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3-Phosphoglycerate Kinase (PGK)

(ATP:3-phospho-D-glycerate 1-phosphotransferase; EC 2.7.2.3)
3-Phosphoglycerate kinase (PGK) catylizes the following reaction:
PGK
Glycerate-3-P + ATP {--------} Glycerate-1,3-P₂ + ADP

PGK has a molecular weight of 47,000. This enzyme does not react with AMP, PEP or glycerate-2,3-P₂.

The amount of enzyme required to convert 1 mM of 1,3-diphosphoglycerate to 3-phosphoglycerate per min at pH

6.9 at 25°C.

1. 0.1 M Triethanolamine¿HCl-NaOH buffer, containing 2 mM MgCl 2 and 1 mM EDTA disodium, pH 7.5

2. 200 mM 3-phospho-D-glycerate

3. 100 mM ATP

4. 12 mM NADH in 10 mM Tris.

1. Set the spectrophotometer (equipped with strip chart recorder and temperature control) at 340 nm and 25°C.

2. Into a quartz cuvette pipette the following:

Triethanolamine buffer 2.60 ml

3-phospho-D-glycerate 0.10 ml

ATP 0.05 ml

NADH/Tris 0.05 ml

3. Add 10 µl of GAPDH and incubate at 25°C for five minutes. Record blank rate at 340 nm.

4. Add 0.1 ml enzyme solution to the cuvette. Mix and record the increase in absorbance at 340 nm for 5 minutes.

5. Calculate ΔE340nm/min

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

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