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 language | English (US) |
|---|---|
| Pages (from-to) | 23-31 |
| Number of pages | 9 |
| Journal | Archives of Biochemistry and Biophysics |
| Volume | 436 |
| Issue number | 1 |
| DOIs | |
| State | Published - Apr 1 2005 |
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Keywords
- Kinetics
- MODY-2
- Molecular modeling
- Mutagenesis
Cite this
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 journal › Article
}
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 -