FACULTY OF APPLIED SCIENCES KUALA LUMPUR CAMPUS

Semester:​ [ / ] May [ ] September [ ] January (please “✓”)

Academic Year: ​2020
Course Code & Title: ​BABS2244 METABOLIC BIOCHEMISTRY
Programme: ​RBS 2 G1
Student’s Name (Registration Number): ​Wong Pui Mun (20WLR08576)
Submission Date: ​15/8/2020

Declaration

I confirm that I have read and shall comply with all the terms and conditions of TAR
College’s plagiarism policy.
I declare that this assignment is free from all forms of plagiarism and for all intents and
purposes is my own properly derived work.

Signature(s): ​
Name(s): Wong Pui Mun
Date: 15/8/2020
Experiment 11: Acyl activation for synthesis reactions.

Title:​ Acyl activation for synthesis reactions.

Objectives:
1. To determine the actual moles of hydroxamate formed.
2. To determine the compound activated in each assay tube per hour.
3. To determine the optical density for acyl activations.
4. To test the expected purity of crude homogenate enzymes.

Introductions:
Coenzyme A is notable for its role in the synthesis of fatty acids and oxidation of pyruvate in
the citric acid cycle. All genomes encode enzymes that use coenzyme A as a substrate, and
around 4% of cellular enzymes depend on it or a thioester, such as acetyl-CoA, as a substrate
(​Salway 2007)​. Coenzyme A is synthesized in a five step process from pantothenate and
cysteine where pantothenate is being obtained from the extracellular medium. Because
coenzyme A is, in chemical terms, a thiol, it can react with carboxylic acids to form
thioesters, thus functioning as an acyl group carrier, usually acetyl (​Gaur​, ​Chitnis​, ​Chauhan
2016)​. Besides, coenzyme A is used as an activator of acetate reaction. Aceto-CoA-kinase
catalyzes the synthesis of acetyl CoA from ATP, acetate, and CoA with the concomitant
formation of AMP and PP. Acetyl CoA formation occurs in two steps. The first involves the
reaction of the enzyme with ATP, acetate, and Mg ions, resulting in the formation of an
enzyme-bound derivative of 5'-phosphoacetyl AMP [acetyl AMP] and PP. In the subsequent
reaction the acetyl moiety is transferred from the enzyme-[acetyl AMP] complex to the
sulfhydryl grouping of CoA, forming acetyl CoA and AMP, and thereby regenerating the free
enzyme. The acetate-activating enzyme is widely distributed in animal tissue, plants, and
yeast, and has been much studied.

The another example is the activation of amino acid by the enzyme Aminoacyl-Transfer
RNA
Synthetases. ​The activation reaction is catalyzed by specific aminoacyl-​tRNA synthetases,
which are also called activating enzymes. The first step is the formation of an aminoacyl
adenylate from an amino acid and ​ATP​. This activated species is a mixed anhydride in which
the carboxyl group of the amino acid is linked to the phosphoryl group of ​AMP​; hence, it is
also known as aminoacyl-AMP (Mark Berg, Stryer 2002). The next step is the transfer of the
aminoacyl group of aminoacyl-​AMP to a particular ​tRNA molecule to form
aminoacyl-tRNA. The sum of these activation and transfer steps is:

The function of the ATP is actually to create a high energy bond that is transferred to the
tRNA molecule. This stored energy will provide the majority of the energy required for
peptide bond formation during translation.
Procedure:
Preparation of enzyme
A medium sized (100-180g) of rat was stunned with a blow on the head and was decapitated
quickly. The liver was then removed at once, weighed, and was cooled in a beaker of ice.
When it was cooled down, the liver was placed in an iced-cold 50 ml beaker and was cut up
into fine pieces and 2 volumes which is twice the weight of the liver in grams of ice cold
0,05M KCl was added. The contents were then being poured into an iced homogenizer. The
homogenizer in ices was maintained by gently turning the plunger of the homogenizer using
hands until an even suspension was obtained. This suspension was immediately used for
assay.

Hydroxamate Assay
The mixtures shown in table 1 were prepared in clinical centrifuge tubes. All the values were
expressed in milliliters. 0.4 ml of extract was added to enzymes assay tube 5 to tube 10. The
contents of each tube were mixed thoroughly and each assay tube was incubated at 37°C for
60 minutes in a water bath. The reaction was terminated by adding 1.4 ml of cool 10% TCA
to each assay tube and each standard which was tube 1 to tube 4. The contents were mixed
thoroughly and 0,6 ml of 2.0 ml FeCl​3 was added to all tubes. After further mixing, the
coagulated protein in tube 5 to tube 10 was centrifuged and the clear supernatant and
standards were decanted into colorimeter tubes. The tubes were allowed to sit for 10 minutes
and the optical densities at 520 mμ against the blank (tube 1) were read and recorded.

Table 1: Contents in each tube.

Substance Tube number

1 2 3 4 5 6 7 8 9 10

0.02M Mix of 15L-amino - - - - 0.2 0.2 0.2 - -

acid

1.0M KF Potassium acetate - - - - - 0.2 0.2 0.2 - -

0.1M ATP - - - - 0.3 0.3 0.3 - 0.3 -

0.1M Tris-Cl - - - - 0.6 0.6 0.6 0.6 0.6 0.6

3.0M NH​2​OH.HCl 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0

0.1M MgCl​2 - - - - 0.3 0.3 0.3 0.3 0.3 0.3

0.1M Hydroxamate - 0.1 0.2 0.4 - - - - - -

standard

H​2​O 2.0 1.9 1.8 1.6 0.2 0.2 - 0.3 0.4 0.7
Results:
Table 2: Absorbance reading of standard tubes at 520 mμ.
Tube Volume of 0.1M Absorbance (OD)
hydroxamate standard

1 0 0

2 0.1 0.040

3 0.2 0.085

4 0.4 0.150

Graph 1: Standard Curve of absorbance (optical density) against hydroxamate concentration

of the standard tubes.

Table 3: Determination of concentration of hydroxamate of each enzyme assay tube based on

the graph 1.
Tube Absorbance (OD) Actual moles of
hydroxamate

5 0.046 1.16

6 0.101 2.16
 7 0.148 3.02

8 0.177 3.55

9 0.042 1.09

10 0.040 1.05

Discussion:
In the first part of the experiment, the optical density (absorbance) of each standard tube
which was tube 1 to tube 4 were obtained by colorimeter at 520 nm. The hydroxamate
concentration for the first tube is 0 and thus the absorbance reading is 0. This tube served as a
blank. A standard curve was constructed in graph 1 that showed the relationship of optical
density over concentration of hydroxamate. Tube 1 was not considered as it served as a
blank. The equation obtained from graph 1 was used to determine the actual concentration of
hydroxamate of each of the enzyme tubes as shown in table 3.

The test tube 5, 7 and 8 contained 0.02M mix of 15 L amino acid. In amino acid activation,
the aminoacyl-tRNA synthetase (enzyme) attracts an amino acid to ATP and thus the active
site can bind the amino acid and ATP together . The ATP then lost two phosphate groups.
The amino acid will then join together in the form of AMP and the enzyme AMP –AA
complex is formed. The tRNA covalently bonds to the amino acid, taking the place of the
AMP, thus an AMP is lost. The high volume of hydroxamic acid was found in tubes 5, 7 and
8 compared to test tube 9 and 10. The volume of hydroxamic acid in test tube 6 is lower than
test tube 5.

Moreover, in test tube 6, 7 and 8 where the tubes contained 1.0 M KF Potassium acetate
indicate an acetate activation reaction. In the acetate activation, the AMP-forming
acetyl-CoA synthetase complex is formed. First, AMP must be bound by the enzyme causing
a conformational change on the active site. This allows the reaction to take place. The active
site is referred to as an A-cluster. A crucial lysine residue must be present in the active site to
catalyze the first reaction where Co-A is bound (Pratt & Cornely 2004). Co-A then rotated in
the active site into the position where acetate can covalently bind to CoA. The covalent bond
is formed between the sulfur atom in Co-A and the central carbon atom of acetate. The
volume of hydroxamic acid is higher in tubes 6, 7, 8 compared to test tube 9 and 10.

Besides, in test tubes 5, 6, 7 and 9, there are 0.3 ml of 0.1M ATP. ATP is a high-energy
molecule that helps to form complex acyl activation. Therefore, the tubes have high
absorbance value in hydroxamic acid which can be found in the product of the reaction. Tube
7 has the highest volume of hydroxamic acid. The presence of ATP acts as an activator of
forming a complex Enz-AMP-acetate or Enz-AMP-AA. Tube 9 has no amino acid or
potassium acetate added in the reaction but it forms hydroxamic acid which can be found in
the liver enzyme. The liver enzyme consists of amino acid or potassium acetate which can
promote the acyl activation. The test tube 8 and 10 has no ATP added; therefore it has lower
volume of hydroxamic acid compared to the rest of test tubes. This is because ATP can
promote the reaction.
When comparing test tube 9 and 10, both of the tubes contain only the liver homogenate.
However, ATP was supplied in test tube 9 while test tube 10 is deprived of it. This can be
obviously seen that test tube 9 has a higher concentration of 0.01M hydroxamate produced
compared to test tube 10 due to the presence of ATP. However, aminoacyl-tRNA synthetase
enzymes utilize ATP as an energy source to attach a tRNA molecule to its specific amino
acid, forming an aminoacyl-tRNA complex and ready for translation at ribosomes (Brown,
Jones-Mortimer & Kornberg 1977). The energy is made available by ATP hydrolysis to
adenosine monophosphate (AMP) as two phosphate groups are removed. Therefore, the
amino acid is essential in order for the reaction to occur and intermediate to form. Although
ATP is not supplied in tube 10, however there is a production of hydroxamate because ATP
is present in small amounts in the fresh liver homogenate where it can be found in
mitochondria.

In addition, test tubes 7 and 8 both were added with identical substrates which were amino
acid, potassium acetate and liver homogenate. By right, both of the tubes should have similar
concentrations of hydroxamate produced however, based on table 3, the concentration of
hydroxamate in test tube 7 is higher than test tube 8. This is due to the fact that test tube 7 is
supplied with 0.1M ATP while test tube 8 was not supplied. ATP acts as an activator for both
acetate activation and amino acid activation; therefore, the presence of ATP helps to promote
the formation of Enz-AMP-acetate intermediate state and leads to a higher activation to
produce a higher volume of hydroxamate. However, reaction still occurs in Tube 8 even
though ATP is not supplied because a small amount of ATP still can be found in the
mitochondria of the liver homogenate. Therefore, due to the less amount of ATP present in
tube 8, there is less hydroxamate produced compared to tube 7.

Conclusion:
In conclusion, out of all the enzyme tubes from tube 5 to tube 10, tube 7 has the highest
concentration of hydroxamate formed due to the presence of all three substrates amino acid,
potassium acetate and liver homogenate with ATP. This is because ATP provides the greatest
activation energy which leads to the highest amount of hydroxamate formed in the product.

References:
1. Pratt C.W. & Cornely, K. 2004, ​Essential biochemistry.​ John Wiley & Sons, United
States.
2. Brown, T.D., Jones-Mortimer, M.C. and Kornberg, H.L. (1977). The enzymic
interconversion of acetate and acetyl-coenzyme A in Escherichia coli. ​Journal of
General Microbiology​, [online] 102(2), pp.327–336. Available at:
https://pubmed.ncbi.nlm.nih.gov/21941/ [Accessed 12 Aug. 2020].
3. Salway​ J.G. 2007, ​Metabolism at a glance​, John Wiley & Sons, ​United States.
4. Gaur​. D, ​Chitnis ​C. E.​, ​Chauhan V. ​S. 2016, ​Advances in malaria research​, John
Wiley & Sons, ​United States.
5. Mark Berg​. J., S​tryer​ L. 2002, ​Biochemistry,​ W.H. Freeman, New York.