Identity of mitochondrial and cytosolic glycerate kinases in rat liver and regulation of their intracellular localization by dietary protein.

Glycerate kinase (ATP: D-glycerate 2-phosphotransferase EC 2.7.1.31) is a key enzyme of glyconeogenesis from serine via hydroxypyruvate. A differential centrifugation of rat liver homogenate and an analysis of the particle fraction by sucrose density gradient centrifugation indicated that 72% and 26% of glycerate kinase are present in mitochondria and cytosol, respectively. A study on the intramitochondrial localization of the enzyme suggested that the mitochondrial glycerate kinase was present in inner membrane and/or matrix. It was found that dietary protein selectively induced mitochondrial glycerate kinase. This result suggested that mitochondrial glycerate kinase had a physiological function for gluconeogenesis from serin. However, the metabolic significance of the cytoplasmic enzyme was still unclear. The properties of solubilized-mitochondrial and cytosolic glycerate kinases were compared. However, no difference between the two enzymes could be found in the kinetic properties, thermal stability, molecular size or electrochemical properties. These results suggested that both enzymes originate from common genetic information. In order to elucidate the regulatory mechanism of the intracellular distribution of glycerate kinase in rat liver, the responses of mitochondrial and cytosolic glycerate kinases to an alteration of dietary protein were studied. The result suggested that an alteration of dietary protein content may regulate the distribution and the translocation of glycerate kinase to mitochondria and cytosol as well as the total amount of glycerate kinase.     

Identity of mitochondrial and cytosolic glycerate kinases in rat liver and regulation of their intracellular localization by dietary protein

Glycerate kinase (ATP : D-glycerate 2-phosphotransferase EC 2.7.1.31) is a key enzyme of gluconeogenesis from serine via hydroxypyruvate. A differential centrifugation of rat liver homogenate and an analysis of the particle fraction by sucrose density gradient centrifugation indicated that 72% and 26% of glycerate kinase are present in mitochondria and cytosol, respectively. A study on the intramitochondrial localization of the enzyme suggested that the mitochondrial glycerate kinase was present in inner membrane and/or matrix. It was found that dietary protein selectively induced mitochondrial glycerate kinase. This result suggested that mitochondrial glycerate kinase had a physiological function for gluconeogenesis from serine. However, the metabolic significance of the cytoplasmic enzyme was still unclear. The properties of solubilized-mitochondrial and cytosolic glycerate kinases were compared. However, no difference between the two enzymes could be found in the kinetic properties, thermal stability, molecular size or electrochemical properties. These results suggested that both enzymes originate from common genetic information. In order to elucidate the regulatory mechanism of the intracellular distribution of glycerate kinase in rat liver, the responses of mitochondrial and cytosolic glycerate kinases to an alteration of dietary protein were studied. The result suggested that an alteration of dietary protein content may regulate the distribution and the translocation of glycerate kinase to mitochondria and cytosol as well as the total amount of glycerate kinase.     

Mitochondrial and cytosolic localization of a single glycerate kinase in rat kidney cortex.

The distribution of glycerate kinase [ATP:D-glycerate 2-phosphotransferase, EC 2.7.1.31] in kidney was studied. This enzyme was found to be present in the renal cortex. By differential centrifugation of the homogenate and sucrose density gradient analysis, it was found that 42% and 60% of the renal glycerate kinase were localized in the cytosol and mitochondria, respectively. The mitochondrial enzyme appeared to be present in the inner membrane and/or matrix. No difference was found between the solubilized-mitochondrial and cytosolic glycerate kinase as regards kinetic properties, thermal stability, electrochemical properties, and molecular size. Immunochemical identity of these enzymes was demonstrated using a rabbit antibody against mitochondrial glycerate kinase purified from rat liver. Although the hepatic enzyme was induced by dietary protein (Kitagawa, Y., Katayama, H., & Sugimoto, E. [1979] Biochim. Biophys. Acta 582, 260--275), the renal enzyme in mitochondria and cytosol was not affected by dietary protein. These results on renal glycerate kinase are compared with those for the hepatic enzyme, and the regulatory mechanism for intracellular distribution of the enzymes is discussed.     

Light and thiol activation of maize leaf glycerate kinase : the stimulating effect of reduced thioredoxins and ATP

Glycerate kinase (EC 2.7.1.31) from maize (Zea mays) leaves was shown to be regulated by light/dark transition. The enzyme more than doubled in activity after either the leaves or isolated mesophyll chloroplasts were illuminated with white light for 10 minutes. Rate of inactivation in the dark was faster in leaves than in the isolated chloroplast fraction. The stimulating effect of light could be mimicked in crude preparations by addition of 10 or 50 millimolar dithiothreitol or 100 millimolar 2-mercaptoethanol. The thiol treatment resulted in 8- to 10-fold activation of glycerate kinase, with the highest rates in the range of 27 to 30 micromoles per mg chlorophyll per hour. Activation was not accompanied by any changes in the apparent M_r value of glycerate kinase as determined by gel filtration (M_r = 47,000). In contrast to maize glycerate kinase, the enzyme from spinach was not affected by either light or thiol exposure.

Partially purified maize glycerate kinase was activated up to 3-fold upon incubation with a mixture of spinach thioredoxins m and f and 5 millimolar dithiothreitol. The thioredoxin and dithiothreitol-treated glycerate kinase could be further stimulated by addition of 2.5 millimolar ATP. The results suggest that glycerate kinase from maize leaves is capable of photoactivation by the ferredoxin/thioredoxin system. The synergistic effect of ATP and thioredoxins in activation of the enzyme supports the earlier expressed view that the ferredoxin/thioredoxin system functions jointly with effector metabolites in light-mediated regulation during photosynthesis.

     

Thiol-dependent regulation of glycerate metabolism in leaf extracts : the role of glycerate kinase in c(4) plants

We have recently reported that the activity of maize leaf glycerate kinase [EC 2.7.1.31] is regulated in vivo by the light/dark transition, possibly involving the ferredoxin/thioredoxin mechanism, and that the stimulating effect of light can be mimicked in vitro by incubation of crude leaf extract with reducing compounds (LA Kleczkowski, DD Randall 1985 Plant Physiol 79: 274-277). In the present study it was found that the time course of thiol activation of the enzyme was substantially dependent on the presence of some low molecular weight inhibitor(s) of activation found both in leaf extracts and mesophyll chloroplasts. Activity of glycerate kinase from maize as well as wheat leaves increased upon greening of etiolated plants and was correlated with the development of photosynthetic apparatus in these species. The maize enzyme was strongly activated by thiols at all stages of development from etiolated to green seedlings. Thiol activation of glycerate kinase was observed for a number of C₄ plants, notably of the nicotinamide adenine dinucleotide phosphate-malic enzyme type, with the strongest effect found for the enzyme from leaf extracts of maize and sorghum (10- and 8-fold activation, respectively). Among the C₃ species tested, only the enzyme from soybean leaves was affected under the same conditions (1.6-fold activation). This finding was reflected by an apparent lack of cross-reactivity between the enzyme from maize leaves and antibodies raised against purified spinach leaf glycerate kinase. We suggest that, in addition to its role as a final step of photorespiration in leaves, glycerate kinase from C₄ species may serve as a part of the facilitative diffusion system for the intercellular transport of 3-phosphoglycerate. Simultaneous operation of both the passive and the facilitative diffusion mechanisms of 3-phosphoglycerate transport in C₄ plants is postulated.     

Purification and characterization of glycerate kinase from the thermoacidophilic archaeonThermoplasma acidophilum: An enzyme belonging to the second glycerate kinase family

Thermoplasma acidophilum is a thermoacidophilic archaeon that grows optimally at 59°C and pH 2. Along with another thermoacidophilic archaeon,Sulfolobus solfataricus, it is known to metabolize glucose by the non-phosphorylated Entner-Doudoroff (nED) pathway. In the course of these studies, the specific activities of glyceraldehyde dehydrogenase and glycerate kinase, two enzymes that are involved in the downstream part of the nED pathway, were found to be much higher inT. acidophilum than inS. solfataricus. To characterize glycerate kinase, the enzyme was purified to homogeneity fromT. acidophilum cell extracts. TheN-terminal sequence of the purified enzyme was in exact agreement with that of Ta0453m in the genome database, with the removal of the initiator methionine. Furthermore, the enzyme was a monomer with a molecular weight of 49 kDa and followed Michaelis-Menten kinetics withK _m values of 0.56 and 0.32 mM forDL-glycerate and ATP, respectively. The enzyme also exhibited excellent thermal stability at 70°C. Of the seven sugars and four phosphate donors tested, onlyDL-glycerate and ATP were utilized by glycerate kinase as substrates. In addition, a coupled enzyme assay indicated that 2-phosphoglycerate was produced as a product. When divalent metal ions, such as Mn²⁺, Co²⁺, Ni²⁺, Zn²⁺, Ca²⁺, and Sr²⁺, were substituted for Mg²⁺, the enzyme activities were less than 10% of that obtained in the presence of Mg²⁺. The amino acid sequence ofT. acidophilum glycerate kinase showed no similarity withE. coli glycerate kinases, which belong to the first glycerate kinase family. This is the first report on the biochemical characterization of an enzyme which belongs to a member of the second glycerate kinase family.     

Handbook of Biosolar Resources : Vol. 1, parts 1 and 2, ‘Basic Principles’ Edited by A. Mitsui and C.C. Black (Editor-in-Chief,O.R. Zaborsky) CRC Press; Boca Raton,USA, 1982 643 pages. $95.00 each part; approx. £113 for both parts

Glycerate kinase from spinach leaves was purified to near homogeneity using PEG/MgCl₂ fractionation, ion exchange, molecular sieving and affinity chromatography. The purified enzyme is a monomer of M_r 40 000, shows a pI-value of 4.8 and a broad pH optimum of 6.5–8.5 and is specific for D-isomer of glycerate. The high activity of crude enzyme (≈ 150 μmol. h^?1.mg chl^?1) indicates that glycerate kinase does not limit the oxidative photosynthetic carbon cycle.     

Characterization of glycerate kinase (2-phosphoglycerate forming), a key enzyme of the nonphosphorylative Entner-Doudoroff pathway, from the thermoacidophilic euryarchaeon Picrophilus torridus

Picrophilus torridus has been shown to degrade glucose via a nonphosphorylative Entner-Doudoroff (ED) pathway. Here we report the characterization of a key enzyme of this pathway, glycerate kinase (2-phosphoglycerate forming). The enzyme was purified 5,100-fold to homogeneity. The 95 kDa homodimeric protein catalyzed the ATP-dependent phosphorylation of glycerate specifically to 2-phosphoglycerate. The enzyme showed highest activity at 60 degrees C and pH 7.3, with ATP as phosphoryl donor and Mg(2+) as divalent cation. By MALDI-TOF analysis, ORF Pto1442 was identified in the genome of P. torridus as the encoding gene, designated gck. Homologs with high sequence identity were identified in the genomes of the archaea Thermoplasma and Sulfolobus spp. and Thermoproteus tenax, for which the operation of nonphosphorylative ED pathways, involving 2-phosphoglycerate forming glycerate kinases, has been proposed.     

A MOFRL family glycerate kinase from the thermophilic crenarchaeon, <Emphasis Type="Italic">Sulfolobus tokodaii</Emphasis>, with unique enzymatic properties

A glycerate kinase gene (ST2037) from the hyperthermophilic crenarchaeon Sulfolobus tokodaii was cloned and expressed in Escherichia coli. The purified homodimeric protein (45 kDa) specifically catalyzed the formation of 2-phosphoglycerate with d-glycerate as substrate. The thermostable enzyme displayed maximum activity (over 20 min) at 90°C and pH 4.5. The maximal activity was in the presence of Co²⁺. The MOFRL family glycerate kinase used AMP as phosphate donor with maximal activity towards GTP. These characteristics of the enzyme suggested its potential in the catalytic production of 2-phosphoglycerate.     

D-glycerate kinase deficiency as a cause of D-glyceric aciduria

D-Glycerate kinase was measured in human livers thanks to a new, sensitive radiochemical assay. The enzyme was extremely unstable in extracts prepared in water, but was partly stabilized in a homogenization mixture containing inorganic phosphate, D-glycerate and EGTA. When extracted in such a stabilizing mixture, glycerate kinase activity amounted to 0.86 +/- 0.21 U/g in control livers and to 0.03 U/g in the liver of a patient with D-glyceric aciduria. In contrast, D-glycerate dehydrogenase (glyoxylate reductase) and triokinase activities were not deficient in the liver of the same patient. It is concluded that D-glycerate kinase deficiency is a cause of D-glyceric aciduria.     

Regulatory Properties of the ADP-Glucose Pyrophosphorylase of the Blue-Green Bacterium Synechococcus 6301

ADP-glucose was found to be the primary sugar nucleotide used for glycogen synthesis by Synechococcus 6301. ADP-glucose pyrophosphorylase was partially purified 12-fold from this blue-green bacterium. The enzyme was activated 8- to 25-fold by glycerate 3-phosphate. Fructose 6-phosphate, fructose 1,6-bisphosphate, 5'-adenylate, and adenosine diphosphate activated the enzyme, but less than glycerate 3-phosphate. The enzyme was inhibited by inorganic phosphate. The I(0.5) of phosphate was 0.072 mm, and in the presence of 2 mm glycerate 3-phosphate, increased to 1.8 mm. The substrate saturation curves for glucose 1-phosphate and ATP were hyperbolic in both the presence and absence of glycerate 3-phosphate or phosphate. The saturation curve for MgCl(2) was sigmoidal; 2 mm glycerate 3-phosphate decreased the sigmoidicity from a Hill slope n value of 5.6 to 2.8, and increased the MgCl(2) optimum from 3 mm to 6 to 7 mm.     

Purification and properties of phosphoglycerate kinase from Thermus thermophilus strain HB8.

(1) A glycolytic enzyme, phosphoglycerate kinase [EC 2.7.2.3], was purified from cells of an extreme thermophile, Thermus thermophilus strain HB8. The enzyme was resistant to heat, and no loss of activity was observed after incubation for 10--20 min at 79 degrees C. (2) Catalytic properties such as pH optimum (pH 6--8.5), kinetic parameters (Km=0.28 mM for ATP, 1.79 mM for glycerate 3-phosphate), substrate specificity and inhibitors of the enzyme were investigated and compared with those of phosphoglycerate kinase from other sources. (3) The enzyme protein consists of a single polypeptide chain of molecular weight 44,600. The isoelectric point is 5.0 The amino acid composition of the enzyme was studied. The contents of ordered secondary structures were estimated to be 29% alpha-helix and 11% pleated sheet from the circular dichroic spectrum of the enzyme protein. (4) The fluorescence spectrum of the enzyme protein showed an emission maximum at 320 nm when excited at 280 nm. The quantum yield was 0.19. Tryptophyl fluorescence was not quenched, in contrast to the fluorescence reported for yeast phosphoglycerate kinase.     

Isolation and characterization of the human D-glyceric acidemia related glycerate kinase gene GLYCTK1 and its alternatively splicing variant GLYCTK2.

Deficiency of human glycerate kinase leads to D-glycerate acidemia/D-glyceric aciduria. Through PCR cloning assisted by in silico approach, we isolated the human glycerate kinase genes--Glycerate Kinase 1 (GLYCTK1) and its alternatively splicing variant--Glycerate Kinase 2 (GLYCTK2), which might be associated with D-glycerate acidemia/D-glyceric aciduria. The locus of GLYCTK gene is mapped to 3p21. PCR amplification in seventeen human tissue cDNAs revealed that both GLYCTK1 and GLYCTK2 are expressed widely almost in all these tissues. The expression of mouse Glyctk in various tissues was demonstrated by in situ hybridization. Both GLYCTK1 and GLYCTK2 proteins are localized in cytosol, and GLYCTK2 proteins are specifically localized in mitochondria. Present results revealed the characteristic expression pattern of murine Glyctk in neural system, skeleton muscle, supporting that glycerate kinase is implicated in D-glycerate acidemia/D-glyceric aciduria.     

Enzymes of fructose metabolism in human kidney.

Activities of enzymes involved in fructose metabolism were measured in samples of human kidney cortex and medulla. The enzymes are ketohexokinase, aldolase, NAD- and NADP-dependent alcohol dehydrogenase, aldehyde dehydrogenase, triokinase and glycerate kinase; hexose biphosphatase and sorbitol dehydrogenase were also investigated. With the exception of glycerate kinase, all enzymes involved in fructose metabolism were found in the human cortex and medulla. The enzyme levels in the medulla were low in comparison with the cortex.     

Determination of picomole amounts of glycerate 3-phosphate, glycerate 2-phosphate, and phosphoenol pyruvate by an enzymatic assay coupled to firefly luciferase/luciferin luminescence

A procedure for the determination of picomole amounts of glycerate 3-phosphate, glycerate 2-phosphate, and phosphoenol pyruvate is described. These metabolites were utilized by the glycolytic enzymes phosphoglycerate mutase, enolase, and pyruvate kinase to generate ATP which was determined by firefly luciferase/luciferin luminescence. The phosphoglycerate mutase used was of the glycerate 2,3-bisphosphate-independent type and was prepared from wheat germ. Stoichiometric conversion of glycerate 3-P, ranging in amount from 9 to 275 pmol, occurred after 25 min preincubation and required a narrow range of added mutase. The application of the procedure for determining these metabolites in suspensions of plant protoplasts is described.     

Functional homology of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase, phosphoglycerate mutase, and 2,3-bisphosphoglycerate mutase

The bisphosphatase domain of the rat liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase has been shown to exhibit a structural similarity to yeast phosphoglycerate mutase and human red blood cell 2,3-bisphosphoglycerate mutase including very similar active site sequences with a histidyl residue being involved in phospho group transfer. The liver bifunctional enzyme was found to catalyze the hydrolysis of glycerate 1,3-bisphosphate to glycerate 3-phosphate and inorganic phosphate. The Km for glycerate 1,3-bisphosphate was 320 microM and the Vmax was 11.5 milliunits/mg. Incubation of the rat liver enzyme with [1-32P]glycerate 1,3-bisphosphate resulted in the formation of a phosphoenzyme intermediate, and the labeled amino acid was identified as 3-phosphohistidine. Tryptic and endoproteinase Lys-C peptide maps of the 32P-phosphoenzyme labeled either with [2-32P]fructose 2,6-bisphosphate or [1-32P]glycerate 1,3-bisphosphate revealed that 32P-radioactivity was found in the same peptide, proving that the same histidyl group accepts phosphate from both substrates. Fructose 2,6-bisphosphate inhibited competitively the formation of phosphoenzyme from [1-32P]glycerate 1,3-bisphosphate. Effectors of fructose-2,6-bisphosphatase also inhibited phosphoenzyme formation. Substrates and products of phosphoglycerate mutase and 2,3-bisphosphoglycerate mutase also modulated the activities of the bifunctional enzyme. These results demonstrate that, in addition to a structural homology, the bisphosphatase domain of the bifunctional enzyme has a functional similarity to phosphoglycerate mutase and 2,3-bisphosphoglycerate mutase and support the concept of an evolutionary relationship between the three enzyme activities.     

Microbial metabolism of C1 and C2 compounds. The role of glyoxylate, glycollate and acetate in the growth of Pseudomonas AM1 on ethanol and on C1 compounds

Succinate (or a product of succinate metabolism) is a catabolite repressor of some enzymes of the serine pathway (hydroxypyruvate reductase, serine-glyoxylate aminotransferase and glycerate kinase) but not of methanol dehydrogenase nor methylamine dehydrogenase. A mutant (PCT64) of Pseudomonas AM1, which is unable to grow on C(1) compounds, lacks glycerate kinase, showing that this enzyme is essential for the operation of the serine pathway. Mutant PCT48, unable to convert acetate into glycollate, has lost the ability to grow both on C(1) compounds and on ethanol. The properties of a third mutant (PCT57) show that Pseudomonas AM1 contains enzymes catalysing the conversion of acetate into glyoxylate. Evidence is presented that hydroxypyruvate reductase is involved in the oxidation of glycollate to glyoxylate during growth on ethanol. A scheme is proposed for the conversion of ethanol and of C(1) compounds into glyoxylate in which acetate (or a derivative) and glycollate are intermediates.     

Purification and characterization of glycerate kinase from a serine-producing methylotroph, Hyphomicrobium methylovorum GM2.

The glycerate kinase of a serine-producing methylotroph, Hyphomicrobium methylovorum GM2, was purified to complete homogeneity and characterized, the first time for an enzyme from a methylotroph. The enzyme was a monomer with a molecular mass about 41-52 kDa. The enzyme was stable against heating at 35 degrees C for 30 min at pH values over 6-10. Maximum activity was observed at pH 8.0 and around 50 degrees C. The Km values for D-glycerate and ATP were 0.13 mM and 0.13 mM, respectively. The enzyme showed high specificity for D-glycerate, and was activated by potassium and ammonium ions. The reaction product of the enzyme was identified as 2-phosphoglycerate.     

Properties of Escherichia coli mutants deficient in enzymes of glycolysis.

Physiological properties of mutants of Escherichia coli defective in glyceraldehyde 3-phosphate dehydrogenase, glycerate 3-phosphate kinase, or enolase are described. Introduction of a lesion in any one of the reversible steps catalyzed by these enzymes impaired both the glycolytic and gluconeogenic capabilities of the cell and generated an obligatory requirement for a source of carbon above the block (gluconeogenic) and one below (oxidative). A mixture of glycerol and succinate supported the growth of these mutants. Mutants lacking glyceraldehyde 3-phosphate dehydrogenase and glycerate 3-phosphate kinase could grow also on glycerol and glyceric acid, and enolase mutants could grow on glycerate and succinate, whereas double mutants lacking the kinase and enolase required l-serine in addition to glycerol and succinate. Titration of cell yield with limiting amounts of glycerol with Casamino Acids in excess, or vice versa, showed the gluconeogenic requirement of a growing culture of E. coli to be one-twentieth of its total catabolic and anabolic needs. Sugars and their derivatives inhibited growth of these mutants on otherwise permissive media. The mutants accumulated glycolytic intermediates above the blocked enzyme on addition of glucose or glycerol to resting cultures. Glucose inhibited growth and induced lysis. These effects could be substantially overcome by increasing the osmotic strength of the growth medium and, in addition, including 5 mM cyclic adenosine 3',5'-monophosphate therein. This substance countered to a large extent the severe repression of beta-galactosidase synthesis that glucose caused in these mutants.