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Literature summary extracted from

  • Hokao, R.; Matsumura, H.; Katsumi, R.; Angkawidjaja, C.; Takano, K.; Kanaya, S.; Koga, Y.
    Affinity shift of ATP upon glycerol binding to a glycerol kinase from the hyperthermophilic archaeon Thermococcus kodakarensis KOD1 (2020), J. Biosci. Bioeng., 129, 657-663 .
    View publication on PubMed

Cloned(Commentary)

EC Number Cloned (Comment) Organism
2.7.1.30 recombinant overexpression of wild-type and mutant enzymes in Escherichia coli strain BL21(DE3) Thermococcus kodakarensis

Crystallization (Commentary)

EC Number Crystallization (Comment) Organism
2.7.1.30 purified recombinant wild-type enzyme in complex with glycerol and AMPPCP, and enzyme mutant K271E free or in complex with glycerol, sitting drop vapor diffusion method, 4-5 mg/ml protein in 5 mM Tris-HCl, pH 7.5, with or without 5 mM glycerol, and 5 mM AMPPCP, is mixed with 0.1 M HEPES, pH 8.0, containing 15% w/v PEG 1000 and 200 mM calcium acetate, 20°C, X-ray diffraction structure determination and analysis at 2.3-3.1 A resolution, molecular replacement using the structure of Tk-GK (PDB ID 2ZF5) as template, modelling Thermococcus kodakarensis

Protein Variants

EC Number Protein Variants Comment Organism
2.7.1.30 K271E site-directed mutagenesis, the mutation disrupts the hexamer formation interface Thermococcus kodakarensis

KM Value [mM]

EC Number KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
2.7.1.30 additional information
-
 additional information Michaelis-Menten kinetics, Tk-GK shows no negative cooperativity for ATP binding, the hexamer formation maintains a high ATP binding affinity Thermococcus kodakarensis
2.7.1.30 0.0597
-
 ATP pH 7.5, 37°C, recombinant wild-type enzyme Thermococcus kodakarensis
2.7.1.30 0.664
-
 ATP pH 7.5, 37°C, recombinant enzyme mutant K271E Thermococcus kodakarensis

Metals/Ions

EC Number Metals/Ions Comment Organism Structure
2.7.1.30 Mg2+ required Thermococcus kodakarensis

Molecular Weight [Da]

EC Number Molecular Weight [Da] Molecular Weight Maximum [Da] Comment Organism
2.7.1.30 97400 101300 recombinant dimeric enzyme mutant K271E, gel filtration Thermococcus kodakarensis
2.7.1.30 98500
-
 recombinant dimeric wild-type enzyme, gel filtration Thermococcus kodakarensis
2.7.1.30 316000
-
 recombinant hexameric wild-type enzyme, gel filtration Thermococcus kodakarensis

Natural Substrates/ Products (Substrates)

EC Number Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
2.7.1.30 ATP + glycerol Thermococcus kodakarensis
-
 ADP + sn-glycerol 3-phosphate
-
 ?
2.7.1.30 ATP + glycerol Thermococcus kodakarensis JCM 12380
-
 ADP + sn-glycerol 3-phosphate
-
 ?
2.7.1.30 ATP + glycerol Thermococcus kodakarensis ATCC BAA-918
-
 ADP + sn-glycerol 3-phosphate
-
 ?

Organism

EC Number Organism UniProt Comment Textmining
2.7.1.30 Thermococcus kodakarensis O93623
-
-
2.7.1.30 Thermococcus kodakarensis ATCC BAA-918 O93623
-
-
2.7.1.30 Thermococcus kodakarensis JCM 12380 O93623
-
-

Purification (Commentary)

EC Number Purification (Comment) Organism
2.7.1.30 recombinant wild-type and mutant enzymes from Escherichia coli strain BL21(DE3) by heat treatment at 90°C for 30 min, followed by ammonium sulfate fractionation, dialysis, and nickel affinity chromatography, then hydroxy apatite chromatography, gel ifltration, and again dialysis Thermococcus kodakarensis

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
2.7.1.30 ATP + glycerol
-
 Thermococcus kodakarensis ADP + sn-glycerol 3-phosphate
-
 ?
2.7.1.30 ATP + glycerol
-
 Thermococcus kodakarensis JCM 12380 ADP + sn-glycerol 3-phosphate
-
 ?
2.7.1.30 ATP + glycerol
-
 Thermococcus kodakarensis ATCC BAA-918 ADP + sn-glycerol 3-phosphate
-
 ?
2.7.1.30 additional information hexameric glycerol kinase (GK) from the hyperthermophilic archaeon Thermococcus kodakarensis KOD1 (Tk-GK) is identified as the substrate-binding form of the enzyme Thermococcus kodakarensis ?
-
-
2.7.1.30 additional information hexameric glycerol kinase (GK) from the hyperthermophilic archaeon Thermococcus kodakarensis KOD1 (Tk-GK) is identified as the substrate-binding form of the enzyme Thermococcus kodakarensis JCM 12380 ?
-
-
2.7.1.30 additional information hexameric glycerol kinase (GK) from the hyperthermophilic archaeon Thermococcus kodakarensis KOD1 (Tk-GK) is identified as the substrate-binding form of the enzyme Thermococcus kodakarensis ATCC BAA-918 ?
-
-

Subunits

EC Number Subunits Comment Organism
2.7.1.30 dimer Tk-GK is a dimer in solution in absence of glycerol Thermococcus kodakarensis
2.7.1.30 hexamer Tk-GK has a hexameric form with a threefold axis in the crystal lattice Thermococcus kodakarensis
2.7.1.30 More in the absence of glycerol, Tk-GK is a dimer in solution. In the presence of its glycerol substrate, it becomes a hexamer consisting of three symmetrical dimers about the threefold axis. Through glycerol binding, all Tk-GK molecules in the hexamer are in closed form as a result of domain-motion. The closed form of Tk-GK has 10fold higher ATP affinity than the open form of Tk-GK. The hexamer structure stabilizes the closed conformation and enhances ATP binding affinity when the glycerol kinase is bound to glycerol Thermococcus kodakarensis

Synonyms

EC Number Synonyms Comment Organism
2.7.1.30 Tk-GK
-
 Thermococcus kodakarensis

Temperature Optimum [°C]

EC Number Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
2.7.1.30 37
-
 assay at Thermococcus kodakarensis

Temperature Stability [°C]

EC Number Temperature Stability Minimum [°C] Temperature Stability Maximum [°C] Comment Organism
2.7.1.30 108
-
 the melting temperatures (Tm) of wild-type Tk-GK and enzyme mutant K271E are 107.8°C and 108.1°C, respectively, for the major transition Thermococcus kodakarensis

Turnover Number [1/s]

EC Number Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
2.7.1.30 4.02
-
 ATP pH 7.5, 37°C, recombinant wild-type enzyme Thermococcus kodakarensis
2.7.1.30 6.74
-
 ATP pH 7.5, 37°C, recombinant enzyme mutant K271E Thermococcus kodakarensis

pH Optimum

EC Number pH Optimum Minimum pH Optimum Maximum Comment Organism
2.7.1.30 7.5
-
 assay at Thermococcus kodakarensis

General Information

EC Number General Information Comment Organism
2.7.1.30 evolution glycerol kinase is a member of the ATPase superfamily, which includes hexokinase, actin, and heat shock protein. These share a common betabetabetaalphabetaalphabetaalpha folding motif and markedly change conformation upon substrate binding because of interdomain motion Thermococcus kodakarensis
2.7.1.30 metabolism glycerol kinase (GK) is a key enzyme of glycerol metabolism. It participates in glycolysis and lipid membrane biosynthesis Thermococcus kodakarensis
2.7.1.30 physiological function glycerol kinase (GK) catalyzes the Mg/ATP-dependent phosphorylation of glycerol to produce glycerol-3-phosphate which is an important metabolic intermediate in glycolysis. In the absence of glycerol, Tk-GK was a dimer in solution. In the presence of its glycerol substrate, it becomes a hexamer consisting of three symmetrical dimers about the threefold axis. Through glycerol binding, all Tk-GK molecules in the hexamer are in closed form as a result of domain-motion. The closed form of Tk-GK has 10fold higher ATP affinity than the open form of enzyme Tk-GK. The hexamer structure stabilizes the closed conformation and enhances ATP binding affinity when the glycerol kinase is bound to glycerol Thermococcus kodakarensis

kcat/KM [mM/s]

EC Number kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment Organism Structure
2.7.1.30 10.15
-
 ATP pH 7.5, 37°C, recombinant enzyme mutant K271E Thermococcus kodakarensis
2.7.1.30 67.34
-
 ATP pH 7.5, 37°C, recombinant wild-type enzyme Thermococcus kodakarensis