Characterization of a monomeric phosphofructokinase from banana — role of magnesium on its regulation

Abstract

A monomeric form of phosphofructokinase (PFK-III) was purified from banana. The enzyme was highly specific for fructose-6-phosphate. However, it displayed a broader specificity towards phosphoryl donor, ATP, UTP, GTP or CTP being equally effective. Heavy metal ions and thiol reagents inhibited the enzyme suggesting the requirement of a SH group for catalytic activity. Well known metabolic effectors of PFK had no effect on the banana enzyme. Citrate at 10 mM inhibited the enzyme by about 60%. Addition of Mg 2+ was essential for activity and among the metal ions tested only Mn 2+ could replace Mg 2+. Kinetic analysis indicated a sequential random mechanism for the enzyme. Though a monomer, initial velocity studies under different experimental conditions suggested: (1) Mg—ATP complex as the phosphorylating substrate and free ATP as the regulator; (2) A dual role for Mg 2+ as a substrate activator and as a modulator of enzyme protein; and (3) Alteration in the affinity of the enzyme towards both the substrates as a result of Mg 2+ binding. Heat inactivation studies indicated that: (a) Mg 2+, by forming a complex with enzyme protein facilitated substrate binding; (b) ATP is bound strongly at the regulatory site; and (c) The binding of Mg—ATP is comparatively weaker.