Investigation of the catalytic mechanism of pyruvate phosphate dikinase

Previous studies have shown that the kinetic mechanism of pyruvate phosphate dikinase (PPDK) from B.symbiosus is bi(ATP,P/sub i/) bi(AMP,PP/sub i/) uni(pyruvate) uni(PEP). Recent studies have shown the same mechanism for the P. shermanii enzyme. Dead end inhibitor and alternate substrate studies indicate that ATP and P/sub i/ binding is random but PP/sub i/ is released from the enzyme before AMP. Mn2+ binding to the enzyme was monitored with 9 and 35 GHz esr. The free enzyme binds one Mn2+/subunit at a high affinity site (Ki ∼ 20μM) and a minimum of one Mn2+/subunit at a low affinity sites. Only one Mn2+/subunit binds at a high affinity site on the E x AMPPNP x P/sub i/ complex. Two possible chemical mechanisms have been considered. The first involves the partial reaction E x ATP x P/sub i/ → E x ADP x PP/sub i/ → EP x AMP x PP/sub i/ and the other E x ATP x P/sub i/ → EPP x AMP x P/sub i/ → EP x AMP x PP/sub i/. They have failed to detect enzyme bound ADP at equilibrium and have failed in their attempt to isolate EPP. They also attempted to distinguish between the two mechanisms with exchange Rxs. PPDK will not catalyze [14C]-AMP ↔ AMPPNP exchange in the presence of P/sub i/, will not catalyze [32P]-P/sub i/ ↔ PP/sub i/ exchange in the presence of AMPCPP, and will not catalyze [14C]-AMP ↔ ATP exchange in the presence of methylene phosphate. Rapid quench techniques will be used to detect ADP or EPP formation