Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P06889 (Mol)
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The phosphoenolpyruvate carboxykinase (PEPCK) from Vibrio costicola catalyzed a 14CO2-oxaloacetate exchange reaction with an unusual nucleotide specificity. ATP gave the higher apparent catalytic efficiency (Vmax/Km, 6.78), followed by GTP (1.30), CTP (0.87) and ITP (0.66). Maximal activity required a divalent cation; CdCl2 and MgCl2 synergistically activated the enzyme, when added in the presence of MnCl2. The sigmoidal saturation curve for MnCl2 (apparent n 2.11) was converted into a hyperbola by 0.01 mM CdCl2 (apparent n 1). The results suggest a double role of the divalent cation in the reaction mechanism, namely as part of the MeATP2- substrate and as free Me2+. Mn2+ would be the best for the first, and Cd2+ for the second role. Preincubation with 0.01 mM CdCl2 increased the activity of the enzyme assayed with MgATP2- through an increase in Vmax; addition of CdCl2 to the reaction mixture elicited further activation, through a 17-fold decrease in the apparent Km for MgATP2-. These results, together with the biphasic curve of activation by CdCl2 when used alone, suggest the existence of two different sites for free Cd2+ on the enzyme.
Biochem Mol Biol Int 1995 Aug
PMID:Effects of divalent cations and nucleotides on the 14CO2-oxaloacetate exchange catalyzed by the phosphoenol pyruvate carboxykinase from the moderate halophile, Vibrio costicola. 853 94

The bloodstream forms of Trypanosoma brucei monomorphic strain 427 serially passaged in rats can differentiate in vitro equally well in HMI-9, HMI-10, SDM-79 or Cunningham's medium into the insect (procyclic) forms by a simple temperature shift from 37 to 26 degrees C in the presence of citrate and cis-aconitate. The procyclic forms thus generated can also continue to multiply at 26 degrees C without replacing the culture medium. The same strain of T. brucei pre-adapted to grow as bloodstream forms in HMI-10 medium at 37 degrees C is also capable of differentiating showing a similar rate of variant surface glycoprotein (VSG) disappearance and appearance of phosphoenolpyruvate carboxykinase (PEPCK) under the same experimental conditions. However, appearance of both procyclin mRNA and procyclin protein is much delayed and the resulting procyclic forms cannot multiply. The culture-adapted bloodstream forms are capable of infecting rats, and the cells thus harvested from the rats can differentiate but cannot multiply in the same manner as the original culture-adapted bloodstream forms. Apparently, a certain variant has been selected during the adaptation of T. brucei bloodstream forms from rat blood to the culture medium. This variant could be a useful tool for identifying the genes involved in differentiation of T. brucei and multiplication of the procyclic forms. Comparison of the protein profiles between the wild-type and the variant during differentiation showed that a major protein band of about 70 kDa remained in the non-dividing variant procyclic forms but vanished in the rapidly dividing wild type procyclic forms. N-terminal determinations indicated that the 70-kDa protein band consists of bovine serum albumin and fetuin. Presumably these two serum proteins are actively taken up by the bloodstream forms via endocytosis. Since the procyclic forms are incapable of endocytosis, the serum proteins may be rapidly diluted in the growing wild type procyclic cells but remain unchanged in the non-dividing procyclic cells of the variant. Further studies are underway in trying to identify the key distinctions between these two lines of cells at the molecular level.
Mol Biochem Parasitol 1995 Jun
PMID:Differentiation of a culture-adapted mutant bloodstream form of Trypanosoma brucei into the procyclic form results in growth arrest of the cells. 853 91

The crystal structure of ATP-dependent phosphoenolpyruvate carboxykinase (ATP-oxaloacetate carboxy-lyase, (transphosphorylating), E.C. 4.1.1.49; PCK) from Escherichia coli strain K12 has been determined using a combination of multiple isomorphous replacement, density modification, and partial model phase combination, and refined to a conventional R-index of 0.204 (Rfree = 0.244) at 1.9 A resolution. Each PCK molecule consists of a 275 residue N-terminal domain and 265 residue C-terminal or mononucleotide-binding domain, with the active site postulated to be within a cleft between the two domains. PCK is an open-faced, mixed alpha/beta protein, with each domain having an alpha/beta folding topology as found in several other mononucleoside-binding enzymes. The putative phosphate-binding site of ATP adopts the P-loop motif common to many ATP and GTP-binding proteins, and is similar in structure to that found within adenylate kinase. However, the beta-sheet topology within the mononucleotide-binding fold of PCK differs from all other families within the P-loop containing nucleoside triphosphate hydrolase superfamily, therefore suggesting it represents the first member in a new family of such proteins. The mononucleotide-binding domain is also different in structure compared to the classical mononucleotide-binding fold (CMBF) common to adenylate kinase, p21ras, and elongation factor-Tu. Several amino acid residues, including R65, K212, K213, H232, K254, D269, K288 and R333 appear to make up the active site of the enzyme, and are found to be absolutely conserved among known members of the ATP-dependent PCK family. A cysteine residue is located near the active-site, as has been suggested for other PCKs, although in the E. coli enzyme C233 is buried and so is most likely not involved in substrate binding or catalysis. Two binding sites of the calcium-analog TB3+ have been determined, one within the active site coordinating to the side-chain of D269, and the other within the C-terminal domain coordinating to the side-chains of E508 and E511.
J Mol Biol 1996 Feb 16
PMID:Crystal structure of Escherichia coli phosphoenolpyruvate carboxykinase: a new structural family with the P-loop nucleoside triphosphate hydrolase fold. 860 5

In human liver, phosphoenolpyruvate carboxykinase (PCK; EC 4.1.1.32) is about equally distributed between cytosol and mitochondria in contrast with rat liver in which it is essentially a cytosolic enzyme. Recently, the isolation of the gene and cDNA of the human cytosolic enzyme has been reported [Ting, Burgess, Chamberlian, Keith, Falls and Meisler (1993) Genomics 16, 698-706; Stoffel, Xiang, Espinosa, Cox, Le Beau and Bell (1993) Hum. Mol. Genet. 2, 1-4]. It was the goal of this investigation to isolate the cDNA of the human mitochondrial form of hepatic PCK. A human liver cDNA library was screened with a rat cytosolic PCK cDNA probe comprising sequences from exons 2 to 9. A cDNA clone was isolated which had overall a 68% DNA sequence and a 70% deduced amino acid sequence identity with the human cytosolic PCK cDNA. Without the flanking 270 bases (=90 amino acids) each at the 5' and 3' end, the sequence identity was 73% on the DNA and 78% on the amino acid level. The isolated cDNA had an open reading frame of 1920 bp; it was 54 bp (equivalent to 18 amino acids) longer than that of human or rat cytosolic PCK cDNA. The isolated cDNA was cloned into the eukaryotic expression vector pcDNAI and transfected into human embryonal kidney cells HEK293; PCK activity was increased by 3-fold in the mitochondria, which normally contain 70% of total PCK activity, but not in the cytosol. The isolated cDNA was also transfected into cultured rat hepatocytes; again, PCK activity was enhanced by about 40-fold in the mitochondria, which normally possess only 10% of total PCK activity, but not in the cytosol. In the rat hepatocytes only the endogenous cytosolic PCK and not the transfected mitochondrial PCK was induced 3-fold with glucagon. Comparison of the amino acid sequences deduced from the isolated cDNA with human and rat cytosolic PCK showed that the additional 18 amino acids were located at the N-terminus of the protein and probably constitute a mitochondrial targeting signal. Northern-blot analyses revealed the human mitochondrial PCK mRNA to be 2.25 kb long, about 0.6 kb shorter than the mRNA of the cytosolic PCK. Primer extension experiments showed that the 5'-untranslated region of mitochondrial PCK mRNA was 134 nucleotides in length.
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PMID:Molecular cloning, sequencing and expression of the cDNA of the mitochondrial form of phosphoenolpyruvate carboxykinase from human liver. 864 61

Expression of the phenylalanine hydroxylase gene in livers and kidneys of rodents is activated at birth and is induced by glucocorticoids and cyclic AMP in the liver. Regulatory elements in a 10-kb fragment upstream of the mouse gene have been characterized. The promoter lacks TAATA and CCAAT consensus sequences and shows only extremely weak activity in transitory expression assays with phenylalanine hydroxylase-producing hepatoma cells. No key elements for regulation of promoter activity are localized within 2 kb of upstream sequences. However, a liver-specific DNase I-hypersensitive site at kb -3.5 comprises a tissue-specific and hormone-inducible enhancer. This enhancer contains multiple protein binding sites, including sites for ubiquitous factors (NF1 and AP1), the glucocorticoid receptor, and the hepatocyte-enriched transcription factors hepatocyte nuclear factor 1 (HNF1) and C/EBP. Mutation revealed that the last two sites are critical not only for basal activity but also for obtaining a maximal hormone response. Efficient transcription from the highly inducible promoter shows absolute dependence upon the enhancer at kb - 3.5, which in turn requires HNF1 and C/EBP as well as hormones. The regulatory region of the mouse phenylalanine hydroxylase gene differs totally from that of humans, even though the genes of both species are expressed essentially in the liver. Furthermore, the phenylalanine hydroxylase gene of mice shows an expression pattern very similar to those of the rodent tyrosine aminotransferase and phosphoenolpyruvate carboxykinase genes, yet each shows a different organization of its regulatory region.
Mol Cell Biol 1996 Jun
PMID:The activity of the highly inducible mouse phenylalanine hydroxylase gene promoter is dependent upon a tissue-specific, hormone-inducible enhancer. 864 24

The action of thyroid hormones on the expression of the mitochondrial ATP synthase beta-subunit gene (ATPsyn beta) is controversial. We detected a binding site for the thyroid hormone receptor between -366 and -380 in the human ATPsyn beta gene by DNase I footprint analysis and band-shift assays. However, expression vectors in which the chloramphenicol acetyl transferase (CAT) reporter gene is driven by the 5' upstream region of ATPsyn beta gene were unresponsive to T3 when transiently transfected to HepG2 or GH4C1 cells. CAT constructs driven by the rat phosphoenolpyruvate carboxykinase (PEPCK) or the growth hormone (GH) promoters were stimulated several fold by T3 in parallel experiments. It is proposed that the biological effects of thyroid hormones on the ATPsyn beta expression occur through indirect mechanisms.
Mol Cell Biochem 1996 Jan 26
PMID:Influence of thyroid hormones on the human ATP synthase beta-subunit gene promoter. 871 24

Patients with an acyl-CoA dehydrogenase deficiency share the disease features of hypoglycemia, hyperammonemia, tissue fatty change, hypoketonemia, carnitine deficiency, and organic acidemia due to apparent disruption of normal fatty acid, glucose, and urea metabolism. Most of the acute clinical episodes occur in young children. These episodes are precipitated by fasting and are often fatal, with the in vivo mechanisms essentially unknown. Since the genes of the rate controlling enzymes of these pathways are tissue and developmentally regulated at the transcriptional level, we measured, throughout neonatal development, the steady-state mRNA levels of long-chain, medium-chain, and short-chain (SCAD) acyl-CoA dehydrogenases, pyruvate carboxylase (PC), phosphoenolpyruvate carboxykinase (PEPCK), carbamyl phosphate synthetase I (CPS), ornithine transcarbamylase (OTC), and argininosuccinate synthetase (AS) in fed or fasted SCAD-deficient BALB/ByJ mice compared to BALB/cBy controls. Overall, our results showed no major effects on expression of acyl-CoA dehydrogenases due to SCAD deficiency, regardless of age or fasting. In SCAD-deficient mice we found depressed mRNA expression and enzyme activity for the urea cycle enzymes CPS and AS at 6 days of age, and found no apparent effects on expression of gluconeogenic enzymes PC or PEPCK. There was a period of overall lower gene expression for most genes at 6 and 15 days, which appears to be in parallel with the developmental period when children with these diseases are most severely affected.
Biochem Mol Med 1996 Apr
PMID:Effects of short-chain acyl-CoA dehydrogenase deficiency on development expression of metabolic enzyme genes in the mouse. 873 88

Hypoglycemia during septic shock is a common and life-threatening sign in the newborn. TNF alpha is an important cytokine in endotoxic shock. The present study was performed to investigate if TNF alpha induces hypoglycemia and if dexamethasone ameliorates the TNF alpha effects in 10 day old rats. TNF alpha induced hypoglycemia and lactacidemia without altering plasma insulin concentration in 10 day old rats. TNF alpha increased GLUT1 mRNA abundance in brain, liver, muscle and fatty tissue, and decreased liver phosphoenolpyruvate carboxykinase (PEPCK) mRNA abundance. Dexamethasone attenuated the hypoglycemia and lactacidemia. Dexamethasone blunted the increase of GLUT1 mRNA abundance and increased the liver PEPCK mRNA abundance. Dexamethasone may be beneficial by promoting gluconeogenesis.
Res Commun Mol Pathol Pharmacol 1996 May
PMID:Dexamethasone attenuates hypoglycemia in ten day old rats treated with TNF alpha. 877 68

The transcription of the yeast FBP1 and PCK1 genes, which encode the gluconeogenic enzymes fructose-1,6-bisphosphatase and phosphoenolpyruvate carboxykinase, is repressed by glucose. Here, we show that this repression is both very strong and exceptionally sensitive to glucose, being triggered by glucose at concentrations less than 0.005% (0.27 mM). This repression remains operative in yeast mutants carrying any one of the three hexose kinases, but is lost in a triple hxk1, hxk2, glk1 mutant. In addition, 2-deoxyglucose can trigger the repression, but 6-deoxyglucose cannot, suggesting that internalization and phosphorylation of the glucose is essential for repression to occur. While gluconeogenic gene transcription is subject to the Mig 1p-dependent pathway of glucose repression, the exquisite response to glucose is maintained in hxk2 and mig1 mutants, suggesting that this pathway is not essential for the response. The response can also be triggered by the addition of exogenous cAMP, suggesting that the Ras/cAMP pathway can mediate repression of the FPB1 and PCK1 mRNAs. However, the response is not dependent upon this pathway because it remains intact in Ras, adenyl cyclase and protein kinase A mutants. The data show that yeast cells can detect very low glucose concentrations in the environment, and suggest that several distinct signalling pathways operate to repress FPB1 and PCK1 transcription in the presence of glucose.
Mol Microbiol 1996 May
PMID:Multiple signalling pathways trigger the exquisite sensitivity of yeast gluconeogenic mRNAs to glucose. 879 72

Glycogen content as well as glycolytic, gluconeogenic and fatty acid synthesis enzyme activities were monitored in young and adult male rats fed diets differing in fat content: 11% (low), 22% (medium) and 42% (high) of total energy from fat. The results showed significant differences in the responses of young and adult rats to changes in dietary fat and carbohydrate. In young animals, increasing dietary fat decreased total liver glycogen phosphorylase (GP), pyruvate kinase (PK), glycerol 3-phosphate dehydrogenase, glucose 6-phosphate dehydrogenase, malic enzyme (ME), ATP-citrate lyase (ATP-CL) and fatty acid synthase (FAS). Increasing dietary fat also affected enzyme levels in other tissues: hexokinase (HK) and pyruvate dehydrogenase (PDH) activities decreased whereas skeletal muscle PK activity increased. The pattern of enzyme changes was similar in livers of fed adults with the exception that liver GP was not affected by dietary manipulations. Overnight food deprivation decreased liver glucokinase (GK), ME, ATP-CL, and FAS activities and increased liver phosphoenolpyruvate carboxykinase (PEPCK) and phosphofructokinase in both young and adult animals. In young animals, food deprivation also: (i) reduced liver GK and PK, (ii) increased kidney PEPCK, (iii) decreased muscle PEPCK and (iv) decreased kidney PDH. Food-deprived adults had increased skeletal muscle PEPCK and kidney glycogen synthetase as well as decreased kidney PEPCK muscle GP activity. These differences suggest that young animals are somewhat more responsive to changes in dietary manipulations. They also show that overnight food restriction causes a more profound metabolic re-organization in younger than in older animals.
Mol Cell Biochem 1996 Jun 07
PMID:Enzymes of carbohydrate metabolism in young and adult rats fed diets differing in fat and carbohydrate. 881 10


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