Yeast hexokinase isoenzyme ScHxk2: stability of a two-domain protein with discontinuous domains.

The hexokinase isoenzyme 2 of Saccharomyces cerevisiae (ScHxk2) represents an archetype of a two-domain protein with the active site located in a cleft between the two domains. Binding of the substrate glucose results in a rigid body movement of the two domains leading to a cleft closure of the active site. Both domains of this enzyme are composed of discontinuous peptide sequences. This structural feature is reflected in the stability and folding of the ScHxk2 protein. Structural transitions induced by urea treatment resulted in the population of a thermodynamically stable folding intermediate, which, however, does not correspond to a molecule with one domain folded and the other unfolded. As demonstrated by different spectroscopic techniques, both domains are structurally affected by the partial denaturation. The intermediate possesses only 40% of the native secondary structural content and a substantial increase in the Stokes radius as judged by circular dichroism and dynamic light scattering analyses. One-dimensional ¹H NMR data prove that all tryptophan residues are in a non-native environment in the intermediate, indicating substantial changes in the tertiary structure. Still, the intermediate possesses quite a high stability for a transition intermediate of about ΔG = -22 kJ mol⁻¹.

Selection and validation of candidate housekeeping genes for studies of human keratinocytes--review and recommendations.

Gene expression analysis using real-time PCR has become an integral part of biomedical research. Appropriate data normalization based on stably expressed housekeeping genes is crucial for reliable results. Thus, candidate housekeeping genes require careful evaluation with regard to the individual experimental system before being selected for studies of human keratinocytes. Future research may be based on published data, as provided by Minner and Poumay in this issue.

The unique hexokinase of Kluyveromyces lactis. Molecular and functional characterization and evaluation of a role in glucose signaling.

The Crabtree-negative yeast Kluyveromyces lactis is capable of adjusting its glycolytic flux to the requirements of respiration by tightly regulating glucose uptake. RAG5 encoding the only glucose and fructose phosphorylating enzyme present in K. lactis is required for the up-regulation of glucose transport and also for glucose repression. To understand the significance of the molecular identity and specific function(s) of the corresponding kinase to glucose signaling, RAG5 was overexpressed and its gene product KlHxk1 (Rag5p) isolated and characterized. Stopped-flow kinetics and sedimentation analysis indicated a monomer-homodimer equilibrium of KlHxk1 in a condition of catalysis, i.e. in the presence of substrates and products. The kinetic constants of ATP-dependent glucose phosphorylation identified a 53-kDa monomer as the high affinity/high activity form of the novel enzyme for both glycolytic substrates suggesting a control of glucose phosphorylation at the level of dimer formation and dissociation. In contrast to the highly homologous hexokinase isoenzyme 2 of Saccharomyces cerevisiae (ScHxk2), KlHxk1 was not inhibited by free ATP in a physiological range of nucleotide concentration. Mass spectrometric sequencing of tryptic peptides of KlHxk1 identified unmodified serine at amino acid position 156. The corresponding amino acid in ScHxk2 is serine 157, which represents the autophosphorylation-inactivation site. KlHxk1 did not display, however, the typical pattern of inactivation under the respective in vitro conditions and maintained a high residual glucose phosphorylating activity. The biophysical and functional data are discussed with respect to a possible regulatory role of KlHxk1 in glucose metabolism and signaling in K. lactis.