Effects of free magnesium and alkali ions on the conformation and glucose-binding strength of yeast hexokinase isozymes.
Titrations of the tryptophan fluorescence of yeast hexokinase (ATP:D-hexose 6-phosphortransferase, EC 2.7.1.1.) isozymes P-I (A) and P-II (B) were performed with Mg2+, Li+, Na+ and K+ as titrant in absence and in presence of glucose, and vice versa, at pH 8.3 and 5.5 at 20 degrees C. Mg2+ quenches the fluorescence of surface tryptophan primarily and does so by producing a conformational change which alters the microenvironment of the tryptophan. For both isozymes Mg2+ exerts a specific ion effect, i.e. significantly larger than the ionic strength (I) effect, which enhances the glucose quenching by causing a conformation change which increases the glucose-binding constant. For the P-I isozyme glucose binding exhibits positive cooperativity at both pH 8.3 and 5.5 when the ionic strength (I) is low, i.e. significantly larger than the ionic strength (I) effect, which enhances the glucose quenching by causing a conformation change which increases the glucose-binding constant. For the P-I isozyme glucose binding exhibits positive sooperativity at both pH 8.3 and 5.5 when the ionic strength (I) is low, i.e. 0.04 or less, regardless of which of the above four cations is present. For P-II, however, glucose binding is non-cooperative at pH 8.3 regardless of I or the cation species and at pH 5.5 and low I with K+ or Mg2+ as the predominant cation present, but there is apparent negative cooperativity at pH 5.5 and low I when Na+ or Li+ predominates. These results are discussed in terms of known structural characteristics of the isozymes.
D001665:Binding Sites; D002412:Cations; D005947:Glucose; D006593:Hexokinase / Q000378:metabolism; D007527:Isoenzymes / Q000378:metabolism; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D009994:Osmolar Concentration; D011485:Protein Binding; D011487:Protein Conformation; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D013050:Spectrometry, Fluorescence; D014364:Tryptophan
D002412:Cations; D007527:Isoenzymes; D014364:Tryptophan; D006593:Hexokinase; D008274:Magnesium; D005947:Glucose