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 journalArticle

8 Citations (Scopus)

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

Fingerprint

Glucokinase
Adenosine Triphosphate
Binding Sites
Glucose
Adenosine Diphosphate Glucose
Glucose-6-Phosphate
Phosphorylation
Hexokinase
Crystallization
Adenosine Triphosphatases
Blood Glucose
Conservation
Brain
Crystal structure
Maintenance
X-Rays
Insulin
X rays

Keywords

  • Kinetics
  • MODY-2
  • Molecular modeling
  • Mutagenesis

Cite this

Marotta, D. E., Anand, G. R., Anderson, T. A., Miller, S. P., Okar, D. A., Levitt, D. G., & Lange, A. J. (2005). Identification and characterization of the ATP-binding site in human pancreatic glucokinase. Archives of Biochemistry and Biophysics, 436(1), 23-31. https://doi.org/10.1016/j.abb.2005.01.018

Identification and characterization of the ATP-binding site in human pancreatic glucokinase. / Marotta, Diane E.; Anand, Gulshan R.; Anderson, Timothy A.; Miller, Stephen P; Okar, David A.; Levitt, David G; Lange, Alex J.

In: Archives of Biochemistry and Biophysics, Vol. 436, No. 1, 01.04.2005, p. 23-31.

Research output: Contribution to journalArticle

Marotta, DE, Anand, GR, Anderson, TA, Miller, SP, Okar, DA, Levitt, DG & Lange, AJ 2005, 'Identification and characterization of the ATP-binding site in human pancreatic glucokinase', Archives of Biochemistry and Biophysics, vol. 436, no. 1, pp. 23-31. https://doi.org/10.1016/j.abb.2005.01.018
Marotta, Diane E. ; Anand, Gulshan R. ; Anderson, Timothy A. ; Miller, Stephen P ; Okar, David A. ; Levitt, David G ; Lange, Alex J. / Identification and characterization of the ATP-binding site in human pancreatic glucokinase. In: Archives of Biochemistry and Biophysics. 2005 ; Vol. 436, No. 1. pp. 23-31.
@article{4c2f0d90b9924c5bba8d64a20c66759d,
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 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.",
keywords = "Kinetics, MODY-2, Molecular modeling, Mutagenesis",
author = "Marotta, {Diane E.} and Anand, {Gulshan R.} and Anderson, {Timothy A.} and Miller, {Stephen P} and Okar, {David A.} and Levitt, {David G} and Lange, {Alex J}",
year = "2005",
month = "4",
day = "1",
doi = "10.1016/j.abb.2005.01.018",
language = "English (US)",
volume = "436",
pages = "23--31",
journal = "Archives of Biochemistry and Biophysics",
issn = "0003-9861",
publisher = "Academic Press Inc.",
number = "1",

}

TY - JOUR

T1 - Identification and characterization of the ATP-binding site in human pancreatic glucokinase

AU - Marotta, Diane E.

AU - Anand, Gulshan R.

AU - Anderson, Timothy A.

AU - Miller, Stephen P

AU - Okar, David A.

AU - Levitt, David G

AU - Lange, Alex J

PY - 2005/4/1

Y1 - 2005/4/1

N2 - 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.

AB - 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.

KW - Kinetics

KW - MODY-2

KW - Molecular modeling

KW - Mutagenesis

UR - http://www.scopus.com/inward/record.url?scp=14744296205&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=14744296205&partnerID=8YFLogxK

U2 - 10.1016/j.abb.2005.01.018

DO - 10.1016/j.abb.2005.01.018

M3 - Article

C2 - 15752705

AN - SCOPUS:14744296205

VL - 436

SP - 23

EP - 31

JO - Archives of Biochemistry and Biophysics

JF - Archives of Biochemistry and Biophysics

SN - 0003-9861

IS - 1

ER -