title: Effects of glucose and magnesium ion on the quenching of yeast hexokinase fluorescence by acrylamide.
abstract: To probe the effects of the substrate, glucose, and the cofactor, Mg2+, on the structure of hexokinase (ATP:D-hexose 6-phosphotransferase, EC 2.7.1.1), titrations of the tryptophan fluorescence of yeast hexokinase isozyme P-II(B) were performed. Acrylamide was used as a quenching titrant in the absence and in the presence of glucose and Mg2+ singly and together at pH 5.5 and 8.3 at 20 degrees C. The four tryptophan residues of the monomeric subunit of yeast hexokinase may be classified as two surface residues, one being highly accessible to dissolved I- and one with restricted accessibility to I-, one glucose-quenchable residue in the cleft, and one buried (Kramp, D.C. and Feldman, I. (1978) Biochim. Biophys. Acta 537, 406--416). The acrylamide data were analyzed by least-squares computer analysis for quenching constants and fractional fluorescence values of the tryptophan residues. The quenching constants measure the accessibilities of the residues to the quencher, while the fractional fluorescences are related to the microenvironments of the fluorophores. At each pH value, glucose altered the quenching constants, but not the fractional fluorescence, of the tryptophan residues. Mg2+ greatly accentuated at this glucose effect, especially for the surface residue near the cleft opening. Comparison of acrylamide- and I-quenching data shows that this particular residue has a positively charged microenvironment. A pH change from 5.5 to 8.3 increased the acylamide-accessibility of the cleft tryptophan but did not seem to influence accessibility of the surface residues or the buried residue significantly, thus strengthening our previous conclusion that the cleft opening is small enough at pH 5.5 to partially restrict entrance of organic molecules and negative ions. However, with saturating glucose present there was a pH effect on the surface residue accessibility. Titrations in 55 vol.% glycerol suggest the presence of transient channels (not just holes) in the hexokinase structure, which allows penetration of the protein by solution. Consequently, the buried tryptophan residue is quenched more strongly by dissolved acrylamide than is attributable to diffusion of quencher through the protein matrix.
mesh_terms: D000178:Acrylamides / Q000494:pharmacology*; D005453:Fluorescence; D005947:Glucose / Q000494:pharmacology*; D005990:Glycerol / Q000494:pharmacology; D006593:Hexokinase / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007527:Isoenzymes / Q000378:metabolism*; D008274:Magnesium / Q000494:pharmacology*; D009994:Osmolar Concentration; D011189:Potassium Chloride; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D012965:Sodium Chloride; D013056:Spectrophotometry, Ultraviolet; D014364:Tryptophan / Q000378:metabolism
chemical_list: D000178:Acrylamides; D007527:Isoenzymes; D012965:Sodium Chloride; D011189:Potassium Chloride; D014364:Tryptophan; D006593:Hexokinase; D008274:Magnesium; D005947:Glucose; D005990:Glycerol