title: Identification and characterization of the ATP-binding site in human pancreatic glucokinase.
abstract: The central role of human pancreatic glucokinase in insulin secretion and, consequently, in maintenance of blood glucose levels has prompted investigation into identification of ATP-binding site residues and examination of ATP- and glucose-binding interactions. Because glucokinase has been resistant to crystallization, computer generated homology models were developed based on the X-ray crystal structure of the COOH-terminal domain of human brain hexokinase 1 bound to glucose and ADP or glucose and glucose-6-phosphate. Human pancreatic glucokinase mutants were designed based upon these models and on ATPase domain sequence conservation to identify and characterize potential glucose and ATP-binding sites. Specifically, mutants Asp78Ala, Thr82Ala, Lys90Ala, Lys102Ala, Gly227Ala, Thr228Ala, Ser336Leu, Ser411Ala, and Ser411Leu were constructed, expressed, purified, and kinetically characterized under steady-state conditions. Compared to their respective wild type controls, several mutants demonstrated dramatic changes in V(max), cooperativity of glucose binding and S(0.5) for ATP and glucose. Results suggest a role for Asp78, Thr82, Gly227, Thr228, and Ser336 in ATP binding and indicate these residues are essential for glucose phosphorylation by human pancreatic glucokinase.
authors.affiliation: Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, Minneapolis, MN 55455, USA.
authors.forename: Diane E
authors.initials: DE
authors.lastname: Marotta
authors.forename: Gulshan R
authors.initials: GR
authors.lastname: Anand
authors.forename: Timothy A
authors.initials: TA
authors.lastname: Anderson
authors.forename: Stephen P
authors.initials: SP
authors.lastname: Miller
authors.forename: David A
authors.initials: DA
authors.lastname: Okar
authors.forename: David G
authors.initials: DG
authors.lastname: Levitt
authors.forename: Alex J
authors.initials: AJ
authors.lastname: Lange
mesh_terms: D000255:Adenosine Triphosphate / Q000737:chemistry / Q000378:metabolism*; D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D003198:Computer Simulation; D018360:Crystallography, X-Ray; D005941:Glucokinase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D005947:Glucose / Q000378:metabolism; D019298:Glucose-6-Phosphate / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D009154:Mutation; D010179:Pancreas / Q000201:enzymology*; D010766:Phosphorylation; D013379:Substrate Specificity
chemical_list: D019298:Glucose-6-Phosphate; D000255:Adenosine Triphosphate; D005941:Glucokinase; D005947:Glucose