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Phospho(enol) Pyruvate Carboxylase (PEPC)

(Orthophosphate:oxalacetate carboxylase-[phosphoralating]; EC 4.1.1.31)

Phospho(enol)pyruvate carboxylase catalyzes the following reaction:

           PEPC
PEP + CO2 ------> oxalacetate + Pi

Carboxylation of PEP to yield OAA and inorganic phosphate (Pi) was first observed in extracts of spinach leaves and reported by Bandurski and Greiner in 1953 (Bandurski, R.S., and Greiner, C.M., J. Biol. Chem., 204, 781, 1953). In further studies, Bandurski (Bandurski, R.S., J. Biol Chem., 217, 137, 1955) purified the enzyme some tenfold and established the basic properties of the enzyme and the reaction. Shortly thereafter, Vennesland and her associates (Tehen, T.T., and Vennesland, B., J. Biol Chem., 213, 533, 1955; Mazelis, M., and Vennesland, B., Plant Physiol. , 32, 591, 1958) showed that PEPC is widely distributed in plants.

Almost from the time of its discovery, PEPC has been assigned a physiological role as the catalyst for a dark CO2 fixation reaction in certain types of plants. It has been recognized for some time (Vickery, H.B., J. Biol. Chem., 205, 369, 1953) that Crassulacean and some other succulent plants accumulate large amounts of carboxylic acids, notably malic acid, as a result of CO2 fixation reactions which occur in the dark.

That amount of enzyme which forms one micromole of oxalacetate from phospho(enol)pyruvate and CO2 at pH 8.0 and 37°C.

0.05 M Tris (hydroxymethyl) aminomethane buffer, pH 8.0 containing the following:
Phosphoenolpyruvate, monocyclohexylammonium salt 2.0 mM
Magnesium Sulfate 10.0 mM
Sodium Bicarbonate 10.0 mM
NADH Disodium salt 0.14 mM
MDH 50 U/ml
Enzyme Solution:
Approximately 1 U/ml in 0.05 M Tris/HCl, pH 7.0. Allow to come to room temperature before use.

Set the spectrophotometer (equipped with strip chart recorder and temperature control) at 340 nm and 37°C.
Into a 1.0 cm cuvette pipette 2.90 ml substrate and incubate in the spectrophotometer at 37°C for five minutes. Record blank if any.
Add 0.1 ml enzyme solution, mix and record change in absorbance at 340 nm for 5-10 minutes.

Activity (U/mg) =	(ΔE_340nm/min)(Total Vol.)(Enz. Diln.)
	(6.22)(Enz. Vol.)(mg Enz./ml)

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