Intrinsic tryptophan fluorescence of bovine liver adenosine kinase, characterization of ligand binding sites and conformational changes.
Bovine liver adenosine kinase is a 45-kDa monomeric protein which exhibits a characteristic intrinsic tryptophan fluorescence with a maximal excitation at 284 nm and an emission peak centered at 335 nm. A total of three tryptophan residues/molecule has been estimated by using a fluorescence titration method. Low values of Stern-Volmer quenching constants in the presence of either acrylamide or iodide (4.2 M-1 or 1.5 M-1, respectively) indicated that the tryptophan residues are relatively buried in the native molecule. Tryptophan residues also showed a high heterogeneity, with a fractional accessible fluorescence value for iodide of 0.65. The enzyme fluorescence was very sensitive to substrate binding, which induced a marked fluorescence quenching, a lower tryptophan accessibility to acrylamide and iodide, and an increase in the tryptophan heterogeneity. ADP or ATP showed a monophasic saturation curve consistent with the existence of one binding site. In contrast, adenosine and AMP gave biphasic saturation curves, suggesting the existence of at least two binding sites, with a high and a low affinity. The presence of MgCl2 increased the affinity of ATP or ADP, whereas the binding of adenosine or AMP was not affected.
Elalaoui
A
A
Université de Montpellier II Sciences et Techniques du Languedoc, URA 488 du CNRS, France.
Divita
G
G
Maury
G
G
Imbach
J L
JL
Goody
R S
RS
D000241:Adenosine / Q000378:metabolism; D000244:Adenosine Diphosphate / Q000378:metabolism; D000248:Adenosine Kinase / Q000737:chemistry / Q000378:metabolism*; D000249:Adenosine Monophosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D002417:Cattle; D004591:Electrophoresis, Polyacrylamide Gel; D008024:Ligands; D008099:Liver / Q000201:enzymology*; D015636:Magnesium Chloride / Q000494:pharmacology; D008970:Molecular Weight; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence; D014364:Tryptophan / Q000737:chemistry*
D008024:Ligands; D015636:Magnesium Chloride; D000249:Adenosine Monophosphate; D000244:Adenosine Diphosphate; D014364:Tryptophan; D000255:Adenosine Triphosphate; D000248:Adenosine Kinase; D000241:Adenosine