Phosphofructokinase-1 from Saccharomyces cerevisiae: analysis of molecularstructure and function by electron microscopy and self-catalysed affinity labelling

Conventional and cryoelectron microscopy portray native octameric yeast pho sphofructokinase-1 (PFK) as consisting of two identical heterotetrameric te trahedron-like moieties being rotated relative to each other. Immunoelectro n microscopy employing subunit-specific IgG identifies alpha-type subunits in the contact zone of the two tetrahedrons, while P-chains are recognized exclusively at the tips of the octamer. The chemical reaction of phosphofru ctokinase with analogues of fructose 6-phosphate followed by autocatalytic phosphoryl transfer from [gamma-P-32]-ATP results in a specific labelling o f the a-subunit. AMP and fructose 2,6-bisphosphate affect labelling by stim ulating the binding of substrate analogue; AMP additionally promotes phosph oryl transfer. No stimulation of labelling is observed with proteolytically modified tetrameric 12-S phosphofructokinase. (C) 1999 Elsevier Science B. V. All rights reserved.