title: Effect of ligand-induced conformational changes on the reactivity of specific sulfhydryl residues in rat brain hexokinase.
abstract: Rat brain hexokinase (ATP:D-hexose 6-phosphotransferase, EC 2.7.1.1) contains 21 cysteine residues. On the basis of the amino acid sequence of the enzyme, these are predicted to be distributed among 14 peptides produced by tryptic digestion. Ten of these peptides, containing cysteine residues derivatized by reaction with the specific sulfhydryl reagent 2-bromoacetamido-4-nitrophenol have been identified in HPLC peptide maps; the four missing peptides are predicted to be relatively large and hydrophobic in character, properties that may have prevented their detection under the present conditions. The sequences encompassed by the 10 identified peptides include 12 of the 21 cysteine residues in the enzyme. The relative reactivity of these sulfhydryl groups with 2-bromoacetamido-4-nitrophenol has been assessed, and is in general accord with what might be predicted on the basis of their accessibility in the previously proposed structure for this enzyme. The effect of various ligands on reactivity of identified sulfhydryl groups has been determined; unique patterns of altered reactivity, resulting from ligand-induced conformational changes, have been observed. Biphasic effects were observed with increasing concentrations of either glucose 6-phosphate (Glc-6-P) or Pi. In both cases, decreased reactivity of sulfhydryls in the N-terminal half of the molecule was observed at low concentrations of the ligand, while further increase in ligand concentration resulted in decreased reactivity of sulfhydryl groups in the C-terminal half. In contrast, sulfhydryls in both N- and C-terminal halves were protected concomitantly by increasing concentrations of Glc. These results are consistent with previous studies that indicated (a) the existence of two sites for binding of Glc-6-P or Pi, a high affinity site in the N-terminal half and a site with lower affinity in the C-terminal half of the brain hexokinase molecule, and (b) binding of Glc to a single site located in the C-terminal half but evoking conformational effects throughout the molecule; the glucose analog, N-acetylglucosamine, previously shown to have more limited effects on conformation, affected reactivity of sulfhydryl groups only in the C-terminal half of the molecule. As reflected by effects on reactivity of sulfhydryl groups, conformational changes induced by binding of nucleotides depends markedly on the specific nature of the purine or pyrimidine base as well as the length and chelation status of the polyphosphate side chain. These results focus attention on specific regions of the molecule (the immediate environment of the sulfhydryl groups) that are affected by the binding of these ligands.
authors.affiliation: Department of Biochemistry, Michigan State University, East Lansing 48824.
authors.forename: J
authors.initials: J
authors.lastname: Hutny
authors.forename: J E
authors.initials: JE
authors.lastname: Wilson
mesh_terms: D000081:Acetamides; D000818:Animals; D001665:Binding Sites; D001921:Brain / Q000201:enzymology*; D003545:Cysteine; D006593:Hexokinase / Q000378:metabolism*; D007700:Kinetics; D008024:Ligands; D008958:Models, Molecular; D009596:Nitrophenols / Q000494:pharmacology*; D010449:Peptide Mapping; D011487:Protein Conformation; D051381:Rats; D013439:Sulfhydryl Reagents / Q000494:pharmacology*
chemical_list: D000081:Acetamides; D008024:Ligands; D009596:Nitrophenols; D013439:Sulfhydryl Reagents; C000309:2-bromoacetamide-4-nitrophenol; D006593:Hexokinase; D003545:Cysteine