Name : Ndumiso

Surname : Ndawonde

Student number : 60516364

Examination period : May /June 2020

ID number : 9704175437084

Module : BCH3701
Question 1
1.1.1. Enzyme chymotrypsin is one of the major digestive enzymes found in the
stomach. It is responsible for cleaving peptide bonds and is synthesized in the
inactive form, chymotrypsinogen, which is then cleaved to form the active
chymotrypsin. The reaction mechanism of chymotrypsin follows that of a Ping-
Pong bi bi mechanism. At the active site, there are three crucial residues
required for catalysis, namely Asp102, His57 and Ser195, all called catalytic
triad. Chymotrypsin employs base catalysis, followed by a nucleophilic attack, for
its catalytic strategy. His57 acts as a base catalyst to enable the oxygen of
Ser195 to make a nucleophilic attack on the carboxyl group of the substrate. This
results in the formation of an unstable intermediate which has an oxygen that is
negatively charged. The charge is stabilised by a nearby Gly193 residue that
neutralizes the charge by hydrogen bonding. Similarly the imidazole ring of His57
is stabilised by Asp102 via electrostatic interactions. The charged His57 then
acts as acid catalyst to facilitate the liberation of the first product, leaving behind
a modified enzyme known as the acyl enzyme
1.2.1 Catalytic triad are set of three coordinated amino acids that can be found
in the active site of some enzymes. Substitution of ser with ala amino acid
may lead to reduction of catalytic power that is Kcat. The same may be
observed also in histidine substitution with ala amino acid as serine-
histidine pair usually acts together to generate a nucleophile of sufficient
 power to attack the carbonyl group of a peptide bond. The effect of
substitution of aspartate with ala amino acid will reduce the Kcat

1.3 in chymotrypsin, this pocket is lined with hydrophobic amino acids, so substrate
proteins with hydrophobic amino acids, such as leucine or isoleucine, bind tightly
and in the correct orientation for the catalytic triad to function. In trypsin, it has a
negatively charged aspartic acid in the pocket, so the substrates that trypsin breaks
apart must have a positively charged amino acid, such as lysine or arginine, in the
proper position. These are digestive enzymes capable of cutting peptide bonds in a
wide range of proteins. In some pathways, such as blood clotting or the immune
system, a serine protease may be so specific that it only can cut a single peptide
bond in a single unique protein substrate protease may be so specific that it only can
cut a single peptide bond in a single unique protein substrate.

1.4
Question 2
2.1

relationship between pH vs Vmax vs stability

6000

5000
Vmax vs stability

4000

Vmax
3000
%stability

2000

1000

0
0 1 2 3 4 5 6 7 8 9 10

Ph

The relationship between this graph of Ph. vs Vmax vs %stability is that the increase in
Ph. leads to decrease of both Vmax and %stability
2.3

1/[S](micro mole) 1/Vo(micro mol/min)

0 0
2 0.005
1 0.0025
0.667 0.0017
0.5 0.0013
0.4 0.0012
0.333 0.00116
0.25 0.00114
0.1667 0.00112
 1/Vo
0.01

0.005 y = 0.0023x + 0.0003

0
-12 -10 -8 -6 -4 -2 0 2 4

-0.005

-0.01

-0.015

-0.02

-0.025
2.4.1 One parameter for determining the catalytic efficiency of a given enzyme is to
determine the kcat/km ratio. Kcat is the turnover number and this describes how many
substrate molecules are transformed into products per unit time by a single enzyme.
The Km value gives us a description of the affinity of the substrate to the active site of
the enzyme. Putting these two together to obtain the ratio allows a way to test how
effective the enzyme is on that particular substrate. The greater the ratio, the higher the
rate of catalysis is; conversely, the lower the ratio, the slower the catalysis is
.
2.5.1 Increasing substrate concentration also increases the rate of reaction to a certain
point. Once all of the enzymes have bound, any substrate increase will have no effect
on the rate of reaction, as the available enzymes will be saturated and working at their
maximum rate.
2.5.2 By increasing the enzyme concentration, the maximum reaction rate greatly
increases.
2.5.3The reduction in temperature reduces the energy of the reactant molecules,
making them move slower and less susceptible to collisions, thereby reducing
the reaction rate.
Question 3
Question 4
4.1. The protease EC number is 3.4.22.69 and its systematic name is peptide
hydrolases.
4.2. The substrate for protease are proteins from any source that is animal meat, egg,
milk, plant sources.
4.3. Both Active site and chymotrypsin has catalytic triad. For chymotrypsin it is serine
195, histidine 57, and aspartate 102.
4.4. Inhibitors that can be used to target this enzyme are HIV-1 protease inhibitors,
which are able to deactivate Mpro. approved inhibitors of HIV-1 protease including
tipranavir (TPV), saquinavir (SQV), ritonavir (RTV), nelfinavir (NFV), lopinavir (LPV),
indinavir (IDV), darunavir (DAR), atazanavir (ATV), and amprenavir (APV)
4.5. Protease use covalent modification as a catalytic strategy. The enzyme employs a
powerful nucleophile to attack the unreactive carbonyl group of the substrate. This
nucleophile becomes covalently attached to the substrate briefly in the course of
catalysis. This occurs in two stages that is acylation to form the acyl-enzyme
intermediate and deacylation to regenerate the free enzyme. First, the highly reactive
serine 195 hydroxyl group attacks the carbonyl group of the substrate to form the acyl-
enzyme intermediate, releasing the alcohol p-nitrophenol (or an amine if the substrate is
an amide rather than an ester). Second, the acyl-enzyme intermediate is hydrolyzed to
release the carboxylic acid component of the substrate and regenerate the free enzyme.
References
https://www.researchgate.net
https://www.ncbi.nlm.nih.gov/books/NBK22526/#A1174
https://www.khanacademy.org/science/high-school-biology/hs-energy-and-transport/hs-
enzymes/a/hs-enzymes-review
https://www.sciencedirect.com/science/article/abs/pii/S002251939890769X
BCH3701 UNISA STUDY GUIDE, 2020
Berg, Tymoczko, Gatto Jr, Stryer. 2019. Biochemistry. 8th edition. Macmillan Education