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At least 19 recordsLinked to original sources
PubMed1978

Adenosine 5'-O-([gamma-18O]gamma-thio)triphosphate chiral at the gamma-phosphorus: stereochemical consequences of reactions catalyzed by pyruvate kinase, glycerol kinase, and hexokinase.

The 2-[18O]phosphorothioate of D-glycerate, chiral at phosphorus, was prepared. The chiral phosphoryl group was transferred enzymically to ADP [by using enolase and pyruvate kinase (ATP:pyruvate 2-O-phosphotransferase; EC 2.7.1.40)] resulting in the synthesis of adenosine 5'-O-([gamma-18O],gamma-thio)triphosphate. This labeled ATP was used as a thiophosphoryl group donor in the reactions catalyzed by glycerol kinase (ATP:glycerol 3-phosphotransferase; EC 2.7.1.30) and by hexokinase (ATP:D-hexose 6-phosphotransferase; EC 2.7.1.1). The product from the latter (glucose 6-phosphorothioate) was converted enzymically into glycerol phosphorothioate. Determination of the relative configurations and diastereoisomeric purities of the samples of glycerol phosphorothioate demonstrates that all three phosphokinases (pyruvate kinase, glycerol kinase, and hexokinase) transfer the thiophosphoryl group with complete stereospecificity, and further shows that these reactions follow an identical stereochemical course.

Adenosine Triphosphate↗
PubMed2001

Subcloning, expression, purification, and characterization of Haemophilus influenzae glycerol kinase.

Glycerol kinase (EC 2.7.1.30) is a bacterial sugar kinase and a member of the sugar kinase/actin/hsc-70 superfamily of enzymes. The enzyme from Escherichia coli is an allosteric regulatory enzyme whose activity is inhibited by fructose 1,6-bisphosphate (FBP) and the glucose-specific phosphocarrier of the phosphoenolpyruvate:glycose phosphotransferase system, IIA(Glc) (previously termed III(Glc)). Comparison of its primary structure with that of the highly similar Haemophilus influenzae glycerol kinase reveals that the amino acid sequence for the binding site for FBP is conserved while the amino acid sequence for the binding site for IIA(Glc) contains differences that are predicted to prevent its inhibition. To test this hypothesis, the H. influenzae glpK gene was assembled from DNA library fragments and subcloned into pUC18. The enzyme is expressed at high levels in E. coli. It was purified to greater than 90% homogeneity by taking advantage of its solubility behavior in a procedure that requires no column chromatography. The initial-velocity kinetic parameters of the purified enzyme are similar to those of the E. coli glycerol kinase. The H. influenzae glycerol kinase is inhibited by FBP but not by IIA(Glc), in agreement with the prediction based on sequence comparison. Sedimentation velocity experiments reveal that inhibition of HiGK by FBP is associated with oligomerization, behavior which is similar to EcGK. The possibility of utilizing mutagenesis studies to exploit the high degree of similarity of these two enzymes to elucidate the mechanism of allosteric regulation by IIA(Glc) is discussed.

Amino Acid Sequence↗
PubMed1998

Mapping the active sites of 3-phosphoglycerate kinase and glycerol kinase with monoammine chromium(III) ATP.

The 12 isomers of monoammine chromium(III) ATP have been used to probe the ATP binding sites of yeast 3-phosphoglycerate kinase and glycerol kinase from Candida mycoderma. Inhibition studies of 3-phosphoglycerate kinase show a dramatic decrease in isomer binding only when the ammonia is in the Delta axial facial anti position. This suggests an open site architecture with only one strong contact point between the coordination sphere and the enzyme surface. These results agree well with the computer modeling studies of bidentate chromium ATP into the nucleotide site determined by X-ray crystallography [McPhillips, T., et al. (1996) Biochemistry 35, 4118-4127]. Both methods describe an open site strongly supporting the validity of the inhibition studies. Inhibition studies of glycerol kinase show significant decreases in binding for all the tested ammonia positions, suggesting a closed site architecture with many contacts between the coordination sphere and the surface of the enzyme. This is in good agreement with X-ray studies [Hurley, T., et al. (1993) Science 259, 673-677] on the Escherichia coli glycerol kinase. Inhibition studies of hexokinase previously reported [Rawlings, J., et al. (1993) Biochemistry 32, 11204-11210] more closely resemble those of 3-phosphoglycerate kinase, suggesting the surprising result that however closely hexokinase and glycerol kinase are related structurally the site around the coordination sphere in hexokinase is functionally open like that of 3-phosphoglycerate kinase.

Adenosine Diphosphate↗
PubMed2005

Tissue-dependent alterations in lipid mass in mice lacking glycerol kinase.

Glycerol kinase (ATP:glycerol-3-phosphotransferase, EC 2.7.1.30, glycerokinase) (Gyk) has a central role in plasma glycerol extraction and utilization by tissues for lipid biosynthesis. Gyk deficiency causes various phenotypic changes ranging from asymptomatic hyperglycerolemia to a severe metabolic disorder with growth and psychomotor retardation. To better understand the potential role of Gyk in tissue lipid metabolism, we determined phospholipid (PL), cholesterol (Chol), and triacylglycerol (TG) mass in a number of tissues from mice lacking Gyk. We report a tissue-dependent response to Gyk gene deletion. Tissues with elevated total PL mass (brain, kidney, muscle) were characterized by the increased mass of ethanolamine glycerophospholipids (EtnGpl), choline glycerophospholipids, and phosphatidylserine (PtdSer). In heart, lipid changes were characterized by a reduction in total PL, including decreased EtnGpl, phosphatidylinositol, and PtdSer mass and decreased TG and FFA mass. In parallel with tissue PL alterations, tissue Chol was also changed, maintaining a normal Chol/PL ratio. Under conditions of Gyk deficiency, we speculate that glycerol-3-phosphate and lipid production is maintained via alternative biosynthesis, including glycolysis, glyceroneogenesis, or by direct acylation of glycerol in brain, muscle, kidney, and liver, but not in heart.

Adipose Tissue↗
PubMed1998

Cloning, sequencing, high expression, and crystallization of the thermophile Thermus aquaticus glycerol kinase.

Glycerol kinase (EC 2.7.1.30) is a key enzyme of glycerol uptake and metabolism in bacteria. Using PCR, we amplified and cloned a glycerol kinase gene, glpK, from Thermus aquaticus. The complete gene has 1488 base pairs, coding for a protein of 496 amino acids with a predicted molecular weight of 54,814. The amino acid sequence deduced from T. aquaticus glpK was found to have identities of 97 and 81%, respectively, with those of Thermus flavus and Bacillus subtilis glpK genes. After overproduction in Escherichia coli, the expressed enzyme was easily purified to homogeneity by DEAE-Toyopearl chromatography. The purified enzyme has been crystallized by the hanging drop vapor diffusion method at 22 degrees C. Comparison of the amino acid sequence with that of the B. subtilis enzyme showed that Ser and Lys are replaced by Ala and Arg, as was seen in mesophile and thermophile enzymes.

Amino Acid Sequence↗
PubMed2004

Glycerol-insensitive Arabidopsis mutants: gli1 seedlings lack glycerol kinase, accumulate glycerol and are more resistant to abiotic stress.

The aim of this study was to investigate the process of glycerol catabolism in germinating Arabidopsis seed. A genetic screen was performed to isolate glycerol-insensitive (gli) mutant seedlings. Three separate mutant loci were identified (gli1, gli2 and gli3). Of these, only gli1 is unable to utilise glycerol. Following germination, gli1 seedlings transiently accumulate glycerol derived from the breakdown of storage oil and are more resistant to hyperosmotic stress, salt stress, oxidative stress, freezing and desiccation. Enzyme assays revealed that gli1 lacks glycerol kinase activity. GLI1 mapped to chromosome 1 near the putative glycerol kinase gene NHO1. Mutations in this gene were identified in three independent gli1 alleles. A cDNA encoding GLI1 was cloned and its function was proven by complementation of an Escherichia coli glycerol kinase (glpK) deletion strain. Quantitative RT-PCR analysis showed that GLI1 is expressed in all tissues, but is transiently upregulated during early post-germinative growth and leaf senescence. These data show that glycerol kinase is required for glycerol catabolism in Arabidopsis and that the accumulation of glycerol can enhance resistance to a variety of abiotic stresses associated with dehydration.

Amino Acid Sequence↗
PubMed2001

Preliminary crystallographic study of Thermus aquaticus glycerol kinase.

Glycerol kinase (GlpK) is an important enzyme which catalyzes the rate-limiting step in a central biochemical pathway involving glycerol metabolism. GlpK from the thermophile Thermus aquaticus has been overexpressed in glpK-deficient Escherichia coli and crystallized by the hanging-drop method. The crystal belongs to the cubic space group I23, with unit-cell parameters a = b = c = 163.94 (3) A. Native data were collected to 2.87 A resolution on a Cu Kalpha rotating-anode X-ray source.

Crystallization↗
PubMed1987

Glycerol metabolism in higher plants: glycerol kinase.

Glycerol kinase activity was identified in extracts of higher plant seeds and seedlings, and was partially purified and characterized from cucumber radicle tissue. The enzyme was localized in the post-mitochondrial supernatant of the cell, and catalyzed the formation of glycerol-3-phosphate. The pH optiumum was 9.0. ATP, CTP, GTP or UTP could be used as the phosphoryl group donor. The Km for glycerol was 55 microM and Km values for the nucleoside triphosphates were 145-620 microM. The Vmax for the reaction was 40-78 pmol product per min. Kinetic data indicate that the enzyme has a sequential mechanism.

Glycerol↗
PubMed1975

Proportional activities of glycerol kinase and glycerol 3-phosphate dehydrogenase in rat hepatomas.

The activities of glycerol 3-phosphate dehydrogenase (EC 1.1.1.8), glycerol kinase (EC 2.7.1.30), lactate dehydrogenase (EC 1.1.1.27), "malic' enzyme (L-malate-NADP+ oxidoreductase; EC 1.1.1.40) and the beta-oxoacyl-(acyl-carrier protein) reductase component of the fatty acid synthetase complex were measured in nine hepatoma lines (8 in rats, 1 in mouse) and in the livers of host animals. With the single exception of Morris hepatoma 16, which had unusually high glycerol 3-phosphate dehydrogenase activity, the activities of glycerol 3-phosphate dehydrogenase and glycerol kinase were highly correlated in normal livers and hepatomas (r = 0.97; P less than 0.01). The activities of these two enzymes were not strongly correlated with the activities of any of the other three enzymes. The primary function of hepatic glycerol 3-phosphate dehydrogenase appears to be in gluconeogenesis from glycerol.

Animals↗
PubMed2000

AluY insertion (IVS4-52ins316alu) in the glycerol kinase gene from an individual with benign glycerol kinase deficiency.

Glycerol kinase deficiency has three distinct forms: an isolated form which may be benign or symptomatic, and a complex form which is symptomatic and part of an Xp21 contiguous gene syndrome. Here we report the case of a male with benign isolated glycerol kinase deficiency who was incidentally identified after observation of pseudohypertriglyceridemia. DNA sequencing of this subject's glycerol kinase gene showed the insertion of an AluY sequence in intron 4 of the glycerol kinase gene. Although Alu insertions have been implicated in other diseases, and a closely related AluY element is found as an insert in the C1 inhibitor gene in patients with hereditary angioedema, this is the first case of glycerol kinase deficiency caused by an Alu insertion.

Adult↗
PubMed1989

Muscle glycerol kinase in Duchenne dystrophy and glycerol kinase deficiency.

The complex glycerol kinase deficiency (GKD) syndrome is an X-linked recessive genetic disorder. The syndrome often includes a myopathy that is similar histologically to Duchenne muscular dystrophy (DMD). The glycerol kinase (GK) locus is in the Xp21 region in the midportion of the short arm of the X chromosome and is in close proximity to the DMD locus. We have investigated GK activity and subcellular distribution of muscle GK in DMD patients and in a patient with the complex GKD syndrome presenting with myopathy. We found no abnormality of muscle GK specific activity or subcellular distribution in DMD. In the patient with the complex GKD syndrome the specific activity and kinetics of muscle GK were normal, but the subcellular distribution of muscle GK was altered. Liver GK had less than 10% of normal activity and showed markedly altered kinetics. These findings indicate that there is no abnormality of muscle GK activity in DMD muscle. Furthermore, the normal GK activity in an individual with the complex GKD syndrome suggests that muscle and liver GK are genetically distinct. These findings support the concept that the complex GKD syndrome results from small deletions that affect closely linked but separate loci for DMD, GK and adrenal hypoplasia.

Adolescent↗
PubMed1982

Glycerol kinase activity and glycerol metabolism of rat granular pneumocytes in primary culture.

Glycerol kinase activity and glycerol utilization by rat granular pneumocytes were determined in order to investigate the rate-limiting step for glycerol incorporation into lung lipids. Granular pneumocytes were isolated in primary culture following trypsinization of rat lungs. Glycerol kinase activity was 8.2 nmol/h per 10(6) cells. Incorporation of [1,3-14C]glycerol into total cell lipids was 0.29 nmol/h per 10(6) cells. In the presence of saturating glycerol concentration, production of 3H2O from [2-3H]glycerol was 13 times greater than incorporation of [14C]glycerol into lipids. Glycerol phosphate dehydrogenase activity in isolated cells was approximately 10 times glycerol kinase activity. In the presence of 5.6 mM glucose, glycerol incorporation into lipids was decreased 79% and detritiation of glycerol was decreased 34%. This effect of glucose was due to a 25% increase in cell glycerol 3-phosphate content, resulting in dilution of the precursor pool and possible inhibition of glycerol phosphorylation. These results indicate that the relatively limited incorporation of glycerol into surfactant phospholipids by lung epithelial cells reflects the relatively high rate of glycerol 3-phosphate oxidation.

Animals↗
PubMed1999

X-Ray structure of glycerol kinase complexed with an ATP analog implies a novel mechanism for the ATP-dependent glycerol phosphorylation by glycerol kinase.

Glycerol kinase (GK) catalyzes the Mg-ATP-dependent phosphorylation of glycerol which yields glycerol 3-phosphate. The 2.8 A new crystal structure of GK complexed with an ATP analog revealed an unexpected position of the gamma-phosphoryl group, which was 7.2 A distant from the 3-hydroxyl group of glycerol, 5.5 A away from the 3-phosphate of the product (glycerol 3-phosphate) and is stabilized by a beta-hairpin structure. Based on the presented crystal structure and the previously determined structures of GK product complexes, we propose a 3-D model of a nucleophilic in-line transfer mechanism for the ATP-dependent phosphorylation of glycerol by GK.

Adenosine Triphosphate↗
PubMed1998

Clinical heterogeneity and novel mutations in the glycerol kinase gene in three families with isolated glycerol kinase deficiency.

Isolated glycerol kinase deficiency (GKD) is an X linked recessive disorder. The clinical and biochemical picture may vary from a childhood metabolic crisis to asymptomatic adult "pseudohypertriglyceridaemia", the result of hyperglycerolaemia. We performed glycerol kinase (GK) gene analysis to study the molecular heterogeneity and genotype-phenotype correlation in eight males from three families with isolated GKD. All patients had hyperglycerolaemia and glyceroluria. Four patients from two families were essentially free of symptoms. Three patients had gastrointestinal symptoms with ketoacidosis or hypoglycaemia or both. One patient had recurrent convulsions as the only acute sign, without evidence that it was correlated with a catabolic state. Fasting tests in two symptomatic patients of family 1 showed hyperketotic states, together with a tendency to hypoglycaemia. The diagnosis was confirmed by a defective 14C-glycerol incorporation into trichloroacetic acid precipitable macromolecules in intact skin fibroblasts. Mutation screening of the GK gene was performed by amplification and direct sequencing of exons using PCR. Three novel mutations were identified: (1) a deletion starting downstream of exon 9, extending to the 3' end of the gene; (2) a nonsense mutation R413X caused by a C1351T transition; and (3) a missense mutation W503R caused by a T1651C transition. In addition, we found differences from the reported sequence: (1) exon 9 actually consists of two exons, which consequently will change the number of GK gene exons from 19 to 20 exons, and (2) nucleotide differences in exon 19. So far, no genotype-phenotype correlation can be established in these GKD families.

Amino Acid Sequence↗
PubMed1990

Glycerol catabolism in Bacillus subtilis: nucleotide sequence of the genes encoding glycerol kinase (glpK) and glycerol-3-phosphate dehydrogenase (glpD).

The glpPKD region of the Bacillus subtilis chromosome was cloned in its natural host in plasmid pHP13. The glpPKD region contains genes required for glycerol catabolism: glpK coding for glycerol kinase, glpD coding for glycerol-3-phosphate (G3P) dehydrogenase and glpP, proposed to code for a positively acting regulatory protein. The cloned 7 kb fragment carries wild-type alleles of glpK, glpD and glpP. It can also complement a strain deleted for the entire glpPKD region. The wild-type alleles were mapped to different subfragments, establishing the gene order glpP-glpK-glpD. The nucleotide sequence of glpK and glpD was determined. Immediately upstream of glpK, an additional open reading frame was found, possibly being part of the same operon. Putative transcription terminators were found in the region between glpK and glpD and downstream of glpD. In a coupled in vitro transcription/translation system, two proteins were found, corresponding in size to those predicted from the deduced amino acid sequences of glycerol kinase and G3P dehydrogenase (54 kDa and 63 kDa, respectively).

Alleles↗
PubMed1985

Glycerol kinase deficiency inhibits glycerol utilization in phosphoglyceride and triacylglycerol biosynthesis.

Glycerol 3-phosphate is an initial metabolite in the biosynthesis of phosphoglycerides and triacylglycerols. Both glycerol and glucose are precursors of glycerol 3-phosphate. Cultured skin fibroblasts from patients with glycerol kinase deficiency utilized glucose, but not glycerol in the biosynthesis of phosphoglycerides and triacylglycerols. Phosphoglyceride and triacylglycerol biosynthesis in glycerol kinase deficiency fibroblasts is not diminished by the inability to use glycerol as a precursor of glycerol 3-phosphate.

Cells, Cultured↗