Characterization of the covalent enzyme intermediates formed during pyruvate phosphate dikinase catalysis

The intermediacy of a pyrophosphorylenzyme (E-PP) and phosphorylenzyme (E-P) in the Clostridium symbiosum pyruvate phosphate dikinase catalyzed interconversion of adenosine 5'-triphosphate (ATP), orthophosphate (P(i)), and pyruvate with adenosine 5'-monophosphate (AMP), inorganic pyrophosphate (PP(i)), and phosphoenolpyruvate (PEP) was examined using transient kinetic techniques. Single-turnover experiments with [gamma-P-32]ATP or [C-14]ATP and PPDK were carried out in the presence and absence of P(i) to test for pyrophosphoryl enzyme and AMP formation, respectively. Formation of the E-PP.AMP complex was found to be followed by P(i) binding and the formation of the E-P.AMP.PP(i) complex. The level of pyrophosphorylenzyme accumulated during a single turnover was found to be dependent on the divalent metal cofactor used (Mn2+ > Co2+ > Mg2+). Single-turnover experiments with [P-32]PEP and PPDK were carried out in the presence and absence of PP(i) and pyruvate to test for phosphorylenzyme formation in the reverse, ATP-forming direction of the reaction. Phosphorylenzyme formed from the reaction of the E.PEP complex was converted in the presence of AMP and PP(i) to free enzyme at a rate exceeding the steady-state turnover rate. The reaction sequence for pyruvate phosphate dikinase was determined to be E + ATP half arrow right over half arrow left E.ATP half arrow right over half arrow left E-PP.AMP half arrow right over half arrow left P(i) E-PP.AMP.P(i) half arrow right over half arrow left E-P.AMP.PP(i) PP(i) half arrow right over half arrow left E-P.AMP AMP half arrow right over half arrow left E-P half arrow right over half arrow left pyruvate E-P.pyruvate half arrow right over half arrow left E-PEP half arrow right over half arrow left E + PEP P-31 NMR analysis of the phosphorylenzyme in the native (-4.0 ppm) and denatured form (-3.9 ppm) revealed a 3-N-phosphohistidine residue. Complexation of Mg2+ resulted in a 0.3 ppm upfield shift of the phosphorus resonance from native phosphorylenzyme while Mn2+ complexation lead to extensive line broadening, indicative of metal cofactor binding in close vicinity to the phosphoryl group. Complexation of the phosphorylenzyme with Mg2+ and oxalate led to extensive line broadening of the P-31 NMR signal, signifying immobilization of the phosphoryl group through coordination to the Mg-oxalate chelate complex.