Elucidation of the molecular interaction between glucokinase and glucokinase regulatory protein

FRANCINI F; BALTRUSCH S; TIEDGE M; LENZEN S

Glasgow

Congreso; 37th EASD Annual Meeting; 2001

Background and Aims: The low-affinity glucose phosphorylating enzyme glucokinase (GK) regulates glycolytic flux at millimolar glucose concentrations in liver and pancreatic beta cells. Hepatic GK activity is modulated on the posttranslational level through an interaction with a regulatory protein (GRP). Most recent studies demonstrate, that the GRP is responsible for the intracellular translocation of GK. We could recently identify an asparagine-leucine consensus motif within the GK protein by phage display library screenings which confer the binding to GRP. It was the aim of this study to characterize the molecular mechanisms underlying the interaction between GK and GRP. Materials and Methods: Site-directed mutagenesis of selected amino acids in the GRP and GK protein was performed by the Altered Sites II in vitro Mutagenesis System. Protein interactions of wild type and mutant proteins were characterized by the MATCHMAKER GAL4 Two-Hybrid System 2 and verified by growth selection and quantitative chemiluminescence ß-galactosidase assays. GK enzyme activities were measured by a photometric assay. Results: An asparagine-leucine consensus motif of GK for interaction with GRP could be localized in three areas of the protein, Leu-309/Asn-313 (A), Asn-350/Leu-355 (B) and Leu58/Asn-204 (C). Binding motif A is part of a proposed nuclear export signal whereas motif C is located within the substrate binding site of GK. Only the double mutation of the amino acids Leu-58/Asn-204 inside the substrate binding site of the GK protein resulted in a complete loss of the interaction with GRP. In addition, we found that also the single mutations of Leu-58 and Asn-204 resulted in a complete loss of binding to GRP. In enzyme activity studies these mutations showed a significant loss of GK affinity for the substrate glucose. In contrast, the double mutation Asn-350/Leu-355 and the single mutation Leu-355 retained a significant interaction of GK/GRP in the range of 50 % of the wild type GK protein. Conclusion: The amino acids Leu-58 and Asn-204 of the GK protein play the most important role for the interaction between GK and GRP. As Asn-204 is localized in the substrate binding site the interaction of GK with GRP affects the catalytic function.