Photoaffinity labeling and photoaffinity cross-linking of phosphofructokinase-1 from Saccharomyces cerevisiae by 8-azidoadeninenucleotides

Phosphofructokinase-1 from Saccharomyces cerevisiae is composed of four alp ha- and four beta -subunits, each of them carrying catalytic and regulatory bindings sites for MgATP. In this paper, various photoaffinity labels, suc h as 8-azidoadenosine 5'-triphosphate, 8-azido-1,N-6-ethenoadenosine 5'-tri phosphate, and 8-N-3-3'(2')-O-biotinyl-8-azidoadenosine 5'-triphosphate hav e been used to study their interaction with the enzyme in the dark and duri ng irradiation. All nucleotidetriphosphates function as phosphate donor for ming fructose 1,6-bisphosphate from fructose 6-phosphate, However, the kine tic analysis revealed distinctly differences between them. Photolabeling ca uses a decrease in enzyme activity to a similar extent, and ATP acts as com petitive effector to inactivation. Three bifunctional diazidodiadeninedinuc leotides (8-diN(3)AP(4)A, mono epsilon -8-diN(3)AP(4)A, and di epsilon -8-d iN(3)AP(4)A) were applied for studying the spatial arrangement of the nucle otide binding sites. No cross-linking of the subunits was obtained by irrad iation of the enzyme with 8-diN(3)AP(4)A. Photolabeling with di epsilon -8d iN(3)AP(4)A resulted in the formation of two alpha-beta crosslinks with dif ferent mobilities in the SDS-polyacrylamide gel electrophoresis, while mono epsilon -8-diN(3)AP(4)A yielded only one alpha-beta cross-link. Because an interfacial location of the catalytic sites between two subunits is less l ikely, we suggest that the formation of cross-linked subunits may be the re sult of specific interactions of the bifunctional photolabels with regulato ry sites at the interface of both subunits. (C) 2001 Academic Press.