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S J Pilkis

Publications and source records attributed to S J Pilkis.

At least 37 records · Page 2Linked to original sources
PubMed1994

The allosteric site of human liver fructose-1,6-bisphosphatase. Analysis of six AMP site mutants based on the crystal structure.

The molecular structure of human liver fructose-1,6-bisphosphatase complexed with AMP was determined by x-ray diffraction using molecular replacement, starting from the pig kidney enzyme AMP complex. Of the 34 amino acid residues which differ between these two sequences, only one interacts with AMP; Met30 in pig kidney is Leu30 in human liver. From this analysis, six sites in which side chains of amino acid residues are in contact with AMP, Ala24, Leu30, Thr31, Tyr113, Arg140, and Met177, were mutated by polymerase chain reaction. The wild-type and mutant forms were expressed in Escherichia coli, purified, and their kinetic properties determined. Circular dichroism spectra of the mutants were indistinguishable from that of the wild-type enzyme. Kinetic analyses revealed that all forms had similar turnover numbers, Km values for fructose 2,6-bisphosphate, and inhibition constants for fructose 2,6-bisphosphate. Apparent Ki values for AMP inhibition of the Leu30 --> Phe and Met177 --> Ala mutants were similar to those of the wild-type enzyme, but the apparent Ki values for the Arg140 --> Ala and Ala24 --> Phe mutants were 7-to 20-fold higher, respectively. The Thr31 --> Ser mutant exhibited a 5-fold increase in apparent Ki for AMP, while mutation of Thr31 to Ala increased the apparent Ki 120-fold. AMP inhibition of the Tyr113 --> Phe mutant was undetectable even at millimolar AMP concentrations. Fructose 2,6-bisphosphate potentiated AMP inhibition of the mutants to the same extent as for the wild-type enzyme, except in the case of the Thr31 --> Ala and Tyr113 --> Phe mutants. Thus, the Met177 --> Ala mutant suggests that the side chain beyond C alpha is not needed for AMP binding, and that the Leu30 --> Phe mutant preserves the AMP contacts with these side chains. Thr31, Tyr113, and Arg140 form key hydrogen bonds to AMP consistent with strong side chain interactions in the wild-type enzyme. Finally, the absence of any effect of fructose 2,6-bisphosphate on AMP inhibition observed in the Thr31 --> Ala mutant may be an important clue relating to the mechanism of synergism of these two inhibitors.

Adenosine Monophosphate↗
PubMed1994

Glucose-6-phosphatase gene expression and activity are modulated in hemorrhagic shock: evidence for a new heat-sensitive activator.

Decreased hepatic fructose 2,6-bisphosphate levels were observed in the early phase of hemorrhagic shock. The lower sugar bisphosphatae level was a result of increased phosphoenolpyruvate levels and decreased glucose-6-phosphate and fructose-6-phosphate levels. The decreased glucose-6-phosphate levels correlated with increased activity of liver glucose-6-phosphatase and a concomitant 2.5-fold increase in glucose-6-phosphatase mRNA abundance. In addition, protein-free filtrate from hemorrhagic shock rats, but not from control rats, increased glucose-6-phosphatase activity. However, when control and hemorrhagic shock protein-free filtrates were heated, they both increased the glucose-6-phosphatase activity of the respective microsomes to the same extent. It is concluded that the early hyperglycemic phase of hemorrhagic shock is due to enhanced glucose-6-phosphatase gene expression and activity and the generation of a heat sensitive activator of the enzyme.

Animals↗
PubMed1994

Evidence for NH2- and COOH-terminal interactions in rat 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase.

The pH kinetic behavior of several rat fructose-2,6-bisphosphatase forms was analyzed. The bisphosphatase maximal velocity of the hepatic 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase was optimal at pH 5, but decreased to 12% of the optimal value in the pH range 7.0-7.5. This decrease depended on deprotonation of a group with a pK of 5.7. In contrast, the separate bisphosphatase domain, a 30-amino acid COOH-terminal truncated form (CT30) of the liver enzyme, and the skeletal muscle bifunctional enzyme exhibited pH-insensitive maximal velocities which were 5-10-fold higher than that of the bisphosphatase of the liver bifunctional enzyme at pH 7.0-7.5. The pK values of the C-2 and C-6 phosphoryl groups were 6.0 and 5.75, respectively, as determined by 31P NMR. Analysis of log kcat/Km versus pH profiles revealed two pK values, one at 6.1, which probably is a substrate pK, and the other at 8.4, which represents an enzyme group. Protein kinase-catalyzed phosphorylation of the liver isoform activated the bisphosphatase, and the pK of the group seen in the kcat profile was increased from 5.7 to 6.4. However, phosphorylation of the CT30 mutant had no effect on the bisphosphatase. The data indicate that NH2- and COOH-terminal interactions in the liver bifunctional enzyme affect the pH dependence of the fructose-2,6-bisphosphatase and its activation by phosphorylation.

Amino Acid Sequence↗
PubMed1994

Isolation of a cDNA for the catalytic subunit of rat liver glucose-6-phosphatase: regulation of gene expression in FAO hepatoma cells by insulin, dexamethasone and cAMP.

cDNA clones coding for the catalytic subunit of rat liver glucose-6-phosphatase (EC 3.1.3.9) were isolated from a rat liver cDNA library in lambda gt11 phage. The sequence of the cDNA and the amino acid sequence derived from it were greater than 90% identical to the corresponding sequences for the mouse and human forms of liver glucose-6-phosphatase. Northern blot analysis of RNA from FAO hepatoma cells revealed that dexamethasone induced the glucose-6-phosphatase mRNA while insulin suppressed its expression. When both hormones were added together insulin completely suppressed the effect of glucocorticoid. cAMP addition alone decreased the abundance of glucose-6-phosphatase mRNA. The results demonstrate multihormonal regulation of gene expression of hepatic glucose-6-phosphatase and support a dominant role for insulin.

Amino Acid Sequence↗
PubMed1994

Mechanism of modulation of rat liver fructose-2,6-bisphosphatase by nucleoside triphosphates.

The mechanism of modulation of fructose-2,6-bisphosphatase of rat liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase by nucleoside triphosphates was studied by employing the Escherichia coli-expressed bisphosphatase domain and a COOH-terminal 30-amino acid truncated form. These forms had Km values for substrate and Ki values for products which were similar to those of the bisphosphatase of the intact bifunctional enzyme, but turnover numbers were 5-fold higher. All forms also exhibited substrate inhibition that was relieved by GTP and ATP. The nucleoside triphosphates bound to the active site, since they were competitive inhibitors at subsaturating substrate concentrations. Guanosine was also a competitive inhibitor at subsaturating substrate concentrations but did not activate at saturating substrate. ATP and GTP had Kd values of 467 and 110 microM, respectively, and 1 mol of nucleoside triphosphate/mol bound per mol of bisphosphatase. The Ki values for guanosine of two mutants, Lys356-->Ala and Arg360-->Ala, were unchanged from that of the wild-type enzyme. However, the Ki for GTP for Arg360-->Ala was 17-fold higher than that of the wild-type enzyme, whereas that for Lys356-->Ala was unchanged. It was concluded that 1) nucleoside triphosphate modulation of the bisphosphatase of the bifunctional enzyme involves a direct interaction with the active site of the bisphosphatase domain; and 2) the activation is caused by the phosphate moieties of GTP and ATP competing with the 2-phospho group of fructose-2,6-bisphosphate for the phosphoenzyme intermediate, thus relieving substrate inhibition.

Adenosine Triphosphate↗
PubMed1994

Preliminary X-ray analysis of a truncated form of recombinant fructose-2,6-bisphosphatase.

The bisphosphatase domain of rat liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase and a C-terminal 30 amino acid truncated form were expressed in high yield in Escherichia coli and purified to homogeneity. The separately expressed bisphosphatase domain and its C-terminal truncated form had kinetic properties similar to the bisphosphatase of the intact bifunctional enzyme, but their turnover numbers were fourfold higher. The truncated enzyme crystallized in space group P1 with two molecules per asymmetric unit. The determined cell dimensions are: a = 41.9 A, b = 43.5 A, c = 57.6 A, alpha = 95.2 degrees, beta = 99.3 degrees, and gamma = 106.2 degrees. These crystals diffract to 2.0 A resolution when exposed to synchrotron radiation and are suitable for crystallographic structure analysis.

Animals↗
PubMed1994

Compensation in pancreatic beta-cell function in subjects with glucokinase mutations.

The relationship between the in vivo insulin secretory responsiveness of the pancreatic beta-cell to glucose and the flux of glucose through the enzyme glucokinase was investigated in six subjects with heterozygous glucokinase mutations and in six matched control subjects. This was done by combining data published previously on the in vivo dose-response relationships between glucose and insulin secretion and on the in vitro enzymatic properties of wild-type and mutant forms of glucokinase. The flux of glucose through glucokinase (GK flux) in these subjects was estimated using a model based on the approximate Michaelis-Menten kinetics of wild-type and mutant forms of the enzyme. In two subjects with glucokinase mutations, which resulted in only a small reduction in enzymatic activity, the decrease in insulin secretion was directly proportional to the decrease in GK flux predicted using a Michaelis-Menten model for both mutant and wild-type glucokinase. However, in four subjects with glucokinase mutations, which resulted in severe reductions in enzymatic activity, insulin secretion was reduced compared with control subjects but less than predicted. This latter result implies the existence of a compensatory change in the beta-cells of such subjects, which results in a relative increase in insulin secretory response. We propose modifications to the simple model relating glucose concentration and GK flux, including glucose-induced overexpression of the normal allele and a role of glucokinase regulatory protein. The modifications take into account the possibility that the degree of compensation may be directly related to the severity of the mutation.

Adolescent↗
PubMed1994

Molecular model of human beta-cell glucokinase built by analogy to the crystal structure of yeast hexokinase B.

Recent studies have shown that mutations in human beta-cell glucokinase that impair the activity of this key regulatory enzyme of glycolysis can cause early-onset non-insulin-dependent diabetes mellitus (NIDDM). The amino acid sequence of human glucokinase has 31% identity with yeast hexokinase, a related enzyme for which the crystal structure has been determined. This homology has allowed us to model the three-dimensional structure of human glucokinase by analogy to the crystal structure of yeast hexokinase B. This model of human glucokinase provides a basis for understanding the effects of mutations on its enzymatic activity. Residues in the active site and on the surface of the binding cleft for glucose are highly conserved in both enzymes. Regions far from the active site are predicted to differ in conformation, and 10 insertions or deletions that range in size from 1 to 7 residues are located on the protein surface between elements of secondary structure. The model structure suggests that human glucokinase binds glucose in a similar manner to yeast hexokinase. The glucose-binding site contains a conserved aspartic acid, two conserved glutamic acids, and two conserved asparagines that form hydrogen bond interactions with the hydroxyls of the glucose similar to those observed in other sugar-binding proteins. Mutation of residues in the predicted glucose-binding site has been found to greatly reduce enzymatic activity. This model will be useful for future structure/function studies of glucokinase.

Amino Acid Sequence↗
PubMed1994

cAMP-independent synergistic effects of insulin and dexamethasone on fructose 2,6-bisphosphate metabolism in H4IIE cells.

Hormonal regulation of fructose 2,6-bisphosphate (Fru-2,6-P2) content was studied in H4IIE cells. These cells were found to be very sensitive to physiological concentrations of insulin. Addition of either insulin or dexamethasone alone increased Fru-2,6-P2 in a time- and dose-dependent manner, and the maximal effect of the hormones was seen at 1 h. Neither hormone had any measurable effect on cAMP levels. The effect of addition of both insulin and dexamethasone on Fru-2,6-P2 was synergistic. Insulin, but not dexamethasone, rapidly increased 6-phosphofructo-2-kinase (6PF-2-K) activity by causing dephosphorylation of the enzyme as judged by a decrease in the Km for fructose-6-phosphate. Addition of both hormones also resulted in a synergistic 10-fold increase in enzyme protein as measured by kinase activity and phosphoenzyme formation. Dexamethasone increased liver 6PF-2-K/Fru-2,6-P2 mRNA abundance by 10- to 12-fold as measured by a ribonuclease protection assay, and insulin increased it by only 4-fold. Effects were observed as early as 1 h after hormone addition, but addition of both hormones together showed no synergy. We conclude that the synergistic effects of insulin and dexamethasone on Fru-2,6-P2 content are mediated by a combination of stimulation of expression of the bifunctional enzyme gene by both hormones and insulin-induced modulation of the activation state of the bifunctional enzyme, both of which are mediated by cAMP-independent mechanisms.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1993

Lack of evidence for a role of Cys-138 as a base catalyst in the skeletal muscle 6-phosphofructo-2-kinase reaction.

The role of Cys-138 in the catalysis of the skeletal muscle 6-phosphofructo-2-kinase reaction was investigated by mutating this residue to serine, glutamine and alanine, expressing the mutants in E. coli with a T7 RNA polymerase-based expression system, and analyzing their kinetic properties. The Cys138Ala mutant had greatly diminished activity, while the Cys138Ser and Cys138Gln mutants had maximal velocities 2-3 fold higher than the wild-type enzyme. It was concluded that Cys-138 does not act as a base catalyst in the kinase reaction, but that it plays a significant structural role in the enzyme's active site.

Amino Acid Sequence↗
PubMed1993

Regulation of rat 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase. Role of the NH2-terminal region.

The role of the NH2-terminal region of the liver and skeletal muscle 6-phosphofructo-2-kinase/fructose 2,6-bisphosphatases was investigated, as well that of a mutant of the liver isoform lacking the first 22 amino acids, by the overexpression of these enzymes in Escherichia coli and the comparison of their kinetic properties. The muscle isoform and the deletion mutant had Km values for fructose 6-phosphate which were 50- and 20-fold higher, respectively, than that of the liver isoform, and the bisphosphatase maximal velocity of the liver deletion mutant was 4-fold higher than that of the native liver isoform. Phosphorylation of the liver isoform increased bisphosphatase activity by 2-3-fold and the Km for fructose 6-phosphate of the 6-phosphofructo-2-kinase by 10-15-fold, but these kinetic effects were greatly diminished for the deletion mutant despite equivalent phosphorylation by cAMP-dependent protein kinase. Arg-173 of the skeletal muscle isoform was found to be functionally equivalent to the residue corresponding to the essential fructose 6-phosphate binding residue of the liver kinase domain, Arg-195. The results suggest that 1) the NH2-terminal regions of the liver and skeletal muscle isoforms are important determinants of fructose 6-phosphate affinity, and 2) the initial 22 amino acids of the liver isoform exert an inhibitory influence on the bisphosphatase and mediate, at least in part, the response of both activities of the enzyme to cAMP-dependent phosphorylation.

Amino Acid Sequence↗
PubMed1993

Expression of the liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase mRNA in FAO-1 cells.

The hormonal regulation of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase gene expression was studied in the rat hepatoma cell line FAO-1. Both 6-phosphofructo-2-kinase and fructose-2,6-bisphosphatase activities were detected in FAO-1 cells, at 68% of the levels found in rat liver. Northern blot analysis showed that FAO-1 cells, like rat liver, contained a predominant species of bifunctional enzyme mRNA, which is 2.2 kb in size. A sensitive RNAase protection assay revealed the presence in FAO-1 cells of an additional mRNA species, which is generated when transcription is initiated from the skeletal muscle promoter of the rat liver/skeletal muscle gene. The liver/skeletal muscle mRNA ratio in FAO-1 cells was 10:1, which is similar to that observed in rat liver. In contrast, in another rat hepatoma cell line, FTO-2B, only the skeletal muscle mRNA was detected. Insulin and dexamethasone induced the liver bifunctional enzyme mRNA in FAO-1 cells by 2-4-fold and 10-20-fold respectively in a concentration- and time-dependent manner, and their effects were antagonized by cyclic AMP. Transcription of the gene in FAO-1 cells, measured by nuclear run-on assays, was also enhanced by dexamethasone and insulin. It is concluded that the FAO-1 cell line is similar to liver with respect to both the preferential use of the liver promoter of the gene and its regulation by hormones, and is therefore an excellent model for the study of the hepatic expression of this gene.

Animals↗
PubMed1993

Isolation of a human liver fructose-1,6-bisphosphatase cDNA and expression of the protein in Escherichia coli. Role of ASP-118 and ASP-121 in catalysis.

A cDNA encoding human liver fructose-1,6-bisphosphatase was isolated from a lambda gt11 library by screening with a rat liver fructose-1,6-bisphosphatase cDNA. The cDNA (1421 base pairs) contains an open reading frame encoding 337 amino acids, corresponding to a protein with an estimated molecular weight of 36,697. Its primary sequence is highly homologous to that of the pig kidney and rat liver enzymes. The human liver cDNA was used to construct a T7 RNA polymerase-transcribed expression vector, and the enzyme was expressed in Escherichia coli BL21 (DE3). Approximately 50% of the expressed human fructose-1,6-bisphosphatase was soluble and enzymatically active, and the enzyme was purified to homogeneity by heat treatment, ammonium sulfate fractionation, and substrate/AMP elution from carboxymethyl-Sephadex. Expressed human liver fructose-1,6-bisphosphatase had a specific activity (9.8 mumol/min/mg of protein) that was half that of the rat liver enzyme, but had an identical Km for substrate. However, the human enzyme was more sensitive to inhibition by fructose-2,6-bisphosphate (Ki = 0.3 microM) and AMP (Ki = 12 microM) than the rat liver form (fructose 2,6-P2, Ki = 4 microM; AMP, Ki = 40 microM). Crystallographic analyses have suggested that Asp-118 and Asp-121 are catalytic residues located in a negatively charged pocket that binds divalent metal cations. These residues were mutated to alanine, and the E. coli-expressed mutant enzymes were purified to homogeneity. The Asp-118-->Ala and Asp-121-->Ala mutants had 1/5000 and 1/20,000 lower Kcat values than the wild-type enzyme, respectively, consistent with their critical role in fructose-1,6-bisphosphatase catalysis.

Amino Acid Sequence↗
PubMed1993

Expression of human liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase in Escherichia coli. Role of N-2 proline in degradation of the protein.

Human liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase is 96% identical to the rat and bovine liver enzymes, and all of the critical catalytic and substrate binding residues in both the kinase and bisphosphatase domains are conserved in the three enzymes. However, in contrast to rat liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase, which is readily expressed in an Escherichia coli T-7 RNA polymerase-based expression system, the human liver bifunctional enzyme could not be expressed in this system. Western blot and slot blot analysis revealed that although both the bifunctional enzyme protein and its mRNA were rapidly induced by the addition of isopropyl-1-thio-beta-D-galactopyranoside, the protein underwent rapid degradation. Deletion of the N-2 proline residue or its mutation to arginine, the corresponding residue in the rat liver enzyme, revealed that this proline residue was responsible for its rapid degradation. The Pro-2-->Arg mutant could be expressed with a high yield (20 mg/liter) in E. coli. The results support the hypothesis that a proline residue at N-2 facilitates bifunctional enzyme degradation in E. coli. The E. coli expressed mutant form was purified to homogeneity by phosphocellulose chromatography, and its kinetic properties were compared with those of the rat liver enzyme. The kinetic properties of the two enzymes were identical except for the presence of substrate (fructose 6-phosphate) inhibition of the human liver enzyme but not of the rat liver enzyme. The ability to express and purify large amounts of human liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase will permit structure/function and x-ray crystal structure studies of the enzyme and ultimately its targeting for drug therapy.

Amino Acid Sequence↗
PubMed1993

Isolation of a cDNA for chicken liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase.

A chicken liver cDNA for 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase was isolated from a Lambda ZAP2 phage library. The chicken liver cDNA codes for a protein that has 89.1, 88.4 and 88.0% amino acid identity with the human, rat and bovine liver isoforms, respectively. The kinetic properties of the rat and chicken liver enzymes, purified to homogeneity after expression in E. coli, were different including negative cooperativity for ATP binding and inhibition by Mg2+ for the chicken liver 6-phosphofructo-2-kinase but not for the rat liver kinase. Differences in the beta-loop ATP signature sequences in the chicken and rat liver kinase domains may explain the kinetic differences and represent the major divergence in the evolution of the enzyme from birds to mammals.

Adenosine Triphosphate↗
PubMed1993

Identification of glucokinase mutations in subjects with gestational diabetes mellitus.

Recent studies have shown that mutations in the glucokinase gene on chromosome 7 can cause an autosomal dominant form of NIDDM with a variable clinical phenotype and onset during childhood. The variable clinical phenotype includes mild fasting hyperglycemia (i.e., a plasma glucose value of > 110 mg/dl, a value that is at least 2-3 SDs above normal), impaired glucose tolerance, gestational diabetes mellitus, as well as overt NIDDM as defined using National Diabetes Data Group or World Health Organization criteria. Because gestational diabetes mellitus was a clinical feature associated with glucokinase mutations, we have screened a group of women with gestational diabetes who also had a first-degree relative with diabetes mellitus for the presence of mutations in this gene. Among 40 subjects, we identified two mutations, suggesting a prevalence of approximately 5% in this group. Extrapolating from this result, the prevalence of glucokinase-deficient NIDDM among Americans may be approximately 1 in 2500.

Amino Acid Sequence↗
PubMed1992

Hepatic 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase. The role of surface loop basic residues in substrate binding to the fructose-2,6-bisphosphatase domain.

Lys-356 has been implicated as a critical residue for binding the C-6 phospho group of fructose 2,6-bisphosphate to the fructose-2,6-bisphosphatase domain of rat liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (Li, L., Lin, K., Correia, J., and Pilkis, S. J. (1992) J. Biol. Chem. 267, 16669-16675). To ascertain whether the three other basic residues (Arg-352, Arg-358, and Arg-360), which are located in a surface loop (residues 331-362) which contains Lys-356, are important in substrate binding, these arginyl residues were mutated to Ala, and each arginyl mutant was expressed in Escherichia coli and purified to homogeneity. The far UV circular dichroism spectra of the mutants were identical to that of the wild-type enzyme. The kinetic parameters of 6-phosphofructo-2-kinase of the mutants revealed only small changes. However, the Km for fructose 2,6-bisphosphate, Ki for fructose 6-phosphate, and Ka for inorganic phosphate of fructose-2,6-bisphosphatase for Arg352Ala were, respectively, 2,800-, 4,500-, and 1,500-fold higher than those for the wild-type enzyme, whereas there was no change in the maximal velocity or the Ki for inorganic phosphate. The Km for fructose 2,6-bisphosphate and Ki for inorganic phosphate of Arg360Ala were 10- and 12-fold higher, respectively, than those of the wild-type enzyme, whereas the maximal velocity and Ki for fructose 6-phosphate were unchanged. In addition, substrate inhibition was not observed with Arg352Ala and greatly reduced with Arg360Ala. The properties of the Arg358Ala mutant were identical to those of the wild-type enzyme. The results demonstrate that in addition to Lys-356, Arg-352 is another critical residue in fructose-2,6-bisphosphatase for binding the C-6 phospho group of fructose 2,6-bisphosphate and that Arg-360 binds the C-2 phospho group of fructose 2,6-bisphosphate in the phosphoenzyme.fructose 2,6-bisphosphate complex. The results also provide support for Arg-352, Lys-356, and Arg-360 constituting a specificity pocket for fructose-2,6-bisphosphatase.

Amino Acid Sequence↗