title: Yeast hexokinase isoenzyme ScHxk2: stability of a two-domain protein with discontinuous domains.
abstract: 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.
authors.affiliation: Institute of Biochemistry and Biotechnology, Martin Luther University Halle-Wittenberg, Kurt-Mothes-Strasse 3, Halle D-06120, Germany.
authors.forename: Hauke
authors.initials: H
authors.lastname: Lilie
authors.forename: Dorit
authors.initials: D
authors.lastname: Bär
authors.forename: Karina
authors.initials: K
authors.lastname: Kettner
authors.forename: Ulrich
authors.initials: U
authors.lastname: Weininger
authors.forename: Jochen
authors.initials: J
authors.lastname: Balbach
authors.forename: Manfred
authors.initials: M
authors.lastname: Naumann
authors.forename: Eva-Christina
authors.initials: EC
authors.lastname: Müller
authors.forename: Albrecht
authors.initials: A
authors.lastname: Otto
authors.forename: Klaus
authors.initials: K
authors.lastname: Gast
authors.forename: Ralph
authors.initials: R
authors.lastname: Golbik
authors.forename: Thomas
authors.initials: T
authors.lastname: Kriegel
mesh_terms: D006593:Hexokinase / Q000737:chemistry*; D008958:Models, Molecular; D019906:Nuclear Magnetic Resonance, Biomolecular; D011489:Protein Denaturation; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry*
chemical_list: D029701:Saccharomyces cerevisiae Proteins; D006593:Hexokinase