CHEM 3453, Homework 4 Student Name:

30 Points
Due Date: 3/21/2021

Problem 4.1 (8 Points, Tinoco, Problem 4.2).

(a) In frog muscle rectus abdominis, the concentrations of ATP, ADP, and phosphate (Pi) are 1.25 ×
10−3 M, 0.50 × 10−3 M, and 2.5 × 10−3 M, respectively. Calculate the Gibbs free energy change
∆r G for the hydrolysis of ATP.

ATP + H2 O −→ ADP + Pi

in muscle. Take the temperature and pH of the muscle as 25◦ C and 7, respectively. The standard
reaction free energy for ATP hydrolysis is -31 kJ/mol.

[ADP ][P i]
∆r G = ∆r G◦ + RT lnQ = ∆r G◦ + RT ln
[AT P ]
0.50 ∗ 10−3 ∗ 2.5 ∗ 10−3
= −31 + 8.314 × 10−3 ∗ 298.15 ∗ ln
1.25 ∗ 10−3
= −31 + 8.314 × 10 ∗ 298.15 ∗ ln1.0 ∗ 10−3
−3

= −48.1 kJ/mol

(b) In muscle, the enzyme creatine phosphokinase catalyzes the following reaction:

Phosphocreatine + ADP −→ creatine + ATP

Calculate the standard free energy change and equilibrium constant for this reaction at 25◦ C. Note
that the standard Gibbs free energy of hydrolysis of phosphocreatine

Phosphocreatine + H2 O −→ creatine + Pi

at 25◦ C and pH 7 is -43.1 kJ/mol.

1) Phosphocreatine + H2 O −→ creatine + Pi, ∆r G◦ = −43.1 kJ/mol

2) ATP + H2 O −→ ADP + Pi, ∆r G◦ = −31 kJ/mol
1) − 2) Phosphocreatine + ADP −→ creatine + ATP, ∆r G◦ = −12.1 kJ/mol

∆r G◦
   
−12.1
Keq = exp − = exp − = 131.8
RT 8.314 ∗ 10−3 ∗ 298.15

1
Problem 4.2 (8 Points, Tinoco, Problem 4.3).

(a) An important step in the glycolytic path is the phosphorylation of glucose by ATP, catalyzed by the
enzyme hexokinase and Mg2+ :

Mg2+ , hexokinase
glucose + ATP −
)−
−−−−
−−−−
−−−−
−−−−
−−*
−− glucose − 6 − P + ADP

with a standard free energy of reaction of -16.7 kJ/mol at 25◦ . In the equal concentration of ADP
and ATP, the phosphorylation of glucose is allowed to proceed to equilibrium. What is the ratio
(glucose-6-P)/(glucose)?

∆ r G◦
   
−16.7
Keq = exp − = exp − = 843.1
RT 8.314 ∗ 10−3 ∗ 298.15
[glucose − 6 − P ][ADP ]
=
[glucose][AT P ]
[glucose−6−P ]
At equal concentration of ADP and ATP, [glucose] is equal to 843.1.

(b) In the absence of ATP, glucose-6-P is unstable at pH 7. In presence of the enzyme glucose-6-
phosphatase, it hydrolyzes to give glucose:
G−6−phosphatase
glucose − 6 − P + H2 O −
)−
−−
−−
−−
−−
−−
−−
−−
−−
−−
−*
− glucose + phosphate

Compute the free energy change and equilibrium constant for this reaction. Combine the reaction in
part (a) and ATP hydrolysis (∆r G◦ = -31 kJ/mol).

Mg2+ , hexokinase
1) glucose + ATP −−
)−−−−
−−−−
−−−−
−−−−
−−*
−− glucose − 6 − P + ADP, ∆r G◦ = −16.7 kJ/mol
2) ATP + H2 O −→ ADP + Pi, ∆r G◦ = −31 kJ/mol
G−6−phosphatase
2) − 1) glucose − 6 − P + H2 O −−
)−−
−−
−−
−−
−−
−−
−−
−−
−−
−*
− glucose + phosphate, ∆r G◦ = −14.3 kJ/mol

∆ r G◦
   
−14.3
Keq = exp − = exp − = 320.2
RT 8.314 ∗ 10−3 ∗ 298.15

2
Problem 4.3 (8 Points, Tinoco, Problem 4.13). You want to make a pH 7.0 buffer using NaOH and
phosphoric acid. The sum of the concentrations of all phosphoric acid species is 0.300 M. At 25◦ , the
equilibrium constants for concentrations given in apparent units of mol/L are:

) H+ + H2 PO−
H3 PO4 * 4 K1 = 7.1 × 10−3
− * +
H2 PO4 ) H + HPO4 2−
K2 = 6.2 × 10−8
HPO2− * + 3−
4 ) H + PO4 K3 = 4.5 × 10−13

(a) Compute the concentrations of all the buffer species.

[H + ][H2 P O4− ] [H+]

K1 = , [H3 P O4 ] = [H2 P O4− ] = 1.41 × 10−5 [H2 P O4− ]
[H3 P O4 ] K1
[H + ][HP O42− ] K2
K2 = , [HP O42− ] = [H2 P O4− ] = 6.2 × 10−1 [H2 P O4− ]
[H2 P O4− ] [H + ]

[H + ][P O43− ] K3
K3 = , [P O43− ] = [HP O42− ] = 4.5 × 10−6 [HP O42− ] = 2.79 × 10−6 [H2 P O4− ]
[HP O42− ] [H + ]
Adding the concentration of all phosphoric acid species

[H3 P O4 ] + [H2 P O4− ] + [HP O42− ] + [P O43− ] = 1.62[H2 P O4− ] = 0.3

So, the concentration of individual phosphoric acid species are

• [H2 P O4− ] = 0.185M

• [H3 P O4 ] = 1.41 × 10−5 [H2 P O4− ] = 2.61 × 10−6 M
• [HP O42− ] = 6.2 × 10−1 [H2 P O4− ] = 0.115M
• [P O43− ] = 2.79 × 10−6 [H2 P O4− ] = 5.17 × 10−7 M

and the sodium concentration is

• [N a+ ] = [H2 P O4− ] + 2 ∗ [HP O42− ] + 3 ∗ [P O43− ] = 0.415M

(b) Use the data in the Table below to calculate K2 at 37◦ C.

reaction pK ∆r H ◦
(in kJ/mol)
H3 PO4 *
) H+ + H2 PO− 4 2.16 -7.95
− * + 2−
H2 PO4 ) H + HPO4 7.21 4.15
HPO2−
4
*
) H+ + PO3−4 12.32 14.7

∆r H 0 1
   
K2,37 1
ln = − −
K2,25 R T2 T1
 
4.15 1 1
= − −
8.314 × 10−3 310.15 298.15
= 0.0648
K2,37
= 1.067
K2,25
K2,37 = 1.067 × K2,25 = 6.62 × 10−8 M

3
Problem 4.4 (6 Points, Tinoco, Problem 6.5). The binding of drug A to protein B was studied by
equilibrium dialysis. In each measurement a 1.00 × 10−6 M solution of the macromolecule was dialyzed
against an excess amount of a solution containing A. After equilibrium was reached, the total concentra-
tion of A on each side of the dialysis membrane was measured using a UV spectrometer. The following
data were obtained for the total concentration of drug A in µM:

Side without protein B Side with protein B

5.1 6.7
10.2 12.8
20.1 23.4
52.2 56.2
105.0 109.1
200.0 204.7

Calculate v for each concentration of drug A. Make a Scatchard plot and evaluate the intrinsic
equilibrium constant Keq and the total number of binding sites per protein B. Evaluate if the independent-
and-identical-sites model is applicable.

From the free and total concentration of the ligand

listed above, we can compute
[A]f ree [A]tot [A]bound v v/[A]
• the concentration of bound ligand,
(µM) (µM) (µM) (105 M−1 )
[A]bound = [A]tot − [A]f ree
5.1 6.7 1.6 1.6 3.137
• number of ligand molecules bound to each macro-
molecule v = [A]bound /[M ] = 106 [A]bound 10.2 12.8 2.6 2.6 2.549

• v/[A] = v/[A]f ree 20.1 23.4 3.3 3.3 1.642

52.2 56.2 4.0 4.0 0.766
All values are tabulated to the right. From these data,
we can make the Scatchard plot. Within experimental 105.0 109.1 4.1 4.1 0.390
error, the data can be represented by a straight line with
200.0 204.7 4.7 4.7 0.235
a slope of 1.03 × 105 M−1 and an intercept with the v-
axis of 5. Thus, the data are in reasonable agreement
with an identical and independent sites model. The in-
trinsic binding constant is 1.03 × 105 M−1 , and there
are about 5 sites per macromolecule.