Time-resolved fluorescence of tryptophans in yeast hexokinase-PI: effect of subunit dimerization and ligand binding.
Time-resolved and steady-state fluorescence measurements have been performed on monomeric and dimeric forms of yeast hexokinase-PI. Observation of similar emission spectra and fluorescence decay parameters for both the forms of the enzyme suggests that tryptophan residue(s) are not likely to be present at the subunit-subunit interface and the process of dimerization does not perturb the local environment of tryptophan(s). The fluorescence decay of tryptophans in enzyme could be fitted to a bi-exponential function with two lifetime components, tau1 approximately 2.2 ns and tau2 approximately 3.9 ns. Binding of glucose, which is known to convert the 'open' conformation of the enzyme to a 'closed' active conformation, results in approximately 30% reduction in emission intensity and a selective decrease in tau1 from approximately 2.2 to approximately 1.1 ns. These effects can be reversed by the addition of trehalose 6-phosphate (an inhibitor of yeast hexokinase), suggesting that the trehalose 6-phosphate inhibits the enzyme by binding to its 'open' inactive conformation rather than competing with glucose to bind to the 'closed' active conformation. Binding of nucleotide ligands (ATP, ADP and adenyl-(beta,gamma-methylene)-diphosphate (AMPPCP)) to the monomeric or dimeric form of enzyme quenched the steady-state fluorescence by approximately 4-8%, but had no measurable effect on the distribution of lifetimes or on their magnitudes. Addition of nucleotides to the enzyme-glucose complex also did not produce any further change in fluorescence decay parameters. These results indicate that it is highly unlikely that the formation of a ternary enzyme-glucose-nucleotide complex from the binary enzyme-glucose complex is accompanied by a large conformational change in the enzyme, as has been surmised in some earlier studies.
Maity
H
H
Tata Institute of Fundamental Research, Colaba, Mumbai, India.
Maiti
N C
NC
Jarori
G K
GK
D019281:Dimerization; D006593:Hexokinase / Q000737:chemistry* / Q000378:metabolism*; D008024:Ligands; D008958:Models, Molecular; D017433:Protein Structure, Secondary; D021122:Protein Subunits; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D013050:Spectrometry, Fluorescence / Q000379:methods; D013997:Time Factors; D014364:Tryptophan / Q000032:analysis*
D008024:Ligands; D021122:Protein Subunits; D014364:Tryptophan; D006593:Hexokinase