Inhibition of N‐acetylglucosamine kinase and N‐acetylmannosamine kinase by 3‐O‐methyl‐N‐acetyl‐D‐glucosamine in vitro

Zeitler, Reinhard; Giannis, Athanassios; Danneschewski, S; Henk, Elfriede; Henk, Thomas; Bauer, Christian; Reutter, Werner; Sandhoff, Konrad

doi:10.1111/j.1432-1033.1992.tb16743.x

Cited by 33 publications

(23 citation statements)

References 16 publications

(6 reference statements)

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“…The inhibition was assessed from the reduced activity relative to control without inhibitors. N-propanoylglucosamine and 3-O-Me-GlcNAc were synthesized and characterized as described earlier (24,25).…”

Section: Methodsmentioning

confidence: 99%

“…Based on the geometry of the active center of hMNK, we postulated that modifications at the C6-position of ManNAc could yield potential inhibitors. N-Propanoylglucosamine and 3-O-Me-GlcNAc were described in previous studies to inhibit MNK activity in cell lysates (24,25). The inhibition effect of these substances was tested in this study with purified recombinant hMNK.…”

Section: ␦1mentioning

confidence: 99%

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Crystal Structures of N-Acetylmannosamine Kinase Provide Insights into Enzyme Activity and Inhibition

Martinez

Nguyen

Hinderlich

et al. 2012

Journal of Biological Chemistry

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Background: UDP-GlcNAc-2 epimerase/ManNAc kinase is the key enzyme for sialic acid biosynthesis. Results: The crystal structure of ManNAc kinase in complex with its substrate ManNAc has been determined at 1.64 Å resolution. Conclusion: Insights into the mechanism of ManNAc phosphorylation have been gained from the substrate-bound structure. Significance: The new structural information presented here offers a basis for designing potential inhibitors of sialic acid biosynthesis.

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Section: Methodsmentioning

confidence: 99%

Section: ␦1mentioning

confidence: 99%

Crystal Structures of N-Acetylmannosamine Kinase Provide Insights into Enzyme Activity and Inhibition

Martinez

Nguyen

Hinderlich

et al. 2012

Journal of Biological Chemistry

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“…After incubation at 25°C for 20 h, 2 l of alkaline phosphatase (1.5 to 7.5 units) in 1.2 mol of glycine-NaOH buffer, pH 10.0, containing 120 nmol of MgCl 2 were added, and the reaction was incubated at 37°C for 30 to 60 min. Three l of each sample were spotted onto plastic-backed cellulose TLC plate (Merck) and developed in 1-propanol, 1 M sodium acetate, pH 5.0, water (7:1:2, v/v/v) for 8 h (43). When the effect of ionic strength was determined, the samples were spotted on Whatman No.…”

Section: Quantitative Assay For Kdn-9-p Synthetase and Neu5ac-9-p Synmentioning

confidence: 99%

Biosynthesis of KDN (2-Keto-3-deoxy-d-glycero -d-galacto -nononic acid)

Angata¹,

Nakata²,

Matsuda³

et al. 1999

Journal of Biological Chemistry

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Although the deaminoneuraminic acid or KDN glycotope (2-keto-3-deoxy-D-glycero-D-galacto-nononic acid)is expressed in glycoconjugates that range in evolutionary diversity from bacteria to man, there is little information as to how this novel sugar is synthesized. Accordingly, biosynthetic studies were initiated in trout testis, an organ rich in KDN, to determine how this sialic acid is formed. These studies have shown that the pathway consists of the following three sequential reactions: 1) Man ؉ ATP 3 Man-6-P ؉ ADP; 2) Man-6-P ؉ PEP 3 KDN-9-P ؉ Pi; 3) KDN-9-P 3 KDN ؉ Pi. Reaction 1, catalyzed by a hexokinase, is the 6-O-phosphorylation of mannose to form D-mannose 6-phosphate (Man-6-P). Reaction 2, catalyzed by KDN-9-phosphate (KDN-9-P) synthetase, condenses Man-6-P and phosphoenolpyruvate (PEP) to form KDN-9-P. Reaction 3, catalyzed by a phosphatase, is the dephosphorylation of KDN-9-P to yield free KDN. It is not known if a kinase specific for Man (Reaction 1) and a phosphatase specific for KDN-9-P (Reaction 3) may exist in tissues actively synthesizing KDN. In this study, the KDN-9-P synthetase, an enzyme that has not been previously described, was identified as at least one key enzyme that is specific for the KDN biosynthetic pathway. This enzyme was purified 50-fold from rainbow trout testis and characterized. The molecular weight of the enzyme was estimated to be about 80,000, and activity was maximum at neutral pH in the presence of Mn 2؉ . N-Acetylneuraminic acid 9-phosphate (Neu5Ac-9-P) synthetase, which catalyzes the condensation of N-acetyl-D-mannosamine 6-phosphate and phosphoenol-pyruvate to produce Neu5Ac-9-P, was co-purified with the KDN-9-P synthetase. Substrate competition experiments revealed, however, that syntheses of KDN-9-P and Neu5Ac-9-P were catalyzed by two separate synthetase activities. The significance of these studies takes on added importance with the recent discovery that the level of free KDN is elevated in human fetal cord but not matched adult red blood cells and (25), , and KDN residue-cleaving sialidases (29 -31), have been identified and partially characterized, and their similarity and dissimilarity to the metabolism of Neu5Acyl residues have been described. In marked contrast, nothing is known concerning how the KDN monosaccharide is synthesized. Two pathways are possible for synthesis of the KDN monomer. First, it could be formed directly from Neu5Ac by deacylation and deamination. Alternatively, it could arise by de novo synthesis via enzymes different from those required for synthesis of Neu5Ac. However, no evidence for the direct conversion of Neu5Ac to KDN has been obtained in any tissue homogenates examined to date. Thus, de novo synthesis by a separate pathway appears to be plausible by analogy with synthesis of other ulosonic acids. For example, 3-deoxy-D-arabino-heptulosonic acid 7-phosphate, a biosynthetic precursor of aromatic amino acids (32), 3-deoxy-D-manno-octulosonic acid, a constituent of bacterial lipopolysaccharides (33, 34) and Neu5Ac (35) are synthesi...

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“…Furthermore, it has been demonstrated that GlcNAc is linked as a monosaccharide to Ser and Thr residues of nuclear [13] and have led to an accurate assay of glucokinase in the presence of GlcNAc kinase [14]. and cytoplasmic proteins [3].…”

Section: N-acetylglucosamine (Glcnac) Is a Prominent Sugar Inmentioning

confidence: 99%

Purification and characterization of N‐acetylglucosamine kinase from rat liver

Hinderlich

Nöhring

Weise

et al. 1998

European Journal of Biochemistry

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N-Acetylglucosamine, a major sugar in complex carbohydrates, enters the pathways of aminosugar metabolism by the action of N-acetylglucosamine kinase (EC 2.7.1.59). In this study we report the purification to homogeneity of GlcNAc kinase from rat liver cytosol using salmine sulfate precipitation, chromatography on phenyl-Sepharose, ATP-agarose and MonoQ, and finally gel filtration on Superdex 200. It was characterized as a dimer of 39-kDa subunits. About 25% of the amino acid sequence of GlcNAc kinase was established by peptide mapping. Part of the ATP-binding site of GlcNAc kinase was identified by sequence comparison with related hexokinases, including the bifunctional enzyme UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase (EC 5.1.3.14/2.7.1.60), the key enzyme of N-acetylneuraminic acid biosynthesis in rat liver.The Cys residues in the active sites of GlcNAc kinase and ManNAc kinase were characterized by chemical modification with N-ethylmaleimide, iodoacetamide and 5,5′-dithiobis(2-nitrobenzoic acid). The finding that the substrates GlcNAc and ManNAc protected their respective enzymes from inhibition by the above sulfhydryl reagents indicates that Cys residues are located in or near the active sites of both enzymes. Use of the specific dithiol-modifying chemical reagents, sodium meta-periodate, sodium metaarsenite/2,3-dimercaptopropanol and diazenedicarboxylic acid bis-N,N′-dimethylamide revealed that the active sites of GlcNAc kinase and ManNAc kinase possess at least one pair of vicinal thiols. Chemical treatment of UDPϪGlcNAc 2-epimerase provided no evidence for the presence of cysteine in the active site of this enzyme. From the incorporation of N-[ 3 H]ethylmaleimide into GlcNAc kinase in the absence and presence of ligands we estimated that the active site of GlcNAc kinase contains two Cys residues.Keywords : N-acetylglucosamine kinase ; UDPϪN-acetylglucosamine 2-epimerase; N-acetylmannosamine kinase ; active-site cysteine ; chemical modification. N-Acetylglucosamine (GlcNAc) is a prominent sugar inGlcNAc kinase is widely distributed in mammalian tissues . Furthermore, it has been demonstrated that GlcNAc is linked as a monosaccharide to Ser and Thr residues of nuclear [13] and have led to an accurate assay of glucokinase in the presence of GlcNAc kinase [14]. and cytoplasmic proteins [3]. GlcNAc, from lysosomal degradation of oligosaccharides or from intestinal degradation of nutriGlcNAc kinase may also play a role in sialic acid biosynthesis, because GlcNAc can be converted into CMP-N-acetylneurtional constituents, is converted into N-acetylglucosamine 6-phosphate (GlcNAc-6-P) by the action of N-acetylglucosamine aminic acid (CMP-Neu5Ac), the precursor of the sialic acids, in vitro [15], whereas several N2 analogs of GlcNAc are metabokinase [4]. GlcNAc-6-P then serves as a substrate in the biosynthesis of UDPϪGlcNAc, which is the substrate of the different lized to sialic acids in vivo [16]. The formation of CMP-Neu5Ac from UDPϪGlcNAc in rat liver is initiated and regulated by the N-ac...

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Inhibition of N‐acetylglucosamine kinase and N‐acetylmannosamine kinase by 3‐O‐methyl‐N‐acetyl‐D‐glucosamine in vitro

Cited by 33 publications

References 16 publications

Crystal Structures of N-Acetylmannosamine Kinase Provide Insights into Enzyme Activity and Inhibition

Crystal Structures of N-Acetylmannosamine Kinase Provide Insights into Enzyme Activity and Inhibition

Biosynthesis of KDN (2-Keto-3-deoxy-d-glycero -d-galacto -nononic acid)

Purification and characterization of N‐acetylglucosamine kinase from rat liver

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