Properties and mechanism of action of pyruvate, phosphate dikinase from leaves

Abstract

1. Sugar-cane leaf pyruvate,Pi dikinase was prepared free of enzymes that would interfere with studies on the stoicheiometry and mechanism of the reaction it catalyses. The reaction was unequivocally shown to involve the conversion of equimolar amounts of pyruvate, ATP and Pi into phosphoenolpyruvate, AMP and PPi. 2. The purified enzyme was stable at pH8·3 only if stored at about 20° in the presence of Mg2+ and a thiol-reducing reagent, care being taken to prevent the oxidation of the thiol. 3. The apparent Michaelis constants for phosphoenolpyruvate and PPi were 0·11mm and 0·04mm respectively and that for AMP was less than 4μm. 4. At pH8·3 the initial velocity of the reaction was about 6 times as fast in the direction towards phosphoenolpyruvate synthesis as in the reverse direction. 5. With the exception of ATP, all the products of the reaction in both directions were inhibitory. 6. The phosphate groups of PPi were derived from Pi and from the terminal phosphate of ATP. 7. Isotope-exchange studies indicated that the reaction proceeds in the following steps: Enzyme+ATP+Pi ⇌ Enzyme–P+AMP+PPi Enzyme–P+pyruvate ⇌ Enzyme+phosphoenolpyruvate