Abstract: SYNOPSIS Hexokinase 2 (Hxk2) from Saccharomyces cerevisiae was one of the first metabolic enzymes described as a multifunctional protein. Hxk2 has a double subcellular localization; it functions as a glycolytic enzyme in the cytoplasm and as a regulator of gene transcription of several Mig1-regulated genes in the nucleus. To get more insights into structure-function relationships of the Hxk2 protein, we followed two different approaches. In the first we deleted the last eight amino acids of Hxk2 and substituted serine 304 with phenylalanine to generate Hxk2wca. Analysis of this mutant demonstrated that these domains play an essential role in the catalytic activity of yeast Hxk2 but has no effect on the regulatory function of this protein. In the second we analysed whether amino acids from lysine 6 to methionine 15 of Hxk2 (Hxk2wrf) are essential to the regulatory role of Hxk2 and if there is an effect on the hexose kinase activity of this protein. Here, we report that the Hxk2wca mutant protein interacts with the Mig1 transcriptional repressor and the Snf1 protein kinase in the nucleus at the level of the SUC2-Mig1 repressor complex. We demonstrate that Hxk2wca maintained full regulatory function because the glucose repression signalling of the wild-type machinery is maintained. We also report that the Hxk2wrf mutant allele is incapable of glucose repression signalling because it does not interact with Mig1 at the level of the SUC2-Mig1 repressor complex. The two mutants, Hxk2wca and Hxk2wrf retain single functions, as a transcriptional factor or as an enzyme with hexose phosphorylating activity, but have lost the original bifunctionality of Hxk2.