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

Research output: Contribution to journalArticlepeer-review

8 Scopus citations

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 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
Volume436
Issue number1
DOIs
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:
Copyright 2017 Elsevier B.V., All rights reserved.

Keywords

  • Kinetics
  • MODY-2
  • Molecular modeling
  • Mutagenesis

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