The document provides an overview of the mechanisms of enzyme action for trypsin, chymotrypsin, and urease. It discusses the structure and catalytic function of these enzymes, detailing the catalytic triad for serine proteases and the role of urease in the hydrolysis of urea. It also highlights ongoing research questions surrounding urease's activation process and its accessory proteins.

1. MECHANISM OF ENZYME
ACTION – TRYPSIN,
CHYMOTRIPSIN & UREASE
RANA SAHA
1st SEMESTER, M.S. (PHARM.)
MEDICINAL CHEMISTRY
NIPER, KOLKATA

2. TRYPSIN – BRIEF INTRODUCTION :
• EC 3.4.21.4
• A protease enzyme discovered in 1876 by
Wilhelm Kuhne.
• In the small intestine, trypsin breaks down
proteins, continuing the process of digestion
that began in the stomach.
• Produced by the pancreas in an inactive form
called trypsinogen.
• Trypsin proteolysis or trypsinization.

3. CHYMOTRIPSIN - BRIEF INTRODUCTION :
• EC 3.4.21.1
• One of the first enzymes to be crystallized.
• A digestive protein that is synthesized in the
pancreas and secreted into the small intestine,
where it helps break down dietary proteins.
• Catalyzes the hydrolysis of peptide bonds at a
rate of about 190 per second.

4. TRYPSIN & CHYMOTRIPSIN – SERINE PROTEASE
• Proteases are hydrolase enzymes that
catalyze the hydrolysis of a peptide
bond in a polypeptide or protein
substrate.
• Proteases can be classified in four
categories.

5. STRUCTURE OF CHYMOTRYPSIN
• α-chymotrypsin, the mature, active
form of the pancreatic enzyme.
• The residues of the catalytic triad and
disulfide bonds are shown as sticks.
• The mature enzyme consists of three
separate polypeptide chains.

6. MECHANISM OF ENZYMATIC ACTION OF
TRYPSIN & CHYMOTRYPSIN
• Chymotrypsin follows a double-displacement (or "ping-pong") pattern, which is
often observed for enzymes that make use of a covalent catalysis strategy.

7. CATALYTIC TRIAD
• A catalytic triad is a set of three coordinated amino acids that can be found in the
active site of hydrolase and transferase enzymes (e.g. proteases, esterases,
lipases).
• Catalytic triads perform covalent catalysis using a residue as a nucleophile.
• The nucleophile is most commonly a serine or cysteine amino acid.
• The reactivity of the nucleophilic residue is increased by the functional groups of
the other triad members.

8. EXAMPLES OF TRIAD

9. MECHANISM OF CATALYTIC TRIAD
• Catalysis is performed in two stages.

10. THE ROLE OF CATALYTIC TRIAD IN THE SERINE
PROTEASE MECHANISM
• Chymotrypsin, a common serine protease, hosts a trio of histidine, aspartate, and
serine residues as its catalytic triad.
• In addition to the triad, the active site of chymotrypsin also features an oxyanion
hole.
• In addition to disrupting the peptide bond with a serine, chymotrypsin does three
other important things: activate the amide bond for the nucleophilic attack,
activate water to make it a good nucleophile, and protonate the amine to make it
a good leaving group.

11. HOW
CHYMOTRYPSIN’S
CATALYTIC TRIAD
WORKS ?
The serine is the only residue that
interacts covalently with the
substrate,
The histidine residue serves as a
proton shuttle.
The aspartate residue stabilizes the
histidine throughout the reaction.

12. UREASE
• Ureases (EC 3.5.1.5), functionally, belong to
the amidohydrolases and
phosphotriesterases superfamily.
• Nickel-dependent metalloenzyme
• The active site of ureases is located in the α -
subunits.
• It catalyses the hydrolysis of urea into
carbon dioxide and ammonia:
• (NH2)2CO + H2O → CO2 + 2NH3

13. FUNCTION OF UREASE

14. HOW DOES UREASE WORK ?
• The active site of ureases consists, besides
the two nickel atoms, of one carbamylated
lysine, four histidines and one aspartate
residue.
• In the active site, the carbamylated lysine
bridges the two nickel atoms, with Ni(1)
further coordinated by two histidines and
Ni(2) by the other two histidines and by an
aspartate residue.

16. UREA HYDROLYSIS
CATALYZED BY
UREASE – HOT
RESEARCH TOPIC
The exact mechanism for urea hydrolysis
catalyzed by urease has been a research hot
topic in the last decades.
And there are some questions yet to be
answered, mostly on –
The role of each accessory protein in the
activation process like UreD, UreF, UreG, and
UreE involved in the assembly of urease’s
active metallocentre, and
the sequence of events and oligomerization
state of each protein in the activation
complex.

17. REFERENCE
1. Lehninger Principles of Biochemistry: 6th Edition
2. Harper's Illustrated Biochemistry, 31st Edition
3. Fundamentals of Biochemistry, Textbook by Charlotte W. Pratt and Donald Voet
4. Biochemistry, Ninth Edition, Lubert Stryer; Jeremy Berg; John Tymoczko;
Gregory Gatto
5. Journal of Advanced Research, Volume 13, September 2018, Pages 3-17
6. The structure-based reaction mechanism of urease, a nickel dependent
enzyme: tale of a long debate - Journal of Biological Inorganic Chemistry,
volume 25, page. 829–845, (2020)

18. THANK YOU.