Identification and characterization of the ATP-binding site in human pancreatic glucokinase

Diane E. Marotta, Gulshan R. Anand, Timothy A. Anderson, Stephen P. Miller, David A. Okar, David G. Levitt, Alex J. Lange

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8 Scopus citations


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 Vmax, cooperativity of glucose binding and S0.5 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.

Original languageEnglish (US)
Pages (from-to)23-31
Number of pages9
JournalArchives of Biochemistry and Biophysics
Issue number1
StatePublished - Apr 1 2005

Bibliographical note

Funding Information:
This work was supported by a research grant from the Juvenile Diabetes Research Foundation International (No. 198236) to A.J.L. Additionally, we wish to thank Ms. Barbara Ketay for her generous gift.

Copyright 2017 Elsevier B.V., All rights reserved.


  • Kinetics
  • MODY-2
  • Molecular modeling
  • Mutagenesis

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