Hkx2 (hexokinase 2) 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 the 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 replaced Ser³⁰⁴ with phenylalanine to generate Hxk2(wca). 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 Lys⁶ to Met¹⁵ of Hxk2 (Hxk2(wrf)) are essential for the regulatory role of Hxk2 and whether there is an effect on the hexose kinase activity of this protein. In the present paper, we report that the Hxk2(wca) 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 have demonstrated that Hxk2(wca) maintained full regulatory function because the glucose-repression signalling of the wild-type machinery is maintained. We also report that the Hxk2(wrf) 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, Hxk2(wca) and Hxk2(wrf) retain single functions, as a transcriptional factor or as an enzyme with hexose-phosphorylating activity, but have lost the original bifunctionality of Hxk2.