MEDF 1012A

Foundation Course For Health Sciences II

Biocatalyst

Dr. Yeung Hang Mee, Po

School of Biomedical Sciences
Email: poyeung@cuhk.edu.hk
Office: Choh-Ming Li Basic Medical Sciences Building 6/F Room 610P

1
 Learning Objectives
• Describe the structures and components of enzymes;

• Understand the enzyme kinetics including Michaelis-Menten

equation and Lineweaver-Burk plot;

• Explain different types of enzyme inhibitions with examples;

• Review multiple factors affecting enzyme’s activity.

 Biocatalyst

• A catalyst speeds up a chemical reaction compared to the

same reaction without the help of catalyst.

• It is not consumed in the reaction and can be recycled.

• It increases the rate of a reaction by lowering the activation

energy requirement of a reaction.
Structures and Components of Enzymes

4
 General Properties of Enzymes

• Most enzymes are proteins:

• They require cofactors
(inorganic) or coenzymes
(organic).

• Some enzymes are ribonucleic

acids (RNA) called ribozymes:
• Ribozymes are capable of
catalyzing specific biochemical
reactions.
• They are found in the
ribosome where they join
amino acids together to form
protein chains.

Reference: sciencedirect.com
 General Properties of Enzymes
• Enzymes have highly specific reactants called
substrates to bind on the active site.

• Trypsin is formed in the small intestine

when its proenzyme form, the trypsinogen
produced by the pancreas, is activated.

• Trypsin cleaves peptide chains mainly at the

carboxyl side of the amino acids lysine or
arginine.

References: Lippincotts Illustrated Reviews Biochemistry 5th Edition.

 General Properties of Enzymes
• Enzymes operate effectively at specific pH and temperature ranges:
• Pepsin (stomach) works effectively at very acidic environment.
• Trypsin (activated at duodenum) works effectively at alkaline environment.
• Alkaline phosphatase (bones and liver) works effectively at alkaline
environment.
• Most of the enzymes (made by proteins) will be denatured at high temperature
due to the change in conformation of its 3D structure.

References: Lippincotts Illustrated Reviews Biochemistry 5th Edition & Becker’s World of the Cell 8th Edition.
 Cofactors
• Cofactors are inorganic
components that are bound
to enzymes for activity.

• An inactive enzyme without

the cofactor is called an
apoenzyme.

• Examples: inorganic metal

ions such as zinc, copper,
ferric ions.
 Coenzymes

• Coenzymes are organic

components that are bound
to enzymes for activity.

• An inactive enzyme without

the coenzyme is also called
an apoenzyme.

• Examples: flavin adenine

nucleotide (FAD),
nicotinamide adenine
dinucleotide (NADH).
 Classifications of Enzymes
• Each enzyme is assigned a four-digit classification number and a
systematic name according to the reaction catalyzed.

References: Biochemistry 7th Edition, W.H. Freeman and Company.

 Classifications of Enzymes
No. Class Type of reaction catalyzed
1 Oxidoreductase Transfer of electron, hydride ions or hydrogen atom.
2 Transferase Group transfer reaction.
3 Hydrolase Hydrolysis (“hydro” means water).
4 Lyase Addition of groups to double bond, or formation of
double bond by removal of group.
5 Isomerase Transfer of groups within molecules to yield isomer.
6 Ligase Formation of C-C, C-S, C-O and C-N bonds by
condensation coupled to ATP cleavage.

Transfer of a PO32- group

References: Biochemistry 7th Edition, W.H. Freeman and Company.

 Activation Energy in Uncatalyzed or
Catalyzed Reaction
• Enzyme can provide an
alternative pathway with 1
lower activation energy for
the chemical reaction.
2

• Options:
• 1? 2?

• ________________________
________________________
________________________

References: Biochemistry 7th Edition, W.H. Freeman and Company.

Enzyme Kinetics

13
Michaelis-Menten Equation

• The Michaelis-Menten equation

reflects the change of velocity of
enzymatic reaction under different
concentrations of substrate provided:

• Part 1: almost a linear phase

since the active sites of all
enzymes are not fully occupied.

• Part 2: a plateau phase since all

enzyme’s active sites are fully
occupied.

Reference: Stryer Biochemistry 6th Edition.

 Michaelis-Menten Equation

1. Vmax: the maximum velocity which

1 the enzyme can reach.

2. Km: a specific value of substrate

concentration which the velocity of
enzymatic reaction reaches to its
half Vmax.

2 3. Km value can indicate if the

substrate added has a high or low
affinity to the particular enzyme.

Reference: Stryer Biochemistry 6th Edition.

 Michaelis-Menten Equation
1. If glucose is the common substrate
for enzyme 1 and enzyme 2:
• Km1: 2 mM
• Km2: 7 mM
Enzyme 1
2. Which enzyme has a higher affinity
Enzyme 2 of glucose? Why?

Answer:
• ____________________________
____________________________
____________________________
____________________________
____________________________
Km1 Km2

Glucose concentration [S] (mM)

Higher Km value Lower affinity

Lower Km value Higher affinity

Transformation of Michaelis-Menten
Equation into Lineweaver Burk Plot

1 =

1 = +

1 = 1
+

Reference: Stryer Biochemistry 6th Edition.

 Transformation of Michaelis-Menten
Equation into Lineweaver Burk Plot
1
1 = 1
Slope = +

For a linear equation:

Y axis value = slope * x axis value + y-intercept
1 (y-intercept)

1 Y axis value = 1
-1
(x-intercept) Slope =

Y-intercept =
1 X-intercept =
-1
Enzyme Inhibitions

19
 Types of Enzyme Inhibitions
• Reversible inhibition:
➢ Dissociation of inhibitor from the enzyme or enzyme-substrate complex is
rapid (a weaker interaction).
➢ Three types:
➢ Competitive
➢ Non-competitive
➢ Un-competitive

• Irreversible inhibition:
➢ Dissociation of inhibitor from the enzyme or enzyme-substrate complex is
very slow (a stronger interaction).
➢ Also known as “suicide inhibition”, i.e. enzyme activity is permanently
inhibited.
 Reversibly Competitive Inhibition
• A competitive inhibitor binds in and competes with substrate for the active site of
the enzyme, which results in an apparent increase in KM without changing the Vmax.

• This happens because the maximal rate of enzymatic reaction (Vmax) can resume if
the substrate concentration is increased (a higher KM with a lower affinity).

• Example: statin for hypercholesterolemia treatment.

Vmax still can achieve if
you increase S!

“他汀類藥物”

Reference: Pearson Education 2011.

 Reversibly Non-competitive Inhibition
• A non-competitive inhibitor can bind to another site outside the active site.
• This leads to lower the Vmax (you cannot resume Vmax by increasing S!)
• KM of the enzyme is less affected since the substrate still can be bound on
the active site, only the product formed is affected.
• Example: cyanide on cytochrome oxidase.

Vmax cannot resume

with increasing S!

Cyanide “山埃”

Reference: Pearson Education 2011.

 Reversibly Un-competitive Inhibition
• An un-competitive inhibitor can bind to another site outside the active site when
the enzyme-substrate complex is formed.
• This leads to lower the Vmax , overall the product will be reduced.
• KM of the enzyme is reduced since this type of inhibitor favors the formation of
enzyme-substrate complex, and the inhibition is more apparent with increasing
substrate concentration.
• Example: monoamine oxidase inhibitor against depression.

Vmax and KM are reduced!

with
With inhibitor

Reference: Pearson Education 2011.

 Enzyme-inhibitor Enzyme-substrate- (ii) Non-competitive type
complex inhibitor complex (Target BOTH free enzyme &
Inhibitor enzyme-substrate complex)
Inhibitor OR

Inhibitor
Inhibitor

Enzyme-inhibitor
complex Enzyme-substrate-
(i) Competitive type inhibitor complex
(Target for FREE enzyme) (iii) Un-competitive type (Target for
enzyme-substrate complex ONLY)
Types of Enzyme Inhibition by Lineweaver-Burk Plot
Normal Non-competitive

VMax

Competitive Un-competitive

KM
KM VMax
 Irreversibly Inhibition
• This type of enzyme inhibitor can bind to the active site and form a
strong bonding (covalent bond) with the enzyme.

• Example: Aspirin.

➢ The COX is an enzyme which

trigger inflammatory responses
when:
➢ The arachidonic acid (AA), a
20-carbon fatty acid in our
body which binds to the active
site of COX including the ser-
529.
➢ Ser-529 means the serine
(amino acid) at the amino acid
sequence no. 529.
Diagram of Cyclo-oxygenase (COX)
 Irreversibly Inhibition
• This type of enzyme inhibitor can bind to the active site and form a
strong bonding (covalent bond) with the enzyme.

• Example: aspirin
➢ The action of aspirin is by:
➢ Addition of acetyl group (acetylation)
on ser-529.

CH3
Factors Affecting Enzyme’s Activity

28
 Rate-determining or Rate-limiting Step
• The metabolic pathway usually involves many steps and the slowest step is defined as
rate-determining or rate-limiting.

• Considering the following examples:

Enzyme 1 Enzyme 2
A B C
Step 1 Step 2
Reactant A Reactant B Product

Ea step 1
Question: which step is the
Ea step 2 rate-limiting step? Why?

Answer:
• ______________________________
______________________________
______________________________
______________________________
______________________________
 Product Inhibition
• The product from a series of enzymatic reactions can inhibit the overall reaction rates.

• Considering the following examples:

Enzyme 1 Enzyme 2
A B C
Reactant A Reactant B Product

• Product inhibition prevents waste that occurs when more of a product is made than
the cell needs.

• It can also prevent harm when having too much of the pathway’s end product may
actually be harmful to us, e.g. cholesterol synthesis by our liver.
 Zymogen

• Zymogen is an inactive precursor of the

enzyme or it is also known as pro-enzyme.

• The conversion of active enzyme from its

zymogen is specific proteolytic cleavage of
zymogen.

• Examples:
• Inactive pepsinogen -> active pepsin
in stomach;
• Inactive trypsinogen in pancreas->
active trypsin at duodenum.

Source: Lehninger Principles of Biochemistry 7th Edition

 Premature Activation of Zymogen

• Premature activation of zymogen is a common phenomenon in acute pancreatitis.

• In normal conditions, the pancreas produces trypsinogen and secretes into duodenum;
the enterokinase (enzyme) in duodenum cleaves the trypsinogen into active trypsin for
dietary protein digestion.

• In acute pancreatitis, the inflammatory response triggers an alternative pathway to

activate trypsinogen into trypsin inside the pancreas, and thus the pancreatic tissues
are auto-digested by the active trypsin.

Source: http://www.scmlifescience.co.kr/en/clinical_test02b.asp
 Summary
1. Most of the enzymes are made of proteins and some are made of ribonucleic acids
(ribozymes).
2. Cofactors are ___________ components and coenzymes are __________
components that are bound to enzymes for activity.
3. There are six different classes for enzyme classifications based on the type of
reaction catalyzed.
4. The _____________________reflects the change of velocity of enzymatic reaction
under different concentrations of substrate provided.
5. Dissociation of inhibitor from the enzyme or enzyme-substrate complex is rapid (a
weaker interaction) is known as ____________ inhibition.
6. Dissociation of inhibitor from the enzyme or enzyme-substrate complex is slow (a
stronger interaction) is known as _______________ inhibition.
7. The metabolic pathway usually involves many steps and the slowest step is defined
as rate-determining or rate-limiting.
8. _______________prevents waste that occurs when more of a product is made than
the cell needs.
9. The conversion of active enzyme from its ____________________is specific
proteolytic cleavage of zymogen.
Preview of Video about DNA & RNA

34
1st BUS Session registration will be
available soon!

35
 References
1. Lehninger Principles of Biochemistry, 7th edition –
Chapter 6 – Enzymes.

2. Lippincotts Illustrated Reviews Biochemistry 5th

edition – Chapter 5 – Enzymes.