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141763
Advanced Biochemistry assignment

3. Rate Enhancement by Urease The enzyme urease enhances the rate of urea hydrolysis at pH
8.0 and 20 0C by a factor of 1014. If a given quantity of urease can completely hydrolyze a given
quantity of urea in 5.0 min at 20 8C and pH 8.0, how long would it take for this amount of urea
to be hydrolyzed under the same conditions in the absence of urease? Assume that both reactions
take place in sterile systems so that bacteria cannot attack the urea.

Answer:
Time to hydrolyze urea = 5min*1014 / (60 min/h)(24 h/day)(365 days/year)
= 9.5 × 108 years = 950 million years

7. Effect of Enzymes on Reactions Which of the listed effects would be brought about by any
enzyme catalyzing the following simple reaction? S ↔P    where    K¿ eq = [P] /[S]
(a) Decreased K eq;
(b) increased k1;
(c) increased K eq;
(d) increased delta G‡ ;
(e) decreased deltaG‡ ;
(f) more negative deltaG0;
(g) increased k2.

Answer is B+E+G .
E is correct because enzymes lower activation energy required in reactions. B+G are both correct
because enzymes don’t change equilibrium in reactions, and catalyze it in both directions.
8. Relation between Reaction Velocity and Substrate Concentration: Michaelis-Menten Equation
(a) At what substrate concentration would an enzyme with a kcat of 30.0s and a Km of 0.0050 M
operate at onequarter of its maximum rate?
(b) Determine the fraction of Vmax that would be obtained at the following substrate
concentrations [S]: ½Km, 2Km, and 10Km.
(c) An enzyme that catalyzes the reaction X ↔ Y is isolated from two bacterial species. The
enzymes have the same Vmax, but different Km values for the substrate X. Enzyme A has a Km
of 2.0 M, while enzyme B has a Km of 0.5 M. The plot below (see Lehninger) shows the kinetics
of reactions carried out with the same concentration of each enzyme and with [X] = 1 M. Which
curve corresponds to which enzyme

Answer
(a) Calculate [S] when V0 = 0.25 Vmax.

According to The Michaelis-Menten equation:

V0 = Vmax[S]/(Km + [S])
According to data, V0 = Vmax when [S]/(Km + [S]) = 0.25
[S] = 1/3 Km
[S] =0.33Km = 0.33(0.0050 M) = 1.7 * 103 M

(b) The Michaelis-Menten equation:

V0/Vmax = [S]/(Km + [S])
[S] = ½ Km, substituting it into the equation results in;
V0 /Vmax = 0.5 Km/1.5Km
V0 /Vmax = 0.33

Also, if we substitute [S] = 2Km;

V0 /Vmax = 2 Km/ (Km+ 2 Km)
V0/Vmax= 0.67
If we substitute [S] = 10Km;
V0 /Vmax = 10 Km/ (Km+ 10 Km)
V0 /Vmax = 0.91

(c) The red curve is the one for enzyme B (because [X] is greater than the Km for B), and the
black curve is for enzyme A.
9. Applying the Michaelis-Menten Equation I
A research group discovers a new version of happyase, which they call happyase*, that catalyzes
the chemical reaction HAPPY ↔ SAD
The researchers begin to characterize the enzyme.
(a) In the first experiment, with [Et] at 4 nM, they find that the Vmax is 1.6Ms. Based on this
experiment, what is the kcat for happyase*? (Include appropriate units.)
(b) In another experiment, with [Et]at 1nM and [HAPPY] at 30 M, the researchers find that
V0=300nMs. What is the measured Km of happyase* for its substrate HAPPY? (Include
appropriate units.)
(c) Further research shows that the purified happyase* used in the first two experiments was
actually contaminated with a reversible inhibitor called ANGER. When ANGER is carefully
removed from the happyase* preparation and the two experiments repeated, the measured Vmax
in (a) is increased to 4.8M s1 , and the measured Km in (b) is now 15 M.
For the inhibitor ANGER, calculate the values of alpha and alpha’.
(d) Based on the information given above, what type of inhibitor is ANGER?

Answer
(a) Using the equation kcat= Vmax/[Et] = 1600 nM s-1/4 nM = 400 s-1
(b) Vmax= Kcat* [Et]
When [Et] = 1nM, Vmax= 400nMs-1
V0/Vmax = 300 nMs-1 / 400 nMs-1 = ¾

Replacing V0/Vmax with ¾ in the Michaelis Menten equation

V0/Vmax = [S]/(Km + [S])
¾ = [S]/(Km + [S])
Km= [S]/3
Since [S] is the concentration of the substrate Happy, which = 30M
Then Km = 30/3 = 10

( c) Vmax variations are a function of Vmax/a'. Because Vmax increased by a factor of 3, a'= 3.
 Also, Km varies as a function of aKm/a'. Because Km increased by * 1.5 ( when ANGER was
removed (So the inhibitor decreased Km by 2/3) and a' = 3, then a=2.
(d) Since ANGER affected alpha and alpha’, then it’s a mixed inhibitor.

10. Applying the Michaelis-Menten Equation II

An enzyme is found that catalyzes the reaction A ↔ B

Researchers find that the Km for the substrate A is 4 M, and the kcat is 20 min–1.

(a) In an experiment, [A] = 6 mM, and V0 = 480 nMmin. What was the [Et] used in the experiment?

(b) In another experiment, [Et] = 0.5 μM, and the measured V0 = 5 μM min. What was the [A] used in
the experiment?

(c) The compound Z is found to be a very strong competitive inhibitor of the enzyme, with an alpha of
10. In an experiment with the same [Et] as in (a), but a different [A], an amount of Z is added that
reduces V0 to 240 nM min. What is the [A] in this experiment?

(d) Based on the kinetic parameters given above, has this enzyme evolved to achieve catalytic
perfection? Explain your answer briefly, using the kinetic parameter(s) that define catalytic perfection.

ANSWER
a) Because [S] is more than a 1000 times greater than Km, I will assumed the measured rate
of the reaction to be Vmax.
Using the equation Vmax = Kcat [Et]
Then [Et] = Vmax/ Kcat = 480nMmin-1/ 20 min-1 = 24 nM.

b) At [Et] = 0.5 μM , Vmax = Kcat * [Et] = 20 min-1 * 0.5 μM, so Vmax= 10 μM min-1
Km = [A], when V0= ½ Vmax …
According to the question, V0= 5 μM min-1 , Which is exactly half of Vmax
So [A] = Km
[A] = 4 μM

c) Because [Et] is the same as in (a), then Vmax= 480 nMmin-1

V0= 240 nMmin-1 ( data from question), which is exactly half of Vmax
So [A] = Km.
When there is an inhibitor with alpha = 10, then Km = 40 μM = [A]

d) No it did not. Kcat/ Km= 0.33/(4*10-6 M-1S-1) = 8.25 * 104 M-1S-1. This is below the
diffusion controlled limit.
11. Estimation of Vmax and Km by Inspection
Although graphical methods are available for accurate determination of the Vmax and Km of an
enzyme-catalyzed reaction (see Box 6–1), sometimes these quantities can be quickly estimated
by inspecting values of V0 at increasing [S]. Estimate the Vmax and Km of the enzyme-
catalyzed reaction for which the following data were obtained.
[S] (M) V0 (M/min)
2.5 10–6 28
4.0 10–6 40
1 10–5 70
2 10–5 95
4 10–5 112
1 10–4 128
2 10–3 139
1 10–2 140

Answer
The velocity didn’t change significantly when [S] increased from 2*10-2 to 1 *10-2, which is an
increase *5. From this, I estimated that Vmax is 140 mM/min.
Km = [S] at ½ Vmax.
½ Vmax = 70 mM/min.
From the table, [S] at 70 mM/min = 1* 10-5 M
So Km= 1* 10-5 M

13. Graphical Analysis of Vmax and Km. The following experimental data were collected during
a study of the catalytic activity of an intestinal peptidase with the substrate glycylglycine:
Glycylglycine +H2O → 2 glycine
[S] (mM) Product formed (micromol/min)
1.5 0.21
2.0 0.24
3.0 0.28
4.0 0.33

8.0
0.40
16.0 0.45
Use graphical analysis ( See Box 6-1) to determine Km and Vmax for this enzyme preparation
and substrate.
Answer
I calculated 1/V0 and 1/[S] from data. These would be plotted on the Lineweaver Burk plot:
V0 1/V0 [S] 1/ [S]
0.21 4.8 1.5 0.67
0.24 4.2 2 0.5

0.28 3.6 3 0.33

0.33 3 4 0.25
0.40 2.5 8 0.13
0.45 2.2 16 0.06

The intercept on Horizontal axis = -1/Km

The intercept on the vertical axis = 1/Vmax
-1/Km = -0.45  Km= 2.2 Mm
-1/Vmax= -2.0  Vmax = 0.50 μM/Min

15. The Turnover Number of Carbonic Anhydrase.

Carbonic anhydrase of erythrocytes (Mr 30,000) has one of the highest turnover numbers known.
It catalyzes the reversible hydration of CO2:
H2O + CO2 ↔ H2CO3
This is an important process in the transport of CO2 from the tissues to the lungs. If 10.0 g of
pure carbonic anhydrase catalyzes the hydration of 0.30 g of CO2 in 1 min at 37 0C at Vmax,
what is the turnover number (kcat) of carbonic anhydrase (in units of min-1 )?

Answer
The turnover number of an enzyme is the number of substrate molecules transformed per unit
time by a single enzyme when the enzyme is saturated with substrate:
Kcat = Vmax / Et
Et = total moles of active sites.
Vmax (Moles of CO2/min)= (0.3g/min) / (44g/min) = 6.8 * 10-3 mol/min
Amount of enzyme (moles) = (10μg) ( 1g/106 μg) / (30000 g/mol) = 3.3 * 10-10 mol

Kcat = 6.8 * 10-3 mol/min / 3.3 * 10-10 mol = 2 * 107 min-1

19. Inhibition of Carbonic Anhydrase by Acetazolamide Carbonic anhydrase is strongly inhibited
by the drug acetazolamide, which is used as a diuretic (i.e., to increase the production of urine)
and to lower excessively high pressure in the eye (due to accumulation of intraocular fluid) in
glaucoma. Carbonic anhydrase plays an important role in these and other secretory processes,
because it participates in regulating the pH and bicarbonate content of several body fluids. The
experimental curve of initial reaction velocity (as percentage of Vmax) versus [S] for the
carbonic anhydrase reaction is illustrated below [see Lehninger] (upper curve). When the
experiment is repeated in the presence of acetazolamide, the lower curve is obtained. From an
inspection of the curves and your knowledge of the kinetic properties of competitive and mixed
enzyme inhibitors, determine the nature of the inhibition by acetazolamide. Explain your
reasoning.

Answer
The graph shows :
1. The inhibitor stops the enzyme from reaching the same Vmax as in the
absence of inhibitor.
2. The shapes of the two curves are similar: at any [S] the two velocities (with
or without inhibitor) are the same.
3. The velocity does not change significantly above [S] = 1 mM, so this means,
at higher [S] concentrations, the velocity shown, is Vmax for each curve.
4. If [S] at which ½ Vmax is estimated, the value would be the same for the
two curves.
 5. Noncompetitive inhibition alters Vmax of enzymes but leaves Km
unchanged. Because of this, acetazolamide acts is acting as a noncompetitive
(mixed) inhibitor of carbonic anhydrase.

21. pH Optimum of Lysozyme The active site of lysozyme contains two amino acid residues
essential for catalysis: Glu35 and Asp52. The pKa values of the carboxyl side chains of these
residues are 5.9 and 4.5, respectively. What is the ionization state (protonated or deprotonated) of
each residue at pH 5.2, the pH optimum of lysozyme? How can the ionization states of these
residues explain the pH-activity profile of lysozyme shown below? (see the figure in Lehninger)

Answer
At pH in the middle of the two pKa values (pH 5.2), the side-chain carboxyl group of Asp52, with the
lower pKa (4.5), is mainly deprotonated (COO- ).
Glu35 , has a higher pKa (5.9), and is protonated (COOH).

At pH below 5.2, Asp52 becomes protonated and the activity decreases.

At pH above 5.2, Glu35 be-comes deprotonated and the activity decreases

Maximum catalytic activity happens at a pH between the pKa values of the two acidic groups. In other
words, when Glu35 is protonated and Asp52 is deprotonated.