Photoaffinity Labeling and Photoaffinity Cross-Linking of Phosphofructokinase-1 from Saccharomyces cerevisiae by 8-Azidoadeninenucleotides

Abstract

Phosphofructokinase-1 from Saccharomyces cerevisiae is composed of four α- and four β-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, monoϵ-8-diN3AP4A, and diϵ-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 diϵ-8-diN3AP4A resulted in the formation of two α-β cross-links with different mobilities in the SDS–polyacrylamide gel electrophoresis, while monoϵ-8-diN3AP4A yielded only one α-β 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.