Structural and Kinetic Studies of Induced Fit in Xylulose Kinase from Escherichia coli

• Published in: Journal of molecular biology 2007-01, Vol.365 (3), p.783-798
• Main Authors: Di Luccio, Eric, Petschacher, Barbara, Voegtli, Jennifer, Chou, Hui-Ting, Stahlberg, Henning, Nidetzky, Bernd, Wilson, David K.
• Format: Article
• Language: English
• Subjects: Adenosine Triphosphatases - chemistry; Amino Acid Sequence; ATPase; BENDING; Carbohydrates - chemistry; Catalysis; CONFORMATIONAL CHANGES; Conserved Sequence; Cryoelectron Microscopy; CRYSTAL STRUCTURE; Crystallography, X-Ray; DIMERIZATION; DIMERS; Enzyme Inhibitors - pharmacology; Escherichia coli; Escherichia coli - enzymology; FGGY kinase; Glycerol Kinase - chemistry; KINETICS; MATERIALS SCIENCE; mechanism; Models, Molecular; Molecular Sequence Data; MUTANTS; Other,OTHER; PENTOSES; PHOSPHATES; Phosphotransferases (Alcohol Group Acceptor) - antagonists & inhibitors; Phosphotransferases (Alcohol Group Acceptor) - chemistry; Phosphotransferases (Alcohol Group Acceptor) - metabolism; Phosphotransferases (Alcohol Group Acceptor) - ultrastructure; Protein Structure, Quaternary; Protein Structure, Secondary; RESOLUTION; Sequence Alignment; Substrate Specificity; SYNERGISM; X-ray structure; xylulokinase
• ISSN: 0022-2836; 1089-8638
• DOI: 10.1016/j.jmb.2006.10.068

The primary metabolic route for d-xylose, the second most abundant sugar in nature, is via the pentose phosphate pathway after a two-step or three-step conversion to xylulose-5-phosphate. Xylulose kinase (XK; EC 2.7.1.17) phosphorylates d-xylulose, the last step in this conversion. The apo and d-xylulose-bound crystal structures of Escherichia coli XK have been determined and show a dimer composed of two domains separated by an open cleft. XK dimerization was observed directly by a cryo-EM reconstruction at 36 Å resolution. Kinetic studies reveal that XK has a weak substrate-independent MgATP-hydrolyzing activity, and phosphorylates several sugars and polyols with low catalytic efficiency. Binding of pentulose and MgATP to form the reactive ternary complex is strongly synergistic. Although the steady-state kinetic mechanism of XK is formally random, a path is preferred in which d-xylulose binds before MgATP. Modelling of MgATP binding to XK and the accompanying conformational change suggests that sugar binding is accompanied by a dramatic hinge-bending movement that enhances interactions with MgATP, explaining the observed synergism. A catalytic mechanism is proposed and supported by relevant site-directed mutants.