Mechanism of action of a dimeric phosphofructokinase from banana: role of magnesium on its kinetics and regulation

Abstract

The dimeric form of banana phosphofructokinase (PFK-II) was purified by polyethylene glycol and ammonium sulphate fractionations, Sepharose 6B molecular sieve and DEAE Sephacell ion exchange chromatography to 72-fold with a yield of 45%. Initial velocity studies suggested that the enzyme followed a sequential ordered reaction mechanism. Heat inactivation studies with substrate and effectors provided evidence for the formation of a stable enzyme-magnesium complex, strong binding of ATP to the enzyme and a comparatively weaker binding of the ATP-Mg complex. Kinetic studies under different experimental conditions strongly suggested (1) the Mg-ATP complex as the phosphorylation substrate, (2) free ATP as the regulator, (3) a dual role for Mg 2+ as an activator of substrate as well as an enzyme modulator, and (4) ATP dependent formation of the enzyme-Mg 2+ complex which modulated the interaction between fructose 6-phosphate and the enzyme.