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

Abstract

1. Sugar-cane leaf pyruvate,P_i 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 P_i into phosphoenolpyruvate, AMP and PP_i. 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 PP_i 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 PP_i were derived from P_i and from the terminal phosphate of ATP. 7. Isotope-exchange studies indicated that the reaction proceeds in the following steps: Enzyme+ATP+P_i ⇌ Enzyme–P+AMP+PP_i Enzyme–P+pyruvate ⇌ Enzyme+phosphoenolpyruvate