Coupled reactions in glycolysis

Coupled reactions in glycolysis
1. Background and theory
The aim of this exercise is to illustrate some of the reactions of glycolysis and how the
thermodynamically unfavourable reactions can be driven by coupling to favourable
reactions. In addition, the use of spectrophotometry to follow the formation or
consumption of NADH is demonstrated.
The reactions under study are:
step 1 of glycolysis:
step 6 of glycolysis:
Glyceraldehyde -
phosphate
dehydrogenase
(GAPDH)
Glyceraldehyde - 3 - phosphate + P i
+ NAD + ← ⎯⎯ ⎯⎯ ⎯⎯ ⎯⎯ ⎯⎯ ⎯⎯ →
1,3 bisphosphoglycerate +NADH
€
step 7 of glycolysis:

In summary:
The optical spectra of NAD + and NADH are shown in figure 1 below. The different
spectra allow the monitoring of the GAPDH reaction where NADH is formed, with
subsequent increase of the absorbance at 340 nm. The other reactions are followed
indirectly from their effect on the equilibrium position of the GAPDH reaction.
If we, for example, start with a NAD + , GAP and phosphate, and initiate the reaction by
the addition of GAPDH, we will observe formation of NADH as the equilibrium is
established. If Mg-ADP and PGK is added at this point, more NADH will be formed
since 1,3-PGA is consumed in the PGK reaction. The reaction stops when both equilibria
are established. More details about the reaction mechanism of GAPDH and the
thermodynamics of glycolysis in Lehninger , pp. 536 and 553.
Figure 1. Optical spectra of NADH and NAD + . The molar absorptivity
(extinction coefficient) of NADH at 340 nm is 6.22 x 10 3 M -1 cm -1
(from Lehninger. P. 517)

2. Materials
2.1 Equipment
Spectrophotometer (preferably a double beam instrument) with recorder.
1 ml quartz cuvette
Pipettors
2.2 Chemicals
2.2.1 Solutions to be prepared by the student
100 mM Tris-HCl pH 7.4 containing 0.5 mM EDTA 100 ml
300 mM sodium phosphate buffer, pH 7.4 50 ml
100 mM MgCl 2 (aqueous) 10 ml
2 mM ADP in Tris buffer 10 ml
2 mM NAD in Tris buffer 10 ml
100 mM glucose (aqueous) 10 ml
0.17 M Sodium arsenate, pH 8.5
Tris buffer: Dissolve the calculated amounts of Tris and Na 2 EDTA in about 80 ml water,
adjust pH to 7.4 with 5 M HCl (small additions when pH approaches the desired value)
and dilute to 100 ml.
Phosphate buffer: dissolve the calculated amount of NaH 2 PO 4 in 40 ml water and adjust
pH to 7.4 with 5 M NaOH; dilute to 50 ml with water.

2.2.2 Solutions available
about 30 mM D,L-glyceraldehyde phosphate
(note that only the D-form serves as substrate!)
about 70 µM glyceraldehyde phosphate dehydrogenase
about 100 µM hexokinase
about 1 µM phosphoglyceratkinas
GAP
GAPDH
HK
PGK
All solutions except the Tris buffer should be kept on ice

3. Procedure
The reactions are monitored by the absorbance at 340 nm where NADH absorbs. Prepare
the substrate mixture in the cuvette and place it in the spectrophotometer. Zero the
instrument and start recording. Record the base line a few minutes, and then make
additions as indicated in the description of the experiments. Be careful to allow the
reaction come to equilibrium after each addition (note that this can take up to 20 min);
note however that all additions do not result in a reaction. Be careful to mix the contents
of cuvette after each addition. Always use a clean pipette tip to avoid any contaminations
of the reagents.
Experiment 1
As a starter, we investigate the complete reaction system. Set the instrument to a total
recording time of about 30 min and set the scale to use 1 absorbance unit corresponding
full scale.
Substrate
Additions
Tris Buffer 805 µl 1. GAPDH 25µl
NAD 100 µl 2. PGK 10 µl
Phosphate buffer 10 µl 3. HK 10 µl
ADP 10 µl
MgCl 2 50 µl
Glucose 25 µl
GAP 40 µl
Then we change the order of the additions to be sure that the system works according to
the description.

Experiment 2
Substrate
Additions
Tris Buffer 950 µl 1. GAPDH 25µl
NAD 100 µl 2. PGK 10 µl
Phosphate buffer 10 µl 3. HK 10 µl
ADP 10 µl 4. Glucose 25 µl
MgCl 2 50 µl
GAP 40 µl
Experiment 3 (change the scale to about 0.2 absorbance units full scale)
Substrate
Additions
Tris Buffer 790 µl 1. GAPDH 25µl
NAD 100 µl 2. PGK 10 µl
Phosphate buffer 10 µl 3. MgCl 2 50 µl
ADP 100 µl
GAP 40 µl
Experiment 4
Substrate
Additions
Tris Buffer 790 µl 1. GAPDH 25µl
NAD 100 µl
Phosphate buffer 10 µl
GAP 40 µl
Arsenate 100 µl

4.
Documentation and reporting
All experimental work is to be recorded in the laboratory notebook by each student. Each
group submits a summary of the observations in experiment 1-4 and a calculation of ∆G o '
for the GAPDH-reaction (compare the result with the literature value in Lehringer
table 14-2). Discuss briefly the effect of arsenate in experiment 4.
The submission of a laboratory complete report will be assigned to some groups. The
report should consist of the following sections:
- Summary
- Introduction (background and aim with the experiments)
- Materials and methods
- Results
- Discussion. This section should also include a summary of the results obtained by
the other groups.