Bacong, Alexa Kim K.

KMBIOKP – N02 September 10, 2021

11900067 PHY-PMD Ma’am Marissa Noel

Assignment 3: Enzymes

1. The following values were obtained for an enzyme in the presence of 3 different
substrates.

Total enzyme concentration [E] = 1.0 mM; Substrate concentration [S] = 1.0 mM. (5 pts)

Substrate Km (mM) Vmax V kcat

(mM/min)

D-glucose 0.0080 1.0 0.99 0.0079

D-fructose 2.0 1.4 0.47 0.94

D- 0.0050 0.50 0.50 0.0025

mannose

Figure 1

(a) Complete the table above by calculating enzyme velocity (v) and turnover number
(kcat) for each substrate.
(b) Which of these substrates binds most tightly to the enzyme? Explain.
Among the substrates with the given values, D-mannose is the substrate that binds
most tightly to the enzyme. Km is a measure of ES binding or the measure of the affinity
of a substrate for an enzyme. A small K m identifies tight binding while a large K m
describes weak binding. Thus, D-mannose with the smallest Km value of all the substrates
is defined as such.
(c) With which substrate does the enzyme exhibit the highest efficiency and specificity?
Explain and show your calculations (if any).
In order to identify the efficiency and specificity of an enzyme, it is best to
determine the kcat/Km ratio. This ratio reflects both the binding catalytic events of the
enzyme against the substrate and, best represents the enzyme’s ability to convert substrate
to product. The greater the value of the ratio, the greater the rate of catalysis. In contrast,
the lower the value of the ratio, the slower the rate of catalysis.

kcat/Km

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 D-glucose 0.9875

D-fructose 0.47

D-mannose 0.5

Figure 2

From the calculated results of the catalytic ratio in Figure 2, it is shown that D-
glucose is the enzyme exhibiting the highest efficiency and specificity.

2. The steady state kinetics of the enzyme aspartase were studied in the absence and presence of
the inhibitor hydroxymethylaspartate (A) ([A] = 2.5 x 10-4 M). The initial rate (v) is given as a
function of substrate concentration. (8 pts)

[S] v (no inhibitor) v (inhibitor present)

(M) (M/min) (M/min)

1x10-4 0.026 0.010

5x10-4 0.092 0.040

1.5x10-3 0.136 0.086

2.5x10-3 0.150 0.120

5x10-3 0.165 0.142

Figure 3

(a) Draw and upload a Lineweaver-Burk plot for the enzyme reaction

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 Lineweaver-Burk Plot
120

100
f(x) = 0.01 x + 5.21
80 R² = 1

60
1\V

40
f(x) = 0 x + 5.06
20 R² = 1

0
0.00E+00 2.00E+03 4.00E+03 6.00E+03 8.00E+03 1.00E+04 1.20E+04
1/[S]

Figure 4

(b) Km in the absence of inhibitor A

K m=V max ( slope )

¿ ( 0.1977261493 ) (0.0033)
−4
¿ 6.5249629 x 10
−4
¿ 6.5 x 10 M
(c) Km in the presence of inhibitor A

K m=V max ( slope )

¿ ( 0.1917729408 ) (0.0095)

−3
¿ 1.82184293 x 10
−3
¿ 1.8 x 10 M
(d) Vmax in the absence of inhibitor A
1
V max =
y −intercept

1
¿
5.0575

¿ 0.1977261493

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 ≈ 0.19772 M / min
(e) Vmax in the presence of inhibitor A
1
V max =
y −intercept

1
¿
5.2145

¿ 0.1917729408

≈ 0.191773 M /min

(f) the type of inhibition = competitive inhibition ( K m increase, V max unaffected)

3. From a Lineweaver-Burk plot of an enzyme, 1/v = 25.0 hr/mol at 1/[S] = 0 and 1/[S] = -
1.13 x 102 L/mol at 1/v = 0. (2 pts)

(a) Calculate Km

1 Km 1 1
V
= ( )( [ ] )
V max S
+
V max

Km 1 1
0= ( )( [ ] )
V max S
+
V max

K 1
−1
( )( [ ] )
= max
V max V max S

m
K¿
−1 K m −1.13 x 102 L
=( )( )¿
V max V max mol

2
−V max −1.13 x 10 L
=( K m)( )
V max mol
1
K m=
L
1.13 x 102
mol

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 mol
K m=0.00884955752
L

−2
K m=8.85 x 10 mol/ L

(b) Calculate Vmax

1 Km 1 1
V
= ( )( ) +
V max [ S ] V max

1 K 1
= m ( 0) +
V V max V max
1 1
=
V V max

hr 1
25.0 =
mol V max

1
V max =
hr
25.0
mol

mol
V max =0.04
hr

4. What advantage can be gained by synthesizing proteolytic enzymes as zymogens? (1 pt)

Synthesizing proteolytic enzymes as zymogens is advantageous as to exist as an

inactive precursor enzyme. This conversion also helps prevent digestion of proteins in the
cells in which they are synthesized.

5. Identify an enzyme used to diagnose a specific human disorder or disease. Discuss its
biochemistry and use in clinical practice. Cite your references. (4 pts)
A deficiency of the galactose-1-phosphate uridylyl transferase (GALT) causes a
rare hereditary disorder known as galactosemia. This disease is characterized by the
body’s inability to convert galactose to glucose. In most cases, galactosemia is inherited
as an autosomal recessive genetic condition.

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 Figure 5. Crystal structure of glucose-1-phosphate bound
nucleotidylated human galactose-1-phosphate uridylyltransferase

Classic galactosemia is the first type of galactosemia which is first identified in

infancy. An infant with the disorder develops jaundice in the eyes, hepatomegaly,
yellowing of the skin and anorexia – all within a few days or weeks from day of birth.
Early course of action taken is through removing lactose from their diet. Despite early
treatment, the infant has a high risk of having developmental delays, speech problems
and abnormalities in motor function.

In addition, the clinical variant galactosemia is a second type of galactosemia

occurring in infants within communities of African Americans and native Africans
located in South Africa.

Diagnosis for the disorder is through detection of both high levels of erythrocyte
galactose-1-phosphate concentration and low erythrocyte galactose-1-phosphate
uridylyltranserase (GALT) enzymatic activity in the blood of the newborn.

References:

Bank, RCSB Protein Data. “RCSB PDB - 5IN3: Crystal Structure of Glucose-1-Phosphate
Bound Nucleotidylated Human Galactose-1-Phosphate Uridylyltransferase.”
Www.rcsb.org, www.rcsb.org/structure/5IN3. Accessed 10 Sept. 2021.

Berry, Gerard T. “Classic Galactosemia and Clinical Variant Galactosemia.” Nih.gov, University
of Washington, Seattle, 9 Mar. 2017, www.ncbi.nlm.nih.gov/books/NBK1518/.

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 enzymes. Zymogen Introduction – Creative Enzymes Blog. www.creative-
enzymes.com/blog/zymogen-introduction/. Accessed 10 Sept. 2021.

“Galactosemia - NORD (National Organization for Rare Disorders).” NORD (National

Organization for Rare Disorders), NORD, 2015, rarediseases.org/rare-
diseases/galactosemia/.

“How to Calculate KCAT and VMAX.” Sciencing, sciencing.com/calculate-kcat-vmax-

7866502.html. Accessed 10 Sept. 2021.

“Purdue University - Department of Chemistry - Page Not Found!” Www.chem.purdue.edu,

www.chem.purdue.edu/courses/chm333/Spring%202013/Lectures/Spring
%202013%20Lecture%2015.pdf.

Aklectures.com, 2021, aklectures.com/lecture/fundamentals-enzymes/catalytic-efficiency-of-

enzymes-kcat-km#:~:text=One%20way%20to%20measure%20the. Accessed 10 Sept.
2021.

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