ATP-Binding Site of Human Brain Hexokinase As Studied by Molecular Modeling and Site-Directed Mutagenesis

Zeng, Chenbo; Aleshin, Alexander E.; Hardie, Jason B.; Harrison, Robert W.; Fromm, Herbert J.

doi:10.1021/bi960750e

Cited by 49 publications

(39 citation statements)

References 33 publications

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“…In human hexokinase I, electron paramagnetic resonance (50) and modeling (17) suggested that Mg 2ϩ interacts with Asp-532, Arg-539, and Asp-657, which are equivalent to Asp-8, Lys-15, and Asp-95 of StHK, through water molecules. Furthermore, the importance of these residues for activity has been confirmed by mutagenesis (51)(52)(53).…”

Section: Resultsmentioning

confidence: 92%

Crystal Structures of an ATP-dependent Hexokinase with Broad Substrate Specificity from the Hyperthermophilic Archaeon Sulfolobus tokodaii

Nishimasu

Fushinobu

Shoun

et al. 2007

Journal of Biological Chemistry

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Hexokinase catalyzes the phosphorylation of glucose to glucose 6-phosphate by using ATP as a phosphoryl donor. Recently, we identified and characterized an ATP-dependent hexokinase (StHK) from the hyperthermophilic archaeon Sulfolobus tokodaii, which can phosphorylate a broad range of sugar substrates, including glucose, mannose, glucosamine, and N-acetylglucosamine. Here we present the crystal structures of StHK in four different forms: (i) apo-form, (ii) binary complex with glucose, (iii) binary complex with ADP, and (iv) quaternary complex with xylose, Mg 2؉ , and ADP. Forms i and iii are in the open state, and forms ii and iv are in the closed state, indicating that sugar binding induces a large conformational change, whereas ADP binding does not. The four different crystal structures of the same enzyme provide "snapshots" of the conformational changes during the catalytic cycle. StHK exhibits a core fold characteristic of the hexokinase family, but the structures of several loop regions responsible for substrate binding are significantly different from those of other known hexokinase family members. Structural comparison of StHK with human N-acetylglucosamine kinase and other hexokinases provides an explanation for the ability of StHK to phosphorylate both glucose and N-acetylglucosamine. A Mg 2؉ ion and coordinating water molecules are well defined in the electron density of the quaternary complex structure. This structure represents the first direct visualization of the binding mode for magnesium to hexokinase and thus allows for a better understanding of the catalytic mechanism proposed for the entire hexokinase family.Phosphorylation of glucose to glucose 6-phosphate is important for both energy metabolism and biosynthesis in the cell. In eukaryotes, the reaction is catalyzed by hexokinases (EC 2.7.1.1), which can phosphorylate several hexoses, including mannose and fructose, in addition to glucose (1). In contrast, bacteria possess glucokinases (EC 2.7.1.2) that are specific for glucose. Bacterial glucokinases can be classified into two groups as follows: (i) glucokinases belonging to the repressors/open reading frames of unknown function/sugar kinases (ROK) 2 family, which is characterized by two signature motifs (2), and (ii) glucokinases without the ROK motifs (3). On the other hand, most Archaea use two types of glucokinase as follows: (i) ADP-dependent glucokinases (4 -9), or (ii) ATP-dependent glucokinases belonging to the ROK family (10, 11).On the basis of amino acid sequence similarity, sugar kinases can be divided into three families as follows: (i) hexokinase family, (ii) galactokinase family, and (iii) ribokinase family (12). Hexokinases and glucokinases belong to the hexokinase family, with the exception of the archaeal ADP-dependent glucokinases (13-15), which are members of the ribokinase family. The crystal structures of several members of the hexokinase family have been reported, including human hexokinase (16 -20), rat and Schistosoma mansoni hexokinase (21), yeast hexokinase (22-25),...

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Section: Resultsmentioning

confidence: 92%

Crystal Structures of an ATP-dependent Hexokinase with Broad Substrate Specificity from the Hyperthermophilic Archaeon Sulfolobus tokodaii

Nishimasu

Fushinobu

Shoun

et al. 2007

Journal of Biological Chemistry

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“…Asn537 is part of a loop, conserved in sequence between ecGlK (Asn14) and yeast hexokinase (Asn91), and is associated with nucleotide binding. A Thr680Val mutant of hexokinase I showed a decrease in k cat of ϳ2,000-fold, while the Thr680Ser mutant only decreased ϳ2.5-fold, showing the importance of this hydrogen bond in catalysis (74).…”

Section: Resultsmentioning

confidence: 94%

“…3). A combination of modeling (3) and electron paramagnetic resonance studies in solution (48) (74). A hydrated Mg 2ϩ binding site has also been suggested for the P. furiosus ADP-dependent glucokinase (34).…”

Section: Resultsmentioning

confidence: 99%

Crystal Structures ofEscherichia coliATP-Dependent Glucokinase and Its Complex with Glucose

Лунин

Schrag

et al. 2004

J Bacteriol

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their narrow specificity for glucose as a substrate. While structural information is available for ADP-dependent glucokinases from Archaea, no structural information exists for the large sequence family of eubacterial ATPdependent glucokinases. Here we report the first structure determination of a microbial ATP-dependent glucokinase, that from E. coli O157:H7. The crystal structure of E. coli glucokinase has been determined to a 2.3-Å resolution (apo form) and refined to final R work /R free factors of 0.200/0.271 and to 2.2-Å resolution (glucose complex) with final R work /R free factors of 0.193/0.265. E. coli GlK is a homodimer of 321 amino acid residues. Each monomer folds into two domains, a small ␣/␤ domain (residues 2 to 110 and 301 to 321) and a larger ␣؉␤ domain (residues 111 to 300). The active site is situated in a deep cleft between the two domains. E. coli GlK is structurally similar to Saccharomyces cerevisiae hexokinase and human brain hexokinase I but is distinct from the ADP-dependent GlKs. Bound glucose forms hydrogen bonds with the residues Asn99, Asp100, Glu157, His160, and Glu187, all of which, except His160, are structurally conserved in human hexokinase 1. Glucose binding results in a closure of the small domains, with a maximal C␣ shift of ϳ10 Å. A catalytic mechanism is proposed that is consistent with Asp100 functioning as the general base, abstracting a proton from the O6 hydroxyl of glucose, followed by nucleophilic attack at the ␥-phosphoryl group of ATP, yielding glucose-6-phosphate as the product.Growth of Escherichia coli by using various fermentable sugars as carbon sources, including glucose, maltose, galactose, and sucrose, primarily involves the phosphoenolpyruvate-dependent phosphotransferase system (PTS) (reviewed in reference 54). However, a secondary, PTS-independent system for utilization of glucose also exists, consisting of glucose uptake by galactose permease (GalP; galactose proton symporter), followed by phosphorylation by glucokinase (GlK; EC 2.7.1.2) to yield the metabolic intermediate glucose-6-phosphate. Although glk mutant strains of E. coli (43) and Bacillus subtilis (63) are not visibly physiologically impaired, this enzyme retains the important function of phosphorylating any free intracellular glucose. Free cytoplasmic glucose may arise from disaccharide hydrolysis, for example, the cleavage of trehalose phosphate in Bacillus subtilis (25), or from metabolism of maltose or isomaltose (61). Indeed, studies of a PTS Ϫ E. coli strain have shown that a growth rate approximately 89% that of wild-type cells can be obtained by overexpression of GalP alone, suggesting that glucose transport, not GlK-dependent phosphorylation, is limiting growth (28). There is considerable industrial interest in enhancing the ability of E. coli to transport and phosphorylate glucose in a PTS-independent manner due to the ability of these strains to direct more carbon flux to aromatic synthesis pathways (20,21,28).Microbial glucokinases can be divided into three families based on sequ...

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“…This is close enough to transfer the ␥-phosphate group of ATP to the C6 hydroxyl. In the analysis of human brain hexokinase I, the mutation of Asp532 in its ATP-binding motif, which structurally corresponds to Asp8 of SgGlkA, to Lys or Glu results in a significant loss of the enzymatic activity (57). In the structure of SgGlkA-glucose-AMPPNP, Asp8 locates between AMPPNP and Asn105 and seems to recognize the ␥-phosphate group of ATP.…”

Section: Resultsmentioning

confidence: 99%

Substrate Recognition Mechanism and Substrate-Dependent Conformational Changes of an ROK Family Glucokinase from Streptomyces griseus

et al. 2012

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Carbon catabolite repression (CCR) is a widespread phenomenon in many bacteria that is defined as the repression of catabolic enzyme activities for an unfavorable carbon source by the presence of a preferable carbon source. In Streptomyces, secondary metabolite production often is negatively affected by the carbon source, indicating the involvement of CCR in secondary metabolism. Although the CCR mechanism in Streptomyces still is unclear, glucokinase is presumably a central player in CCR. SgGlkA, a glucokinase from S. griseus, belongs to the ROK family glucokinases, which have two consensus sequence motifs (1 and 2). Here, we report the crystal structures of apo-SgGlkA, SgGlkA in complex with glucose, and SgGlkA in complex with glucose and adenylyl imidodiphosphate (AMPPNP), which are the first structures of an ROK family glucokinase. SgGlkA is divided into a small ␣/␤ domain and a large ␣؉␤ domain, and it forms a dimer-of-dimer tetrameric configuration. SgGlkA binds a ␤-anomer of glucose between the two domains, and His157 in consensus sequence 1 plays an important role in the glucose-binding mechanism and anomer specificity of SgGlkA. In the structures of SgGlkA, His157 forms an HC3-type zinc finger motif with three cysteine residues in consensus sequence 2 to bind a zinc ion, and it forms two hydrogen bonds with the C1 and C2 hydroxyls of glucose. When the three structures are compared, the structure of SgGlkA is found to be modified by the binding of substrates. The substrate-dependent conformational changes of SgGlkA may be related to the CCR mechanism in Streptomyces.

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ATP-Binding Site of Human Brain Hexokinase As Studied by Molecular Modeling and Site-Directed Mutagenesis

Cited by 49 publications

References 33 publications

Crystal Structures of an ATP-dependent Hexokinase with Broad Substrate Specificity from the Hyperthermophilic Archaeon Sulfolobus tokodaii

Crystal Structures of an ATP-dependent Hexokinase with Broad Substrate Specificity from the Hyperthermophilic Archaeon Sulfolobus tokodaii

Crystal Structures ofEscherichia coliATP-Dependent Glucokinase and Its Complex with Glucose

Substrate Recognition Mechanism and Substrate-Dependent Conformational Changes of an ROK Family Glucokinase from Streptomyces griseus

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