Differences in nucleotide-binding site of isoapyrases deduced from tryptophan fluorescence.
Comparative studies of intrinsic and extrinsic fluorescence of apyrases purified from two potato tuber varieties (Pimpernel and Desirée) were performed to determine differences in the microenvironment of the nucleotide binding site. The dissociation constants (K(d)) of Pimpernel apyrase for the binding of different fluorescent substrate analogs: methylanthranoyl (MANT-), trinitrophenyl (TNP-), and epsilon -derivatives of ATP and ADP were determined from the quenching of Trp fluorescence, and compared with K(d) values previously reported for Desirée enzyme. Binding of non-fluorescent substrate analogues decreased the Trp emission of both isoapyrases, indicating conformational changes in the vicinity of these residues. Similar effect was observed with fluorescent derivatives where, in the quenching effect, the transfer of energy from tryptophan residues to the fluorophore moiety could be additionally involved. The existence of energy transfer between Trp residues in the Pimpernel enzyme was demonstrated with epsilon -analogues, similar to our previous observations with the Desirée. From these results we deduced that tryptophan residues are close to or in the nucleotide binding site in both enzymes. Experiments with quenchers like acrylamide, Cs(+) and I(-), both in the presence and absence of nucleotide analogues, suggest the existence of differences in the nucleotide binding site of the two enzymes. From the results obtained in this work, we can conclude that the differences found in the microenvironment of the nucleotide binding site can explain, at least in part, the kinetic behaviour of both isoenzymes.
Espinosa
V
V
Departamento de Bioquímica y Biología Molecular, Facultad de Ciencias Químicas y Farmacéuticas, Universidad de Chile, Santiago, Chile.
Kettlun
A M
AM
Zanocco
A
A
Cardemil
E
E
Valenzuela
M A
MA
D020106:Acrylamide / Q000737:chemistry; D000244:Adenosine Diphosphate / Q000031:analogs & derivatives / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism; D001081:Apyrase / Q000737:chemistry / Q000378:metabolism*; D001665:Binding Sites; D002586:Cesium / Q000737:chemistry; D007454:Iodides / Q000737:chemistry; D007527:Isoenzymes / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D009711:Nucleotides / Q000737:chemistry / Q000378:metabolism*; D038761:Photobleaching; D011198:Solanum tuberosum / Q000737:chemistry / Q000201:enzymology*; D013050:Spectrometry, Fluorescence; D013379:Substrate Specificity; D014364:Tryptophan / Q000737:chemistry*
D007454:Iodides; D007527:Isoenzymes; D009711:Nucleotides; D002586:Cesium; D020106:Acrylamide; D000244:Adenosine Diphosphate; D014364:Tryptophan; D000255:Adenosine Triphosphate; D001081:Apyrase