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.