Home LiteratureArticle Details
PMID: 1854332 Published · ppublish English Comparative Study Journal Article Research Support, Non-U.S. Gov't Research Support, U.S. Gov't, P.H.S.

Expression and site-directed mutagenesis of hepatic glucokinase.

The Biochemical journal ·Vol. 277 ( Pt 1) ·1991-07-01 ·Pages 159-63

Lange AJ, Xu LZ, Van Poelwijk F, Lin K, Granner DK, Pilkis SJ

Abstract

Soluble rat liver glucokinase was expressed at high levels at 22 degrees C in the BL21(DE3)pLysS strain of Escherichia coli. Aspartate-211 of yeast hexokinase has been implicated as a catalytic residue from crystallographic data. The corresponding residue in rat liver glucokinase, aspartate-205, was mutated to alanine and the expressed mutant had 1/500th of the activity of the wild type, with no change in the Km values for glucose or ATP. The results support a role for this residue as a base catalyst in the glucokinase reaction and, most probably, a similar role in the reactions of all members of the hexokinase family.

MeSH Terms
Animals Base Sequence Cloning, Molecular Escherichia coli/genetics Gene Expression Glucokinase/genetics,isolation & purification,metabolism Kinetics Liver/enzymology Molecular Sequence Data Molecular Weight Mutagenesis, Site-Directed Oligonucleotide Probes Rats Recombinant Proteins/isolation & purification,metabolism
Chemicals
Oligonucleotide Probes Recombinant Proteins Glucokinase
Authors & Affiliations
6 authors, click to expand affiliations / ORCID
Lange A J
Department of Physiology and Biophysics, SUNY, Stony Brook, NY 11794.
Xu L Z
Van Poelwijk F
Lin K
Granner D K
Pilkis S J
References (24)
24 references, click to expand
  1. Protein measurement with the Folin phenol reagent.
    J Biol Chem. 1951 Nov;193(1):265-75 PMID: 14907713
  2. Hormonal control of hexokinase activity in animal tissues.
    Biochim Biophys Acta. 1970 Sep 22;215(3):461-76 PMID: 5507368
  3. Expression of mammalian liver glycolytic/gluconeogenic enzymes in Escherichia coli: recovery of active enzyme is strain and temperature dependent.
    Protein Expr Purif. 1990 Nov;1(2):169-76 PMID: 1967024
  4. T7 lysozyme inhibits transcription by T7 RNA polymerase.
    Cell. 1987 Apr 24;49(2):221-7 PMID: 3568126
  5. Pancreatic islet glucose metabolism and regulation of insulin secretion.
    Diabetes Metab Rev. 1986;2(3-4):163-214 PMID: 2943567
  6. Use of bacteriophage T7 RNA polymerase to direct selective high-level expression of cloned genes.
    J Mol Biol. 1986 May 5;189(1):113-30 PMID: 3537305
  7. Expression of the bisphosphatase domain of rat liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase in Escherichia coli.
    Proc Natl Acad Sci U S A. 1988 Sep;85(18):6642-6 PMID: 2842783
  8. Inhibition of restriction endonuclease Nci I cleavage by phosphorothioate groups and its application to oligonucleotide-directed mutagenesis.
    Nucleic Acids Res. 1986 Dec 22;14(24):9679-98 PMID: 3027659
  9. The rapid generation of oligonucleotide-directed mutations at high frequency using phosphorothioate-modified DNA.
    Nucleic Acids Res. 1985 Dec 20;13(24):8765-85 PMID: 3001650
  10. Mutations in the active site of Escherichia coli phosphofructokinase.
    Nature. 1987 Jun 4-10;327(6121):437-9 PMID: 2953977
  11. Expression of rat liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase and its kinase domain in Escherichia coli.
    Proc Natl Acad Sci U S A. 1989 Oct;86(19):7316-20 PMID: 2552438
  12. Expression of rat hepatic glucokinase in Escherichia coli.
    Biochem Biophys Res Commun. 1989 Dec 15;165(2):817-25 PMID: 2688646
  13. Rat glucokinase gene: structure and regulation by insulin.
    Proc Natl Acad Sci U S A. 1989 Jul;86(13):4838-42 PMID: 2662183
  14. Complete amino acid sequence of rat brain hexokinase, deduced from the cloned cDNA, and proposed structure of a mammalian hexokinase.
    Proc Natl Acad Sci U S A. 1989 Apr;86(8):2563-7 PMID: 2704734
  15. The amino acid sequence of rat liver glucokinase deduced from cloned cDNA.
    J Biol Chem. 1989 Jan 5;264(1):363-9 PMID: 2909525
  16. Molecular cloning of glucokinase cDNA. Developmental and dietary regulation of glucokinase mRNA in rat liver.
    J Biol Chem. 1987 May 5;262(13):6032-8 PMID: 3553185
  17. Use of T7 RNA polymerase to direct expression of cloned genes.
    Methods Enzymol. 1990;185:60-89 PMID: 2199796
  18. Rapid action of insulin and cyclic AMP in the regulation of functional messenger RNA coding for glucokinase in rat liver.
    J Biol Chem. 1984 Jan 10;259(1):343-6 PMID: 6323405
  19. Structure of a complex between yeast hexokinase A and glucose. II. Detailed comparisons of conformation and active site configuration with the native hexokinase B monomer and dimer.
    J Mol Biol. 1980 Jun 25;140(2):211-30 PMID: 7001032
  20. Regulation of glucokinase in liver.
    Curr Top Cell Regul. 1976;11:1-50 PMID: 187384
  21. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications.
    Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350-4 PMID: 388439
  22. DNA sequencing with chain-terminating inhibitors.
    Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463-7 PMID: 271968
  23. Rat hepatic glucokinase: improved purification and some properties.
    Arch Biochem Biophys. 1972 Apr;149(2):349-60 PMID: 4579946
  24. Expression of rat liver fructose-1,6-bisphosphatase in Escherichia coli.
    Biochem Biophys Res Commun. 1991 Apr 15;176(1):137-44 PMID: 1850252
Article Info
Journal
The Biochemical journal
Abbr.
Biochem J
ISSN
0264-6021
DOI
10.1042/bj2770159
Published
1991-07-01
Pages
159-63
Language
English
Region
England
NLM ID
2984726R
PMCID
PMC1151205
Subset
IM
Grants
NIDDK NIH HHS · DK 35107 · United States
External Links
PubMed source DOI: 10.1042/bj2770159 PMC Full Text View journal details
Analysis Services
Analysis Services

Contact

No. 2 Wenbo Road, Zhangqiu District, Jinan, Shandong

Qilu Normal University · Genelibs Bioinformatics Lab

750 Shunhua Rd, Jinan

2F, Bldg F, University Science Park

Tel: 0531-88819269

WeChat Official Account

Follow our WeChat subscription account for real-time updates and the latest in medical and biological research.


Business Email

E-mail: product@genelibs.com

Copyright © 2026.Genelibs Jinan Honghui Biotechnology Co., Ltd. 鲁ICP备20007159号-2