Identification and characterization of the ATP-binding site in human pancreatic glucokinase.
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.
Marotta
Diane E
DE
Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, Minneapolis, MN 55455, USA.
Anand
Gulshan R
GR
Anderson
Timothy A
TA
Miller
Stephen P
SP
Okar
David A
DA
Levitt
David G
DG
Lange
Alex J
AJ
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
D019298:Glucose-6-Phosphate; D000255:Adenosine Triphosphate; D005941:Glucokinase; D005947:Glucose