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N-Acetylglucosamine is a major component of com-plex carbohydrates. The mammalian salvage pathway of N-acetylglucosamine recruitment from glycoconjugate deg-radation or nutritional sources starts with phosphorylation byN-acetylglucosamine kinase. In this studywe describe the identification of two active site cysteines of the sugar kinase by site-directed mutagenesis and computer-based structure prediction. | Eur. J. Biochem. 269 4212-4218 2002 FEBS 2002 doi 10.1046 j.1432-1033.2002.03117.x Structure and function of V-acetylglucosamine kinase Identification of two active site cysteines Markus Berger Hao Chen Werner Reutter and Stephan Hinderlich Institut fur Molekularbiologie und Biochemie Freie Universitat Berlin Berlin-Dahlem Germany N-Acetylglucosamine is a major component of complex carbohydrates. The mammalian salvage pathway of N-acetylglucosamine recruitment from glycoconjugate degradation or nutritional sources starts with phosphorylation by N-acetylglucosamine kinase. In this study we describe the identification of two active site cysteines of the sugar kinase by site-directed mutagenesis and computer-based structure prediction. Murine N-acetylglucosamine kinase contains six cysteine residues al 1 of which were mtitled to serine residues. The strongest reduction of enzyme activity was found for the mutant C131S I olkweal yy C14SS. Dtter-mination of the kinetic properties of the cysteine mutants showed that the decreased enzyme activities were due to a strongly decreased affinity to either N-acntylglucosamine for C131S or ATPforC143S. A l c sn eken l lleKỊtLrn ls eiCỊir m of N-acetylglucosamine kinase showed a high homology to glucokinase. A model of the three-dimensional structure of N-acetylglucosamine kinase based on the known structure of glucokinase was therefore generated. This model confirmed that both cysteines are located in the active site of N-acetylglucosamine kinase with a potential role in the binding of the transfered y-phosphate group of ATP within the catalytic mechanism. Keywords N-acntylglucosamine kinase N-acntylglucos-amine aminosugar metabolism ATP binding domain cysteine residue. N-Acntylglucosamine kinase GlcNAc kinase EC 2.7.1.59 catalyzes the phosphorylation of N-acetylglucosamine GlcNAc to GlcNAc 6-phosphate. GlcNAc rrom lyso--omal degradation of oligosaccharides or nutritional sources is a mum subtlTine or the cynthyeis df E DP-GceN .
