Binding of nucleotides AMP and ATP to yeast phosphofructokinase: Evidence for distinct catalytic and regulatory subunits

Abstract

The binding of ATP to yeast phosphofructokinase, as monitored by flow dialysis, is heterogeneous and is adequately described by assuming two independent classes of three binding sites each per enzyme molecule. Under similar conditions, the binding of 5′AMP is homogeneous and a binding stoichiometry of three 5′AMP molecules per enzyme molecule is evaluated. Displacement experiments show that only the ATP molecules bound to the first class of three tight binding sites are displaced by an excess of 5′AMP. Thus, these tight ATP binding sites can be identified as “regulatory sites” in agreement with kinetic data. Furthermore, molecular weight determinations and electrophoresis results are consistent with an heterologous α 3 β 3 structure of the enzyme oligomer. Therefore the present binding data suggest that yeast phosphofructokinase is constituted by three “catalytic” and three “regulatory” subunits.