Photoaffinity labeling and photoaffinity cross-linking of phosphofructokinase-1 from Saccharomyces cerevisiae by 8-azidoadeninenucleotides.
Phosphofructokinase-1 from Saccharomyces cerevisiae is composed of four alpha- and four beta-subunits, each of them carrying catalytic and regulatory bindings sites for MgATP. In this paper, various photoaffinity labels, such as 8-azidoadenosine 5'-triphosphate, 8-azido-1,N6-ethenoadenosine 5'-triphosphate, and 8-N3-3'(2')-O-biotinyl-8-azidoadenosine 5'-triphosphate have been used to study their interaction with the enzyme in the dark and during irradiation. All nucleotidetriphosphates function as phosphate donor forming fructose 1,6-bisphosphate from fructose 6-phosphate. However, the kinetic analysis revealed distinctly differences between them. Photolabeling causes a decrease in enzyme activity to a similar extent, and ATP acts as competitive effector to inactivation. Three bifunctional diazidodiadeninedinucleotides (8-diN3AP4A, monoepsilon-8-diN3AP4A, and diepsilon-8-diN3AP4A) were applied for studying the spatial arrangement of the nucleotide binding sites. No cross-linking of the subunits was obtained by irradiation of the enzyme with 8-diN3AP4A. Photolabeling with diepsilon-8-diN3AP4A resulted in the formation of two alpha-beta cross-links with different mobilities in the SDS-polyacrylamide gel electrophoresis, while monoepsilon-8-diN3AP4A yielded only one alpha-beta cross-link. Because an interfacial location of the catalytic sites between two subunits is less likely, we suggest that the formation of cross-linked subunits may be the result of specific interactions of the bifunctional photolabels with regulatory sites at the interface of both subunits.
Knoche
M
M
Institut für Biochemie, Fachbereich Chemie und Pharmazie, Johannes Gutenberg-Universität Mainz, Germany.
Mönnich
K
K
Schäfer
H J
HJ
Kopperschläger
G
G
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000378:metabolism*; D001665:Binding Sites; D015153:Blotting, Western; D002918:Chymotrypsin / Q000378:metabolism; D003432:Cross-Linking Reagents / Q000737:chemistry / Q000378:metabolism*; D003624:Darkness; D004591:Electrophoresis, Polyacrylamide Gel; D005635:Fructosediphosphates / Q000378:metabolism; D005636:Fructosephosphates / Q000378:metabolism; D007700:Kinetics; D008027:Light; D008274:Magnesium / Q000378:metabolism; D010732:Phosphofructokinase-1 / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D019920:Photoaffinity Labels / Q000737:chemistry / Q000378:metabolism*; D021122:Protein Subunits; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000378:metabolism
D003432:Cross-Linking Reagents; D005635:Fructosediphosphates; D005636:Fructosephosphates; D019920:Photoaffinity Labels; D021122:Protein Subunits; C027618:fructose-6-phosphate; D000255:Adenosine Triphosphate; D010732:Phosphofructokinase-1; C068652:1-phosphofructokinase; D002918:Chymotrypsin; D008274:Magnesium; C029063:fructose-1,6-diphosphate