Differential scanning calorimetric study of rat brain hexokinase: domain structure and stability.
Rat brain hexokinase (ATP:D-hexose 6-phosphotransferase, EC 2.7.1.1) has been studied by differential scanning calorimetry. In "high-ionic-strength" buffer (50 mM Tris-Cl, 0.5 mM EDTA, 10 mM monothioglycerol, pH 8.5), and assuming two-state behavior with calorimetric enthalpy equal to van't Hoff enthalpy, the endotherm could be deconvoluted into two transitions with Tm values of about 48 and 51 degrees C and enthalpies of about 109 and 112 kcal/mol, respectively. A similar endotherm was seen when glucose or glucose 6-phosphate was present, except that Tm values for both transitions were increased. The glucose analog, N-acetylglucosamine, had no observable effect on the endotherm, which is in agreement with previous studies indicating that this ligand, unlike glucose and glucose 6-phosphate, does not induce conformational changes that lead to increased stability of the enzyme. In "low-ionic-strength" buffer (5 mM Tris-Cl, 0.5 mM EDTA, 10 mM monothioglycerol, pH 8.5), the transitions were partially resolved even in the absence of ligands, with Tm values of about 49 and 55 degrees C. Due to difficulties with erratic baseline behavior under the low-ionic-strength conditions, enthalpies were not routinely determined, but these appeared to be similar to those seen in high-ionic-strength buffer. Also similar was the increase in stability, as reflected by the increase in Tm for both transitions, when glucose or glucose 6-phosphate was present. Correlation of these transitions with specific regions of the molecule was established by analysis of enzyme in which the domain corresponding to the first transition was selectively denatured by a partial scan in the calorimeter. Subsequent rescanning of these samples showed only the second transition, confirming the selective denaturation of the domain corresponding to the first transition and retention of the folded structure of the second domain. Discrimination between denatured (first transition) and undenatured (second transition) domains was based on the markedly increased susceptibility of the denatured region to tryptic digestion; regions of the molecule that retained their folded structure and resistance to proteolysis were identified by immunoblotting techniques using monoclonal antibodies recognizing epitopes having defined locations within the overall sequence. Based on this analysis, the first transition corresponds to unfolding of the C-terminal half of the molecule, with the second transition resulting from unfolding of the more stable N-terminal half. The order of unfolding could be reversed in the presence of ATP-Mg2+ and N-acetylglucosamine, conditions which have been shown to result in selective stabilization of the C-terminal domain.
Department of Biochemistry and Neuroscience Program, Michigan State University, East Lansing 48824.
T K
TK
White
J Y
JY
Kim
J E
JE
Wilson
D000818:Animals; D000911:Antibodies, Monoclonal; D001921:Brain / Q000201:enzymology*; D002152:Calorimetry, Differential Scanning / Q000379:methods; D004795:Enzyme Stability; D000939:Epitopes / Q000032:analysis; D006593:Hexokinase / Q000378:metabolism*; D007700:Kinetics; D008024:Ligands; D008970:Molecular Weight; D009994:Osmolar Concentration; D010446:Peptide Fragments / Q000032:analysis / Q000276:immunology / Q000302:isolation & purification; D051381:Rats; D014357:Trypsin
D000911:Antibodies, Monoclonal; D000939:Epitopes; D008024:Ligands; D010446:Peptide Fragments; D006593:Hexokinase; D014357:Trypsin