally labile than the wild type testis enzyme. RLN-RT2K was more sensitive to the denaturant. These results suggest that the N-terminus of the liver enzyme could interact with the kinase domain of the testis enzyme, regulating the kinase activity but was unable to affect the phosphatase domain. These differences could be explained by the large differences in net charges of the terminal tails.

Allosteric Regulation↗

PubMed1997

Chemical mechanism of the fructose-6-phosphate,2-kinase reaction from the pH dependence of kinetic parameters of site-directed mutants of active site basic residues.

A bifunctional enzyme, fructose-6-phosphate 2-kinase-fructose 2, 6-bisphosphatase, catalyzes synthesis and degradation of fructose 2, 6-bisphosphate. Mutants of basic residues, including Lys51, Arg78, Arg79, Arg136, Lys172, and Arg193, immediately around the active site of rat testis fructose 6-P,2-kinase were constructed, and their steady state kinetics, ATP binding, and the effect of pH on the kinetics were characterized. All mutants showed a several-fold increase in KMgATP, much larger increases in KFru 6-P, and decreased V compared to those of the wild type enzyme (WT). Replacement of Lys172 and Arg193 with Ala and Leu, respectively, also produced mutants with large KFru 6-P values. Substitution of Lys51, which is located in a Walker-A motif (GXXGXGKT, amino acids 45-52), with Ala or His resulted in enzymes with increased KMgATP values and unable to bind Fru 6-P. The dissociation constants for 2'(3')-O-(N-methylanthraniloyl)-ATP (mantATP) and ATP of all these mutants except Lys51 were similar. Lys51 mutants were unable to bind mantATP. The pH dependence of V and the V/Ks for MgATP and Fru 6-P suggest a mechanism in which reactants and enzyme combine irrespective of the protonation state of groups required for binding and catalysis, but only the correctly protonated enzyme-substrate complex is catalytically active. A chemical mechanism is suggested in which a general base accepts a proton from the 2-hydroxyl of Fru 6-P concomitant with nucleophilic attack on the gamma-phosphate of MgATP. Phosphoryl transfer is also facilitated by interaction of the gamma-phosphate with a positively charged residue that neutralizes the remaining negative charge. The dianionic form of the 6-phosphate of fructose 6-P is required for binding, and it is likely anchored by a positively charged enzyme residue. A comparison of the pH dependence of kinetic parameters for Ala or His mutant proteins at Lys51, Lys172, and Arg79 suggests that Lys51 interacts with the gamma-phosphate of MgATP and that several other arginines likely participate in transition state stabilization of the transferred phosphoryl. The active site general base has yet to be identified.

Adenosine Triphosphate↗

PubMed1997

The active sites of fructose 6-phosphate,2-kinase: fructose-2, 6-bisphosphatase from rat testis. Roles of Asp-128, Thr-52, Thr-130, Asn-73, and Tyr-197.

To investigate the role in catalysis and/or substrate binding of the Walker motif residues of rat testis fructose 6-phosphate, 2-kinase:fructose-2,6-bisphosphatase (Fru 6-P,2-kinase:Fru-2,6-Pase), we have constructed and characterized mutant enzymes of Asp-128, Thr-52, Asn-73, Thr-130, and Tyr-197. Replacement of Asp-128 by Ala, Asn, and Ser resulted in a small decrease in Vmax and a significant increase in Km values for both substrates. These mutants exhibited similar pH activity profiles as that of the wild type enzyme. Mutation of Thr-52 to Ala resulted in an enzyme with an infinitely high Km for both substrates and an 800-fold decreased Vmax. Substitution of Asn-73 with Ala or Asp caused a 100- and 600-fold increase, respectively in KFru 6-P with only a small increase in KATP and small changes in Vmax. Mutation of Thr-130 caused small changes in the kinetic properties. Replacement of Tyr-197 with Ser resulted in an enzyme with severely decreased binding of Fru 6-P with 3-fold decreased Vmax. A fluorescent analog of ATP, 2'(3')-O-(N-methylanthraniloyl)ATP (mant-ATP) served as a substrate with Km = 0.64 microM, and Vmax = 25 milliunits/mg and was a competitive inhibitor with respect to ATP. When mant-ATP bound to the enzyme, fluorescence intensity at 440 nm increased. mant-ATP binding of the wild type and the mutant enzymes were compared using the fluorometric method. The Kd values of the T52A and D128N enzymes were infinitely high and could not be measured, while those of the other mutant enzymes increased slightly. These results provide evidence that those amino acids are involved in substrate binding, and they are consistent with the crystallographic data. The results also suggest that Asp-128 does not serve as a nucleophile in catalysis, and since there are no other potential nucleophiles in the active site, we hypothesize that the Fru 6-P,2-kinase reaction is mediated via a transition state stabilization mechanism.

Amino Acids↗

PubMed1996

The crystal structure of the bifunctional enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase reveals distinct domain homologies.

BACKGROUND: Glucose homeostasis is maintained by the processes of glycolysis and gluconeogenesis. The importance of these pathways is demonstrated by the severe and life threatening effects observed in various forms of diabetes. The bifunctional enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase catalyzes both the synthesis and degradation of fructose-2,6-bisphosphate, a potent regulator of glycolysis. Thus this bifunctional enzyme plays an indirect yet key role in the regulation of glucose metabolism. RESULTS: We have determined the 2.0 A crystal structure of the rat testis isozyme of this bifunctional enzyme. The enzyme is a homodimer of 55 kDa subunits arranged in a head-to-head fashion, with each monomer consisting of independent kinase and phosphatase domains. The location of ATPgammaS and inorganic phosphate in the kinase and phosphatase domains, respectively, allow us to locate and describe the active sites of both domains. CONCLUSIONS: The kinase domain is clearly related to the superfamily of mononucleotide binding proteins, with a particularly close relationship to the adenylate kinases and the nucleotide-binding portion of the G proteins. This is in disagreement with the broad speculation that this domain would resemble phosphofructokinase. The phosphatase domain is structurally related to a family of proteins which includes the cofactor independent phosphoglycerate mutases and acid phosphatases.

Amino Acid Sequence↗

PubMed1996

A mechanism of regulation of hepatic Fru 2,6-P2 concentration upon refeeding: involvement of xylulose 5-P and cyclic-AMP.

In order to determine the mechanism for delayed increase in Fructose 2,6-P2 in livers of refed rats, the time course of changes in various metabolites upon refeeding NIH or high sucrose diet was investigated. Kinetics of increase in Fructose 2,6-P2 and Xylulose 5-P were similar but different from hexose 6-P or glycogen in the livers of 48 h starved rats refed with NIH diet. The increase in the Fructose 2,6-P2 level was a result of a combination of changes in Fructose 6-P,2-kinase and Fructose 2,6-bisphosphatase activity ratios, indicating dephosphorylation of the bifunctional enzyme and decreased cAMP. A similar correlation between Fructose 2,6-P2 and Xylulose 5-P and dephosphorylation was observed with refeeding high sucrose diet and also with 16 h starved rats. These kinetic results are consistent with the idea that a specific protein phosphatase 2A, activated by Xylulose 5-P, dephosphorylates Fructose 6-P,2-kinase:Fructose 2,6-bisphosphatase and also decreased protein kinase A activity, resulting in increased hepatic Fructose 2,6-P2.

Animals↗

PubMed1996

A mechanism for fatty acid inhibition of glucose utilization in liver. Role of xylulose 5-P.

The glucose-stimulated rise in Fru-2,6-P2 in liver results from xylulose 5-P activation of a specific protein phosphatase 2A which dephosphorylates Fru-6-P,2-kinase:Fru-2,6-bisphosphatase (Nishimura, M., and Uyeda, K. (1994) J. Biol. Chem. 269, 26100-26106). In order to determine the role of xylulose 5-P in regulating Fru-2, 6-P2 in liver, the effect of fatty acids, various hexoses, and hormones was examined in perfused rat liver and in intact rats. When 24-h starved rat livers were perfused with acetate, butyrate, or propionate, Fru-2,6-P2 and xylulose 5-P decreased to the same extent and at similar rates. The activity ratios of the kinase and the phosphatase changed in a reciprocal manner, indicating that the phosphorylated form of the enzyme was increased by the fatty acids perfusion. The fatty acids caused the similar changes in the metabolites and the phosphorylation state of the bifunctional enzyme in livers of fed animals. Fructose, galactose, or mannose perfusion in starved rat liver increased both Fru-2,6-P2 and xylulose 5-P and converted the bifunctional enzyme to the dephospho form. Both the Fru-2,6-P2 and xylulose 5-P levels in rats fed a high fat diet decreased over 50% compared to that in control rats. These results indicated a close correlation between Fru-2,6-P2 and xylulose 5-P levels and the phosphorylation state of fructose 6-P, 2-kinase:fructose 2,6-bisphosphatase. Fatty acid inhibition of glucose metabolism can be explained by a decrease in xylulose 5-P, which lowers xylulose 5-P-activated protein phosphatase 2A activity, resulting in more phosphorylated form of the bifunctional enzyme and consequently lower Fru-2,6-P2.

Animals↗

PubMed1996

Islet fructose 6-phosphate, 2-kinase:fructose 2,6-bisphosphatase: isozymic form, expression, and characterization.

Polymerase chain reaction analysis of the mRNA isolated from rat islets demonstrated that the major isozyme of Fructose 6-P,2-kinase:Fructose 2,6-bisphosphatase was the heart type enzyme, and that the liver type enzyme was not detectable. The islet enzyme was expressed in Escherichia coli and purified to homogeneity. The islet enzyme showed the highest Fructose 6-P,2-kinase activity (478 milliunits/mg) compared to the other isozymes and Fructose 2,6-Pase activity (39 milliunits/mg). Fructose 6-P,2-kinase showed KmF6P = 17 microM, which is within the range of in vivo Fru 6-P concentrations in islets. 6-P-Gluconate was a potent inhibitor of Fructose 2,6-Pase. The data suggest that Fructose 6-P,2-kinase activity of the bifunctional enzyme was high and Fructose 2,6-Pase activity was inhibited under physiological variations of blood glucose concentration.

Animals↗

PubMed1996

Enzymatic and fluorescence studies of four single-tryptophan mutants of rat testis fructose 6-phosphate,2-kinase:fructose 2,6-bisphosphatase.

In order to determine environments around four tryptophan residues, located in the N-terminus, in the kinase and in the phosphatase domains of rat testis Fru 6-P,2-kinase:Fru 2,6-bisphosphatase, mutant enzymes containing a single tryptophan were constructed by site-directed mutagenesis. The kinetic constants of these mutant enzymes were similar to those of the wild-type enzyme. The sum of the fluorescence intensities of the enzymes was 1.5 x that of the wild-type enzyme, and Trp 299, Trp 64, Trp 15, and Trp 320 contributed 38%, 28%, 17%, and 17%, respectively. The fluorescence polarization of the wild-type enzyme was significantly lower than any of the mutant enzymes, suggesting proximity of two tryptophan residues in the wild-type enzyme. The polarization in the presence of Fru 6-P affected only Trp 15, which suggested that it is located near the Fru 6-P binding site, but Trp 64 is not. Inactivation of both enzyme activities and unfolding of these enzymes in guanidine were monitored by activity assays and fluorescence intensities and maxima. Both Fru 6-P,2-kinase and Fru 2,6-bisphosphatase activities of all these enzymes were inactivated between 0.7 and 1 M guanidine. Enzymes containing Trp 64 or Trp 15 showed biphasic fractional unfolding curves, but those of Trp 299 or Trp 320 showed gradual steady changes. Fluorescence quenching by iodide indicated that Trp 64 was not accessible and that other Trp residues were only slightly accessible to solvent. These results suggest that all the Trp residues are in heterogeneous environments and that none are exposed on the protein surface.

Animals↗

PubMed1995

Purification and characterization of a novel xylulose 5-phosphate-activated protein phosphatase catalyzing dephosphorylation of fructose-6-phosphate,2-kinase:fructose-2,6-bisphosphatase.

We have shown previously (Nishimura, M., Fedorov, S., and Uyeda, K. (1994) (J. Biol. Chem. 269, 26100-26106) that the administration of high concentrations of glucose stimulates dephosphorylation of Fru-6-P,2-kinase: Fru-2,6-bisphosphatase in perfused liver, and xylulose (Xu) 5-P activates the dephosphorylation reaction. To characterize the protein phosphatase, we have purified the Xu 5-P-activated protein phosphatase to homogeneity from livers of rats injected with high glucose. Several protein phosphatases in the livers were separated by DEAE-cellulose chromatography, but only one peak of the enzyme was activated by Xu 5-P. The protein phosphatase was inhibited by okadaic acid (IC50 = 1-3 nM) and did not require Mg2+ or Ca2+, suggesting that the enzyme was type 2A. The enzyme was a heterotrimer (M(r) = 150,000) and consisted of structural (A, 65 kDa) catalytic (C, 36 kDa), and regulatory (B, 52 kDa) subunits. Amino acid sequences of five tryptic peptides derived from the B subunit showed similarity with those of the B alpha isoform of rat protein phosphatase 2A, but five out of 73 residues were different. The protein phosphatase catalyzed dephosphorylation of Fru-6-P,2-kinase:Fru-2,6-Pase, phosphorylase alpha, and pyruvate kinase, and the Km values were 0.8 microM, 3.7 microM, and 2.2 microM, respectively. Among these substrates dephosphorylation of only the bifunctional enzyme was activated by Xu 5-P, and the K alpha value for Xu 5-P was 20 microM. Xu 5-P was the only sugar phosphate which activated the PP2A among all the sugar phosphates examined. These results demonstrated the existence and isolation of a unique heterotrimeric protein phosphatase 2A in rat liver which catalyzed the dephosphorylation of Fru-6-P,2-kinase:Fru-2,6-Pase and was activated specifically by Xu 5-P. The Xu 5-P-activated protein phosphatase 2A explains the increased Fru 2,6-P2 level in liver after high glucose administration.

Amino Acid Sequence↗

PubMed1995

Hexose phosphate binding sites of fructose 6-phosphate,2-kinase:fructose 2,6-bisphosphatase.

A previous chemical modification study [Kitamura et al. (1989) J. Biol. Chem. 264, 6344-6438] has shown that N-bromoacetylethanolamine phosphate labeled specifically Cys107 of rat liver Fru 6-P,2-kinase:Fru 2.6-Pase and the corresponding Cys of the bovine heart enzyme, leading to inactivation of kinase activity. Since Fru 6-P provided protection against the inactivation, this region of the enzyme was thought to be a Fru 6-P binding site of the kinase enzyme. To examine this possibility, oligonucleotide-directed mutagenesis has been used to alter several residues in expressed rat testis Fru 6-P,2-kinase:Fru 2,6-Pase. The change of Lys100, Lys103, and Asp112 caused at most a 2-fold increase in KmF6P and a 2-3-fold increase in KmATP, suggesting that these residues are not involved in the direct binding of Fru 6-P. However, change of Arg102 to Leu and to Lys resulted in a 325x and 22x, respectively, increase in KmF6P, and change of Arg102 to Glu resulted in nearly complete loss of the kinase activity. Change of Cys105 to Ala or Ser increased KmF6P about 10x. The Vmax of all these mutated enzymes except the one that changed Arg102 to Glu (R102E) was increased 10% to 85%. The kinetic parameters of Fru 2,6-Pase were not altered by these changes. R102E formed several polymeric forms of the enzyme, including a tetramer. Both R102E and an additional derivative that substituted Lys for Arg102 (R102K) were slightly more susceptible to guanidine inactivation than the wild-type enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed1995

Expression of bovine heart fructose 6-phosphate,2-kinase:fructose 2,6-bisphosphatase and determination of the role of the carboxyl terminus by mutagenesis.

Bovine heart fructose 6-P,2-kinase:fructose 2,6-bisphosphatase was expressed in Escherichia coli. In order to determine the role of the carboxyl-terminal peptide, 49 and 78 amino acids from the C-terminus were deleted using oligonucleotide-directed mutagenesis. The expressed wild-type and mutant enzymes were purified to homogeneity, and the steady-state kinetics of the mutant enzymes were compared to those of the wild-type enzyme. Deletion of 49 residues (Del 49) resulted in a 35% decrease in KmFru6P, a 36% increase in Vmax, and a 2-fold increase in Kcat/Km of the kinase. There was no change in the kinetic properties of the phosphatase activity. Deletion of 78 residues (Del 78) resulted in a 4.5-fold decrease in KmFru6P, a 2.5-fold increase in Vmax, a 12-fold increase in kcat/Km of the kinase, and a 3-fold increase in kcat/Km of the phosphatase. Phosphorylation of the wild-type and Del 49 enzymes resulted in decreased KmFru6P and activation of the kinase without affecting the phosphatase activity. Thermal inactivation rates of the wild-type and Del 49 enzymes were similar, but the rate of Del 78 was more rapid. The phosphorylated wild-type and Del 49 enzymes were more sensitive to thermal inactivation than the dephospho forms. Urea inactivation of the kinase and phosphatase of wild-type and Del 49 were similar, but Del 78 was more sensitive to urea. All phosphorylated enzymes were more susceptible to urea inactivation.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed1995

Crystallization and preliminary X-ray analysis of fructose 6-phosphate, 2-kinase:fructose 2,6-bisphosphatase.

Diffraction-quality crystals of the bifunctional enzyme fructose 6-phosphate, 2-kinase:fructose 2,6-bisphosphatase from rat testis have been obtained. The crystals were grown in the presence of ATP gamma S, fructose 6-phosphate, the detergent n-octylglucoside, and the precipitant polyethylene glycol 4000. The crystals have the symmetry of the trigonal space group P31/221 with a = b = 83.0 A and c = 130.6 A. Flash-frozen crystals diffract to beyond 2.2 A, and native data have been collected.

Adenosine Triphosphate↗

PubMed1994

Glucose-stimulated synthesis of fructose 2,6-bisphosphate in rat liver. Dephosphorylation of fructose 6-phosphate, 2-kinase:fructose 2,6-bisphosphatase and activation by a sugar phosphate.

The effect of glucose on hepatic fructose (Fru) 2,6-P2 in starved rats was investigated. When livers were perfused with high glucose (40 mM), hexose-P in the liver increased immediately reaching the maximum within in 2 min, but Fru 2,6-P2 after a lag period of 4 min increased linearly. The activation of Fru 6-P,2-kinase and inactivation of Fru 2,6-Pase also showed a similar lag period. Determination of the phosphate contents of the bifunctional enzyme after 10 min of glucose perfusion revealed that 90% of the enzyme was in the dephospho form while only 10% of the control liver enzyme was dephosphorylated. Comparison of crude extracts of liver perfused with either high glucose or normal glucose (5.6 mM) showed that high glucose livers contained 50% higher protein phosphatase activity, which dephosphorylated the bifunctional enzyme. Subcellular fractionation of the extract showed that activation of the protein phosphatase occurred in the cytosol. Desalting of the cytosolic fraction resulted in a 50% loss of the protein phosphatase activity. The low molecular weight activator in the cytosol was isolated, and by various chemical and enzymatic methods it was identified as xylulose 5-P. The activation of protein phosphatase by xylulose 5-P showed a highly sigmoidal saturation curve. The rate of formation of xylulose 5-P in the perfused liver showed a lag period of approximately 2 min, and after 4 min its concentration reached 10 microM, the minimum concentration necessary for the activation of the protein phosphatase. We conclude that the mechanism of glucose-induced Fru 2,6-P2 synthesis was not due to increased Fru 6-P as generally thought but occurred as a result of dephosphorylation of Fru 6-P,2-kinase:Fru 2,6-Pase. Moreover, the dephosphorylation was enhanced by increased xylulose 5-P, which activated a specific protein phosphatase. The results suggest a mechanism for coordinated regulation of glycolysis and the pentose shunt pathway that is mediated by xylulose 5-P.

Animals↗

PubMed1994

Effect of adding phosphorylation sites for cAMP-dependent protein kinase to rat testis 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase.

In contrast to liver and heart 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatases, the testis isozyme lacks a phosphorylation site for cAMP-dependent protein kinase. In order to determine the effect of phosphorylation site location for the protein kinase on rat testis bifunctional enzyme, consensus amino acid sequences (RRXS) were added at different distances from the N-terminus by site-directed mutagenesis. The expressed wild-type enzyme (WT) and mutant enzymes containing a phosphorylation site at Ser7 (mutant enzyme RT2KS7, where RT2K = rat testis 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase), Ser15 (RT2KS15), or Ser30 (RT2KS30) were purified to apparent homogeneity. All the mutant enzymes served as substrates for the protein kinase, and the phosphate incorporation was over 90%. The Km values of protein kinase A for RT2KS7, RT2KS15, and RT2KS30 were 250 microM, 110 microM, and 50 microM, respectively, and the relative rates were 1, 8, and 23. Various kinetic parameters of dephospho and phospho forms of these enzymes were determined. The kinetic constants of the dephospho form of RT2KS30 were similar to those of WT, but those of RT2KS15 and RT2KS7 showed an 8-fold increase in KmFru6P, an approximately 30% decrease in the Fru-6-P,2-kinase activity, and a 3-fold increase in fructose-2,6-bisphosphatase activity. Phosphorylation of RT2KS30 resulted in a shift in the Fru-6-P saturation curve from Michaelis-Menten kinetics to sigmoidal, with increased KmFru6P and activation of fructose-2,6-bisphosphatase. The kinetic constants of RT2KS15 and RT2KS7 were not altered by phosphorylation. All the mutant enzymes were more sensitive to heat inactivation than was WT.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗