Investigations of the partial reactions catalyzed by pyruvate phosphate dikinase

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Abstract
The kinetic mechanism of pyruvate phosphate dikinase (PPDK) from Bacteroides symbiosus was investigated with several different kinetic diagnostics. Initial velocity patterns were intersecting for AMP/PPi and ATP/Pi substrate pairs and parallel for all other substrate pairs. PPDK was shown to catalyze [14C]pyruvate .dblarw. phosphoenolpyruvate (PEP) exchange in the absence of cosubstrates, [14C]AMP .dblarw. ATP exchange in the presence of Pi/PPi but not in their absence, and [32P]Pi .dblarw. PPi exchange in the presence of ATP/AMP but not in their absence. The enzyme was also shown, by using [.alpha..beta.-18O,.beta.,.beta.-18O2]ATP and [.beta..sbd.18O,.gamma.,.gamma.,.gamma.-18O3]ATP and 31P NMR techniques, to catalyze exchange in ATP between the .alpha..beta.-bridge oxygen and the .alpha.-P nonbridge oxygen and also between the .beta..gamma.-bridging oxygen and the .beta.-P nonbridge oxygen. The exchanges were catalyzed by PPDK in the presence of Pi but not in its absence. These results were interpreted to support a bi(ATP,Pi) bi(AMP,PPi) uni(pyruvate) uni(PEP) mechanism. AMP and Pi binding order was examined by carrying out dead-end inhibition studies. The dead-end inhibitor adenosine 5''-monophosphorothioate (AMPS) was found to be competitive vs AMP, noncompetitive vs PPi, and uncompetitive vs PEP. The dead-end inhibitor imidodiphosphate (PNP) was found to be competitive vs PPi, uncompetitive vs AMP, and uncompetitive vs PEP. These results showed that AMP binds before PPi. The ATP and Pi binding order was studied by carrying out inhibition, positional isotope exchange, and alternate substrate studies. The dead-end inhibitor adenylyl imidodiphosphate (AMPPNP) was shown to be noncompetitive vs Pi, which ruled out an ordered mechanism in which Pi binds first. The positional isotope exchanges rates observed with [.alpha.,.beta.-18O,.beta.,.beta.-18O2]ATP as a function of Pi concentration followed a normal saturation curve, eliminating an ordered mechanism in which ATP binds first. The initial velocities of the PPDK-catalyzed reaction of Pi and of the two alternate substrates arsenate and thiophosphate were measured as a function of ATP concentration. A Lineweaver-Burk plot of these data indicated a random mechanism. Product inhibition studies were carried out by using initial velocity and equilibrium isotope exchange techniques. The results from these studies indicate that the ATP/Pi and AMP/PPi binding steps are at steady state and that AMP forms an abortive complex. Two possible chemical mechanism are described, one of which involves the intermediacies of a covalent pyrophosphoryl-enzyme and a covalent phosphoryl-enzyme and the other of which involves the intermediacies of ADP and a covalent phosphoryl-enzyme.

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