Methodology for Enzymes

Phosphoenol Pyruvate Carboxylase
(Orthophosphate: Oxaloacetate Carboxylase Phosphorylative EC 4.1.1.31)

This enzyme is involved in the fixation of carbon-dioxide in C4 plants. It has a high affinity to CO2 molecule. The reaction catalyzed by this enzyme is given below.
Phosphoenol Pyruvate + CO2
PEP Case
Oxaloacetic acid


Sorghum, maize, sugarcane etc. are some of C4 plants.





Principle
The oxaloacetic acid formed by the action of PEP case is coupled with MDH and followed by NADH oxidation. Since the sources of PEP case contain enough MDH there is no need to add MDH exogenously.





Materials
» Tris HCl Buffer 0.1M (pH 7.8)
» Magnesium Chloride (10mM) 203.5mg/50mL water.
» Sodium Bicarbonate (10mM) 84mg/100mL water
» Phosphoenolpyruvate (5mM) 23.28mg/100mL water
» NADH (0.4mM) 5.32mg/10mL water
» Enzyme extract - see under MDH


Procedure
1.  Mix various recipes as shown below:
Sr. No.

Reagents

Test
mL
Blank
mL
1
Tris-HCl buffer pH 7.8
0.8
1.3
2
Magnesium chloride
0.5
0.5
3
Sodium bicarbonate
0.5
0.5
4
Phosphoenol pyruvate
0.5
0.5
5
Enzyme extract
0.2
0.2
6
NADH
0.5
0
2.  Proceed steps 2 to 4 as in MDH
.

Calculation
Calculate as for MDH.



References
1.  Ting I P and Osmond, C B (1973) Plant Physiol 51 439.
2.  Sadasivam, S and Gowri, G (1981) Photosynthetica 15 453.