Modulation of human glucokinase intrinsic activity by SH reagents mirrors post-translational regulation of enzyme activity |
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| Institution: | 1. Urologic Oncology Branch, Center for Cancer Research, National Cancer Institute, NIH, Bethesda, MD 20892, USA;2. Radiation Biology Branch, Center for Cancer Research, National Cancer Institute, NIH, Bethesda, MD 20892, USA;3. Chemical Genomics Center, National Center for Advancing Translational Sciences, National Institutes of Health, Rockville, MD 20850, USA;4. Mitochondrial Medicine Laboratory, Department of Pathology, University of Alabama at Birmingham, Birmingham, AL 35294, USA;5. Department of Cell Biology, University of Geneva, 1211 Geneva 4, Switzerland;6. Developmental Therapeutics Branch, Center for Cancer Research, National Cancer Institute, NIH, Bethesda, MD 20892, USA;7. University of New Mexico Health Sciences Center, Albuquerque, NM 87131, USA;8. Leidos Biomedical, Frederick National Laboratory for Cancer Research, Frederick, MD 24060, USA;9. Ludwig Institute for Cancer Research, New York, NY 10017, USA;10. The Wistar Institute, Philadelphia, PA 19104, USA;11. Department of Chemistry, Vanderbilt University, Nashville, TN 37240, USA;1. Chair for Molecular Physical Chemistry, Heinrich Heine University Düsseldorf, 40225 Düsseldorf, Germany;2. Institute for Pharmaceutical and Medicinal Chemistry, Heinrich Heine University Düsseldorf, 40225 Düsseldorf, Germany;1. Koch Institute for Integrative Cancer Research, Cambridge, MA, USA;2. Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA;3. Department of Pathology, Yale University School of Medicine, New Haven, CT, USA;4. Department of Dermatology, Yale University School of Medicine, New Haven, CT, USA;5. Whitehead Institute for Biomedical Research, Cambridge, MA 02139, USA;6. Dana-Farber Cancer Institute, Boston, MA 02215, USA;7. Broad Institute, Cambridge, MA 02139, USA;1. Department of Biochemistry and Molecular Biology, University of Chicago, Chicago, IL, USA;2. Department of Microbiology, University of Chicago, Chicago, IL, USA |
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| Abstract: | The low-affinity glucose phosphorylating enzyme glucokinase plays a key role in the process of glucose recognition in pancreatic B-cells. To evaluate mechanisms of intrinsic regulation of enzyme activity human pancreatic B-cell and liver glucokinase and for comparison rat liver glucokinase were expressed in E. coli bacteria. A one-step purification procedure through metal chelate affinity chromatography revealed 58 kDa proteins with high specific activities in the range of 50 U/mg protein and Km values around 8 mM for the substrate d-glucose with a preference for the α-anomer. There were no tissue specific differences, no species differences in the electrophoretic mobility, and no differences of the kinetic properties of these well conserved enzymes. The deletion of the 15 tissue-specific NH2-terminal amino acids of the human glucokinase resulted in a catalytically active enzyme whose kinetic properties were not significantly different from those of the wild-type enzymes. The human and rat glucokinase isoforms were non-competitively inhibited by the sulfhydryl group reagents alloxan and ninhydrin with Ki values in the range of 1 μM. The inhibition of glucokinase enzyme activity was reversed by dithiothreitol with an EC50 value of 9 μM for alloxan and of 60 μM for ninhydrin. d-Glucose provided protection against alloxan-induced inhibition of human and rat glucokinase isoenzymes with half-maximal effective concentrations between 11 and 16 mM. The enzyme inhibition by alloxan was accompanied by a change in the electrophoretic mobility with a second lower molecular 49 kDa glucokinase band which can be interpreted as a compact glucokinase molecule locked by disulfide bonds. Quantification of free sulfhydryl groups revealed an average number of 3.6 free sulfhydryl groups per enzyme molecule for the native human glucokinase isoforms. Alloxan decreased the average number of free sulfhydryl groups to 1.9 per enzyme molecule indicating that more than one SH side group is oxidized by this compound. The extraordinary sensitivity of the SH side groups of the glucokinase may be a possible mechanism of enzyme regulation by interconversion of stable (active) and unstable (inactive) conformations of the enzyme. In pancreatic B-cells the glucose-dependent increase of reduced pyridine nucleotides may stabilize the enzyme in the 58 kDa form and provide optimal conditions for glucose recognition and glucose-induced insulin secretion. |
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