The energetically difficult production of phosphoenolpyruvate from pyruvate and ATP is overcome by use of both high-energy phosphoanhydride groups in ATP. The value of AG0' for hydrolysis of MgATP to MgADP and P: is -7.8 kcal mol-1, and for hydrolysis to AMP and MgPPb it is -10.3 kcal mol-1 (Arabshahi and Frey, 1998). The energy of both phosphoanhydride bonds is used in the action of pyruvate phosphate dikinase (EC 18.104.22.168) to produce phosphoenolpyruvate from pyruvate and ATP in bacteria and plants. The overall reaction is described by eq. 10-9.
Phosphoenolpyruvate + MgPP; + AMP + 2H
As the reaction stands in eq. 10-9, the free energy available from the cleavage of ATP into AMP and PPi is AG'0 = -10.3 kcal mol-1 (Frey and Arabshahi, 1998), whereas -14 kcal mol-1 are required to phosphorylate pyruvate. On this basis, the reaction is not spontaneous as written (AG°' = +3 kcal mol-1). However, the hydrolysis of PP; by inorganic pyrophosphatase will generate an additional -4.6 kcal mol-1 (Jencks and Regenstein, 1970), making the production of phosphoenolpyruvate spontaneous in vivo.
The enzymatic mechanism follows a stepwise course according to eqs. 10-10a to 10-10c (Wood et al., 1977). Because the three steps (a, b, and c) take place independently, the covalent intermediates E-PPMg and E-P can be isolated.
E + MgATP -, " E-PPMg.AMP - " E—PPMg + AMP (1040a)
E—PPMg + Pj ^ ' E—PPMg.Pj -, " E—P.MgPPj ^ " E—P + MgPPj
E—P + Pyruvate " E—P.Pyruvate -. E + Phosphoenolpyruvate (1040c)
Moreover, the enzyme catalyzes the exchange of [14C]AMP into ATP, by virtue of eq. 10-10a, and of 32PP into ATP, by virtue of eqs. 10-10a and 10-10b. Reaction of the enzyme with MgATP produces E-PPMg; reaction of E-PPMg with P: produces E-P; and reaction of E-P with pyruvate produces phosphoenolpyruvate. The pyrophosphoryl and phosphoryl groups are bonded to a histidine residue, and the steady-state kinetics is hexa-uni ping pong, scheme 10-5 (Thrall et al., 1993).
ATP AMP Pj PP. Pyruvate P-enolpyruvate
E E.ATP^^ E-PP.AMP E-PP E-PP.Pj E-P.PPj E-P E-P.Pyruvate E
The chemical mechanism in fig. 10-6 traces the fates of the and y-phosphoryl groups in the reaction, in which the P-phosphoryl of ATP becomes the phosphate group in phosphoenolpyruvate, and the y-phosphoryl group is transferred to phosphate in the formation of pyrophosphate. This is one of the few examples of enzymatic nucleophilic substitution at the P-phosphorus of ATP. The chemistry of the process is outlined in fig. 10-6, which shows how the P-phosphate of ATP becomes covalently bonded to the active site histidine
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