Name: Nabeel Yousaf Roll No:0091 BH-CHEM 2018-22

Explain the mechanistic pathway of Chymotrypsin catalysis?

Introduction:

Chymotrypsin is an enzyme used for digesting proteins.

Chymotrypsin is found in the duodenum that selectively cleaves (cuts)

off pieces of amino acids from the protein chain.

Specifically chymotrypsin cleaves phenylalanine, tyrosine, and

tryptophan bonds, or in other words the aromatic amino acids. It cleaves
these amino acids starting from the C-terminus of the protein.

Chymotrypsin structure:

The primary structure shows that disulfide bonds are the crucial role to
the protein folding. The protein is spherical and itself consists of three
polypeptide chains. There is also a pocket in the enzyme other than the
active site and known as S1 pocket which, in the case of chymotrypsin, is
lined with relatively hydrophobic residues such as Ser-189, Ser-214, Trp-
215, Gly-216, and Gly-226.

The active site includes Ser-195, His-57, and Asp-102 (the catalytic
triad). Ser-195 is hydrogen bonded to the His-57 and it in turn is
 hydrogen bonded to the Asp-102 residue. The His-57 role is to position
the serine residue and polarize the hydroxyl group so it can be
deprotonated to the alkoxide ion. In the presence of the substrate, this
accepts a proton by acting as a base. Asp-102 orients the His-57 and
stabilizes it through hydrogen bonding and electrostatics

Mechanism of action

Step 1: When substrate (polypeptide) binds, the side of chain of the residue next to the peptide
bond to be cleaved nestles in a hydrophobic pocket on the enzyme, positioning the peptide bond
for attack. Histidine extracts one proton from serine to form an alkoxide ion. This serine ion
reacts with the substrate.

Step 2: In chymotrypsin, the carboxylate R-group of Asp102 forms a hydrogen bond with R
group of His 57. When this happens, it compresses this hydrogen bond and shifts electron
density to the other nitrogen atom (not involved in the H-bond) in the R-group of His57 becomes
a very strong base. This allows His 57 to deprotonate SeR195 and turn it into a strong
nucleophile that can attack the substrate.

Oxygen develops a partially negative charge in the oxyanion hole

Step 3: Instability of the negative charge on the substrate carbonyl oxygen when will leads to
collapse of the tetrahedral intermediate, re-formation of a double bond with carbon which breaks
the peptide bond between the carbon and amino acid group. The amino leaving group is
protonated by His57, facilitating its displacement. Once the oxyanion hole stabilizes the negative
charge, the bond breaks because the proton from Histidine is binding to nitrogen to make it less
likely to carbon. The leaving group is stabilized and the acyl-enzyme is formed

Step 4: The amine component is departed from the enzyme (metabolized by the body) and binds
to serine. This completes the first stage (acylation of enzyme). The first product has been made.

Step 5: A water molecule is added where the N terminus was. Histidine deprotonates the water
to form a hydroxyl group. This hydroxyl group attaches to carbon from the carboxyl side and
destabilizes the acyl intermediate. The bond is broken.

Step 6: An incoming water molecule is deprotonated by acid-base catalysis, generating a

strongly nucleophilic hydroxide ion. Attack of hydroxide on the ester linkage of the acylenzyme
generates a second tetrahedral intermediate.
Step 7: collapse of the tetrahedral intermediate form the second product, a carboxylate anion,
and displace Ser195. The proton from Histidine goes back to Serine.

Step 8: The carboxylic acid is released and the enzyme is reformed to catalyze the next reaction
with the original active site.
Which mechanism involved in digestion of protein, absorption and
transport of amino acids to the cell?

Protein Digestion

Dietary proteins are very large complex molecules that cannot be absorbed from the
intestine.To be absorbed, dietary proteins must be digested to small simple molecules
(amino acids), which are easily absorbed from the intestine.

I-Digestion in the stomach

Protein digestion begins in the stomach by gastric juice.

1- Role of gastric HCl

• It causes denaturation of proteins.

• It converts proteins to metaproteins, which are easily digested.
• It activates pepsinogen to pepsin.
• It makes pH in the stomach suitable for the action of pepsin.

2- Pepsin

• It is an endopeptidase acting on central peptide bond in which amino group

belongs to aromatic amino acids e.g. phenyl alanine, tyrosine and tryptophan.
• It is secreted in an inactive form called pepsinogen.
• Its optimum pH: 1.5-2.2
• It is activated by HCl then by autoactivation.

3- Rennin

• It is a milk-clotting enzyme.
• It is present in stomachs of infants and young animals.
• Its optimum pH: 4
• It acts on casein converting it to soluble paracasein, which in turn binds calcium ions
forming insoluble calcium paracaseinate. Calcium paracaseinate is then digested by
pepsin.
4- Gelatinase

It is an enzyme that liquefies gelatin.

The end products of protein digestion in the stomach are proteoses, peptones and large
polypeptides.

II- Digestion in the small intestine

Digestion of proteins is completed in the small intestine by proteolytic enzymes present in

pancreatic and intestinal juices.

A. Pancreatic Juice

1- Trypsin

• It is an endopeptidase that hydrolyzes central peptide bond in which the

carboxyl group belongs to basic amino acids e.g. arginine, lysine and
histidine.
• It is secreted in an inactive form called trypsinogen.
• Its optimum pH: 8
• It is activated by enterokinase enzyme then by autoactivation.

2- Chymotrypsin

• It is an endopeptidase that hydrolyzes central peptide bond in which the carboxyl

group belongs to aromatic amino acids.
• It is secreted in an inactive form called chymotrypsinogen.
• It is activated by trypsin.
• Its optimum pH: 8

3- Elastase

• It is an endopeptidase acting on peptide bonds formed by glycine, alanine and

serine.
• It is secreted in an inactive form called proelatase.
• It is activated by trypsin.
• It digests elastin and collagen.
• Its optimum pH: 8

4- Carboxypeptidase

• It is an exopeptidase that hydrolyzes the terminal (peripheral) peptide bond at the

carboxyl terminus (end) of the polypeptide chain.
• It is secreted in an inactive form called procarboxypeptidase.
• It is activated by trypsin.
 • Its optimum pH: 7.4

B. Intestinal Juice

1- Aminopeptidase

• It is an exopeptidase that acts on the terminal peptide bond at the amino terminus of
the polypeptide chain.
• It releases a single amino acid

2- Tripeptidase

• It acts on tripeptides
• It releases a single amino acid and dipeptide

3- Dipeptidase

• It acts on dipeptides
• It releases 2 amino acids

The end products of protein digestion in the small intestine are amino acids

Protein Absorption

- It is an active process that needs energy.

- Energy needed is derived from hydrolysis of ATP.
- It occurs in small intestine.
- Absorption of amino acids is rapid in the duodenum and jejunum, but slow in the ileum.

Mechanisms of amino acids absorption

There are two mechanisms for amino acids absorption.

1- Carrier proteins transport system
2- Glutathione transport system ( Glutamyl cycle)

1- Carrier proteins transport system

• It is the main system for amino acid absorption.

• It is an active process that needs energy.
• The energy needed id derived from ATP.
• Absorption of one amino acid molecule needs one ATP molecule.
• There are 7 carrier proteins, one for each group of amino acids.
• Each carrier protein has to sites one for amino acid and one for Na+.
• It co-transports amino acid and Na+ from intestinal lumen to cytosol of
intestinal mucosa cells.
• The absorbed amino acid passes to the portal circulation, while Na + is
extruded out of the cell in exchange with K+ by sodium pump.
2- Glutathione transport system ( Glutamyl cycle)

• Glutathione is used to transport amino acids from intestinal lumen to cytosol of

intestinal mucosa cells.
• It is an active process that needs energy.
• The energy needed id derived from ATP.
• Absorption of one amino acid molecule needs 3 ATP molecules.
• Glutathione reacts with amino acid in the presence of glutamyl transpeptidase to
form glutamyl amino acid.
• glutamyl amino acid releases amino acid in the cytosol of intestinal mucosa cells with
formation of 5-oxoproline that is used for regeneration of glutathione to begin
another turn of the cycle.
Describe the biosynthesis of triglycerides and ketone bodies
and its utilization?

Triglycerides

Triglycerides consist of a single glycerol molecule and three fatty acids. Triglycerides form by
condensation (dehydration) reactions between the hydroxyl (OH) groups of the glycerol and the
carboxyl (COOH) group of three fatty acids. Triglycerides are esters being derived from an
alcohol and a fat

Glycerol (blue) is an alcohol derivative of glyceraldehyde and has three hydroxyl groups. It acts
as the backbone of the structure. Fatty acids (red) – there are more than 70 types of fatty acid but
they all have long hydrocarbon tails and a terminal carboxyl group (COOH). The variety of fatty
acids determine the properties of each triglyceride.

Synthesis of triglycerols

Liver and adipose tissue are the major sites of triacylglycerol (TAG) synthesis. The TAG synthesis in
adipose tissue is for storage of energy whereas in liver it is mainly secreted as VLDL and is transported.
The TAG is synthesized by esterification of fatty acyl CoA with either glycerol-3- phosphate or dihydroxy
acetone phosphate (DHAP).

The glycerol part of the fat is derived from the metabolism of glucose. DHAP is an intermediate of
glycolysis.

Glycerol-3-phosphate may be formed by phosphorylation of glycerol or by reduction of dihydroxy

acetone phosphate (DHAP)

In adipose tissue, glycerol kinase is deficient and the major source is DHAP derived from glycolysis.In
liver, glycerol kinase is active. The fatty acylCoA molecules transfer the fatty acid to the hydroxyl groups
of glycerol by specific acyltransferases

Minor Pathway

In addition to these two pathways, in the intestinal mucosal cells the TAG synthesis occurs by the MAG
pathway.The 2-MAG absorbed is re-esterified with fatty acylCoA to form TAG.
Digestion of Triglycerols

Pancreatic lipase can easily hydrolyze the fatty acids esterified to the 1st and 3rd carbon atoms of
glycerol forming 2- monoacylglycerol and two molecules of fatty acid.

Then an isomerase shifts the ester bond from position 2 to 1. The bond in the 1st position is then
hydrolyzed by the lipase to form free glycerol and fatty acid.

The binding of co-lipase to the triacylglycerol molecules at the oil water interface is obligatory for the
action of lipase.

The co-lipase is secreted by the pancreas as an inactive zymogen (molecular weight 11,000). It is
activated by trypsin.

Biosynthesis and utilization of Ketone bodies:

Ketone bodies- Introduction

The compounds namely acetone, acetoacetate and β-hydroxybutyrate (or 3-
Hydroxybutyrate) are known as ketone bodies. Only the first two are true ketone* bodies
while β-hydroxybutyrate does not possess a keto (C=O) group. Ketone bodies are water
soluble and energy yielding. Acetone, however, is an exception, since it cannot be
metabolized.
In humans and most other mammals, acetyl-CoA formed in the liver during oxidation of fatty
acids can either enter the citric acid cycle or undergo conversion to the “ketone bodies,”
acetone, acetoacetate, and β-hydroxybutyrate, for export to other tissues. (The term “bodies”
is a historical artifact; the term is occasionally applied to insoluble particles, but these
compounds are quite soluble in blood and urine.) Acetone, produced in smaller quantities than
the other ketone bodies, is exhaled.

Acetoacetate and β-hydroxybutyrate are transported by the blood to tissues other than the
liver (extrahepatic tissues), where they are converted to acetyl-CoA and oxidized in the citric
acid cycle, providing much of the energy required by tissues such as skeletal and heart muscle
and the renal cortex. The brain, which preferentially uses glucose as fuel, can adapt to the
use of acetoacetate or β-hydroxybutyrate under starvation conditions, when glucose is
unavailable. The production and export of ketone bodies from the liver to extrahepatic
tissues allows continued oxidation of fatty acids in the liver when acetyl-CoA is not being
oxidized in the citric acid cycle.

Ketogenesis

The synthesis of ketone bodies is termed as ‘ketogenesis’. Ketogenesis occurs only in the
mitochondria of liver cells. It occurs when there is a high rate of fatty acid oxidation in the
liver.
The enzymes for ketone body synthesis are located in the mitochondrial matrix. Acetyl CoA,
formed by oxidation of fatty acids, pyruvate or some amino acids, is the precursor for ketone
bodies. Ketogenesis occurs through the following reactions:

Step 1

Initially, 2 acetyl-CoA are condensed to form acetoacetyl-CoA, catalyzed by the

enzyme thiolase. In this step, 1 CoA is cleaved, thereby providing enough energy for the
synthesis of the product.
Step 2The next step, which is catalyzed by β-hydroxy-β-methylglutaryl-CoA synthase (HMG-
CoA synthase), uses water to add another molecule of acetyl-CoA to the beta carbon of the
acetoacetyl-CoA.

This step produces β-hydroxy-β-methylglutaryl-coenzyme A (HMG-CoA), which is a branched

6-carbon compound and an intermediate in the synthesis of cholesterol in the cytosol.

Step 3

One acetyl-CoA is cleaved by HMG-CoA lyase, producing acetoacetate.

Step 4

Acetoacetate can now either be reduced to D-β-hydroxybutyrate by D-β-hydroxybutyrate

dehydrogenase in a NADH+H+–dependent reaction, or undergo spontaneous decarboxylation
to form acetone.

Beta-hydroxybutyrate is the ketone body with the highest blood concentration during periods
of fasting or starvation. The figure below summarizes ketogenesis reactions.
Utlilization of Ketone Bodies:

• Ketone bodies are utilized for energy needs in the extrahepatic tissues

such as brain, heart, skeletal muscle and kidney.

• The two ketone bodies-acetoacetate and β-hydroxybutyrate

serve as important sources of energy for the peripheral
tissues.

• The tissues which lack mitochondria (e.g. erythrocytes) however,

cannot utilize ketone bodies.

• During prolonged starvation, ketone bodies are the major fuel source for the

brain and other parts of CNS.

• The ketone bodies can meet 50-70% of the brain’s energy needs.

Step 1

Formation of acetoacetate from β-hydroxybutyrate

β-hydroxybutyrate is converted to acetoacetate by β-hydroxybutyrate dehydrogenase.

In the 1st step of utilization (unless this has already occurred), beta-hydroxybutyrate is oxidized
to acetoacetate, the 2nd most common ketone body in the blood. This is an NAD+–dependent
reaction and is catalyzed by beta-hydroxybutyrate dehydrogenase.

Step 2

Acetoacetate is then activated to acetoacetyl-COA through 1 of 2 mechanisms:

2. Formation of acetoacetyl CoA

 • 3-ketoacyl-CoA transferase transfers the CoA-group of the succinyl-CoA (a by-product
of the citric acid cycle) to the carboxyl group of the acetoacetate, yielding acetoacetyl-
CoA as well as succinate.
• Catalyzed by acetoacetyl-CoA synthetase, CoA undergoes an ATP–dependent reaction
with the carboxyl group of acetoacetate, forming water, ATP, and acetoacetyl-CoA.

Step 3

Formation of acetyl CoA

In the final step, acetoacetyl-CoA is cleaved by thiolase with the use of 1 CoA to form 2 acetyl-
CoA.

Acetyl-CoA is now available for energy production in the citric acid cycle and for the synthesis of
necessary reducing equivalents to keep the respiratory chain running.
Explain what Respiratory carriers are and how they play
their role and biological oxidation and reduction?
Electron carriers, sometimes called electron shuttles, are small organic molecules that
readily cycle between oxidized and reduced forms and are used to transport electrons during
metabolic reactions. There are two electron carriers that play particularly important roles during
cellular respiration: NAD+ (nicotinamide adenine dinucleotide, shown below) and FAD (flavin
adenine dinucleotide). Both NAD + and FAD can serve as oxidizing agents, accepting a pair of
electrons, along with one or more protons, to switch to their reduced forms. NAD + accepts two
electrons and one H+ to become NADH, while FAD accepts two electrons and two H + to become
FADH2. NAD+ is the primary electron carrier used during cellular respiration, with FAD
participating in just one (or two sometimes two) reactions.

As shown in the image above, NAD + is a small organic molecule whose structure includes the
RNA nucleotide adenine. (FAD is a similar type of molecule, although its functional groups are
different.) Both molecules are B vitamin derivatives, with NAD + produced from niacin and FAD
produced from riboflavin. NAD + and FAD are coenzymes, organic molecules that serve as
helpers during enzyme-catalyzed reactions, and they receive electrons and protons as part of
these reactions. Specifically, both NAD + and FAD serve as cofactors for enzymes
called dehydrogenases, which remove one or more hydrogen atoms from their substrates.

ENZYMES INVOLVED IN OXIDATION AND REDUCTION REACTIONS

Are called as Oxidoreductases which include : oxidases, dehydrogenases, hydroperoxidases and

oxygenases.

➢ Oxidases use oxygen as an electron acceptor .

➢ Dehydrogenases can’t use O2 as an electron acceptor
➢ Hydroperoxidases use H2O2 as a substrate Oxygenases catalyze the direct transfer of O2 into
the substrate
➢ Oxidases & dehydrogenases are involved in respiration; hydroperoxidases neutralize free
radicals & oxygenases are involved in biotransformation reactions.
 OXIDASES

Catalyze the removal of hydrogen from a substrate with the involvement of oxygen as a H –
acceptor, forming water or hydrogen peroxide.
Exist in two different forms :
• some of them are copper containing such as, Cytochrome oxidase , the terminal
component of ETC which transfer the e - finally to O2 .
• Other are flavoproteins such as , L – amino acid oxidase (FMN linked) and Xanthine
oxidase (FAD linked)

DEHYDROGENASES

Perform 2 main functions:

• Transfer hydrogen from one substrate to another in a coupled Oxidation /Reduction reaction
• As components of Electron transport chain such as cytochromes Dehydrogenases use
coenzymes – nicotinamides & riboflavin - as hydrogen carriers
• Nicotinamides can be in the form of NAD + or NADP+  Riboflavin can be – FMN or FAD same as
oxidases

HYDROPEROXIDASES

Includes 2 sets of enzymes : catalases and peroxidases

Peroxidases reduce H2O2 at the expense of several other substances H2O2 + AH2 → 2H2O + A
Catalases uses H2O2 as electron acceptor & electron donor 2H2O2 → 2H2O Peroxisomes are rich in
oxidases and catalases.

OXYGENASES

Catalyze the incorporation of O2 into substrates in 2 steps

• Oxygen is bound to the active site of the enzyme

• Bound O2 is reduced or transferred to the substrate Consist of two sets of enzymes 1.

Dioxygenases : incorporate both atoms of oxygen into the substrate ; A + O2 → AO2 2.

Monooxygenases : incorporates one atom of oxygen into the substrate & the other is reduced to water
A – H + O2 + ZH2 → A – OH + H2O + Z

Quiz CHEM-1201

How the transport of fatty acid into mitochondria take place through carnitine shuttle

Carnitine, derived from an amino acid, is found in nearly all cells of the body. Its name is derived from
the Latin carnus or flesh.  Carnitine plays a critical role in energy production. It transports long-chain
fatty acids into the mitochondria so they can be oxidized ("burned") to produce energy.  Carnitine is
concentrated in tissues that utilize fatty acids as a dietary fuel

The carnitine shuttle is responsible for transferring longchain fatty acids across the barrier of the inner

mitochondrial membrane to gain access to the enzymes of beta-oxidation.

The carnitine shuttle consists of three enzymes:

Carnitine palmitoyl transferase 1 (CPT1A & CPT1B)

Carnitine acylcarnitine translocase (SLC25A20)

Carnitine palmitoyl transferase 2 (CPT2)) Carnitine

Carnitine acyltransferase I, which is located on the outer mitochondrial membrane, transfers the
fatty acyl group from fatty acyl‐CoA to the hydroxyl (OH) group of carnitine. The acyl‐ carnitine
then moves across the intermembrane space to a translocase enzyme, which, in turn, moves
the acyl‐carnitine to carnitine acyltransferase II, which exchanges the carnitine for Coenzyme A.