MEDF1010

Foundation Course for Health Sciences I

Biocatalyst
CHEUNG Hoi Hung Albert
Assistant Professor
School of Biomedical Sciences
Phone: 3943 9796
Email: [email protected]
Office: Rm707A, 7/F, LKS-IBSB 1
 Learning Outcomes

• Describe the structure and components of enzymes, and the

roles of coenzymes in enzymatic reactions

• Describe the mechanisms regulating enzymatic reactions

• Compare and contrast reversible vs. irreversible, competitive vs.

non-competitive enzyme inhibitions, and to explain their
properties using Lineweaver-Burk plot

• Discuss the use of enzyme, enzyme competitors and inhibitors

as therapeutics

2
Almost all biological
functions are supported
by chemical reactions
catalyzed by enzymes

Enzyme is a biological catalyst which speeds up

reactions.

Carbonic anhydrase Rate enhancement:

107
3
 Enzyme activity is rarely expressed
as enhancement rate, but
expressed as international unit (IU)

1 IU (or U): the amount of enzyme that catalyzes 1 mol of

substrate to product per minute

katal (or kat): the amount of enzyme that catalyzes 1 mole of

substrate to product per second

1 IU = ? Katal

The specific activity of an enzyme is a measure of the number

of IU/mg (or IU/mL) protein 4
 Enzyme classification
• Enzymes can be classified by the types of reactions they catalyze
• 7 major classes, with subclasses under each major class
• Each enzyme is assigned a 4-digit code number (e.g. 2.7.1.1 for hexokinase)
Class Reaction Examples
1. Oxidoreductases Transfer of electrons (hydride ions or H atoms) Dehydrogenases, peroxidases

2. Transferases Group transfer reactions Hexokinase, transaminases

3. Hydrolases Hydrolysis reactions Alkaline phosphatase, trypsin

4. Lyases (synthases) Cleavage of C – C, C

– O, C– N, or other bonds by Fumarase, dehydratases
elimination, leaving double bonds or rings, or addition of
groups to double bonds
5. Isomerases Transfer to groups within molecules to yield isomeric forms Triose phosphate isomerase,
phosphoglucomutase
6. Ligases (synthetases) Formation of C– C, C
– S,C
– O, and C– N bonds by Pyruvate carboxylase, DNA
condensation reactions coupled to cleavage of ATP or ligases
similar cofactor
7. Translocases Catalyse the movement of ions or molecules across NADH:ubiquinone reductase
(added in 2018) membranes or their separation within membranes (H+ translocating) 5
https://iubmb.qmul.ac.uk/enzyme/
 Reaction and substrate specificity
Most enzymes are highly specific for
• the type of reaction catalyzed
• nature of the substrate(s)

Highly specific enzymes:

e.g. catalase (substrate: H2O2), urease (substrate: urea)

Some enzymes have broader substrate specificity:

e.g. chymotrypsin, trypsin, elastase (serine protease)

Substrate specificity of serine

protease is determined by the
Val
active site
Thr

Asp
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Active site

• cleft or crevice for catalysis

• constitutes only a small part of total

volume of an enzyme
• consists of amino acids from different
parts of the linear sequence
• contains the residues that directly
participate in the making and breaking of
bonds
• binds the substrates and additional
functional molecule, if any
• may require cofactor / coenzyme

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 Coenzyme / cofactor
Activities of some enzymes require helper molecules called:
- coenzyme (organic molecules)
e.g. derivatives of vitamin B, coenzyme A
- cofactors (inorganic elements)
e.g. Ca2+, iron, copper, manganese

Coenzymes and co-factors may be:

- loosely and transiently bound to enzymes; or
- tightly and permanently bound to enzymes as prosthetic groups

Coenzymes are often modified during

enzymatic reaction, and then recycled by
another enzyme (e.g. NAD+).

Some coenzymes (e.g. Co-A) assist in

transport of intermediates from one enzyme
to another during a sequence of reactions. Pyruvate dehydrogenase complex 8
Some coenzymes are transient carriers for functional groups

Vitamin B6 and the coenzyme derivatives

B6 derivatives are involved in >100 reactions

in carbohydrate and lipid metabolism

Pyridoxal phosphate is needed in amino acid

synthesis, catabolism and interconversion (as
a transient carrier of amino group during amino
acid metabolism).

B6 requirement increases with protein intake.

(active form) 9
Vitamins and minerals are important supplements in formula milk

Many vitamins are precursors

of coenzymes
Vitamin B6
• B1 (thiamine pyrophosphate)
deficiency may
• B2 (flavin mononucleotide)
cause neurologic
symptoms and • B6 (pyridoxal phosphate)
anemia • Niacin (NAD+)
• Pantothenic acid (CoA)
• Biotin (biocytin)
• Vitamin B12 (deoxyadenosyl
cobalamin)

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Minerals are co-factors for enzymes

Mineral deficiencies can result in many

disorders including anemia and goiter.
How does enzyme work?
 Active site of enzyme binds the
substrate and catalyzes the reaction

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Models of enzyme action

Uncatalyzed reaction

Lock-and-key

Induced fit
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 Enzymes lower the
activation energy for
transition state formation

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Energy barrier between formation of product from substrate

There is an activation
energy (ΔGǂ) for formation
of the transition state (ǂ)

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Enzymes increase reaction rates by lowering activation energy

ΔGB: Binding energy

The binding energy is the free energy that

is released by the formation of weak
interactions between a complementary
substrate and enzyme.

The binding energy is maximized since only

the correct substrate can interact with an
enzyme and is released when the enzyme
facilitates formation of the transition state.

Note: Enzymes increase reaction rate, but do not affect equilibrium (why?) 17
Enzyme Kinetics

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19
 S P
1. Mix enzyme + substrate
2. Record product formation at different time points
3. Repeat the experiment with a different substrate concentration (keep enzyme
concentration unchanged)
4. Calculate initial rate or velocity of the reaction at different [S]

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 Initial velocity vs substrate concentration

Michaelis-Menten equation:

 When there is no S, [S] = 0, V0 = ?

 When [S] gets bigger and bigger, 𝑉0 approaches Vmax
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 Derivation of Michaelis-Menten equation

For any chemical reaction,

k
A+B C+D

Reaction rate V = k [A][B], where k is called rate constant

For an enzymatic reaction S → P ,

k1 k2
E+S ES E+P
k-1
As the amount of P is very small at the beginning, the reverse reaction E + P → ES is
ignored.

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Note: for reference only (needn’t remember how to derive)
 k1 k2
E+S ES E+P
k-1

Rate of ES formation = k1 ( [Et] – [ES] ) [S] Et : Total enzyme

Rate of ES breakdown = k-1 [ES] + k2 [ES]

At steady-state, [ES] is constant, so the rate of ES formation = the rate of ES breakdown

k1 ( [Et] – [ES] ) [S] = k-1 [ES] + k2 [ES]

k1 [Et][S] = ( k1[S] + k-1 + k2 ) [ES]

k1 [Et][S]
[ES] =
k1[S] + k-1 + k2

[Et][S]
[ES] =
[S] + (k-1 + k2)/k1 23
 k1 k2
E+S ES E+P
k-1

[Et][S]
[ES] =
[S] + (k-1 + k2)/k1

k−1 + k2 [Et][S]
Define Km = [ES] = (1)
k1 Km+ [S]

Initial velocity V0 is determined by the breakdown of ES to form product, which is

determined by [ES]:
V0 = k2 [ES] (2)

Substitute equation (1) to equation (2):

k2[Et][S]
V0 = (3)
Km+ [S]
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 k1 k2
E+S ES E+P
k-1

k2[Et][S]
V0 = (3)
Km+ [S]

Maximum velocity occurs when the enzyme is saturated, that is, when [ES] = [Et], therefore

Vmax = k2 [Et] (4)

Substitute equation (4) to equation (3), we obtained the Michaelis-Menten equation:

Note: Km has the same unit as [S] 25

 When Km = [S],

𝟏
𝑽𝟎 = 𝑽𝒎𝒂𝒙
𝟐

Km is the substrate concentration at which the initial reaction

rate is half of the maximal rate of the enzymatic reaction

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What does the value of Km tell us
about the enzymatic reaction?

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If ES E + P is the rate limiting reaction,

Then k2 « k1 ; Or k2 « k-1

k2 + k-1 k-1
Km = 
k1 k1
= Dissociation constant for ES
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 HA ⇌ H+ + A−
The dissociation constant is usually written as the equilibrium constant:
+ −
[𝐻 ][𝐴 ]
𝐾𝑎 =
[𝐻𝐴]

k-1
ES ⇌ E + S
k1

Rate of forward reaction = k-1 [ES]

Rate of reverse reaction = k1 [E][S]

At equilibrium: Rate of forward reaction = Rate of reverse reaction

k-1 [ES] = k1 [E][S]
k−1 [E][S]
=
k1 [ES]
≈ Km Dissociation constant 29
Because Km approximates dissociation constant of the ES
complex, it is usually used to as a measure of the affinity of
the enzyme for its substrate.

Effect of substrate concentration on

(initial) reaction velocities for two
enzymes:

- Enzyme 1 has a small Km

- Enzyme 2 has a large Km

=> Enzyme 1 has a higher affinity to
the substrate because it requires
less [S] to reach half Vmax

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31
 Lineweaver-Burk Double Reciprocal Plot

𝟏 𝑲𝒎 + [𝑺]
Take reciprocal =
𝑽𝟎 𝑽𝒎𝒂𝒙 [𝑺]

𝟏 𝑲𝒎 [𝑺]
= +
𝑽𝟎 𝑽𝒎𝒂𝒙 [𝑺] 𝑽𝒎𝒂𝒙 [𝑺]

𝟏 𝑲𝒎 𝟏 𝟏
= · +
𝑽𝟎 𝑽𝒎𝒂𝒙 [𝑺] 𝑽𝒎𝒂𝒙

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Effect of substrate concentration on initial velocity

𝟏 𝑲𝒎 𝟏 𝟏
= · +
𝑽𝟎 𝑽𝒎𝒂𝒙 [𝑺] 𝑽𝒎𝒂𝒙
y x

Michaelis-Menten plot Double-reciprocal

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(Lineweaver-Burk) plot
 k1 k2
E+S ES E+P
k-1

Maximum velocity (Vmax) occurs when the enzyme is saturated, that is, when [ES] = [Et],
therefore
Vmax = k2 [Et]

If ES → E + P is the rate-limiting reaction, then k2 is called

kcat (turnover number)

kcat : the number of substrate molecules converted to

product in a unit time by a single enzyme molecule when
the enzyme is saturated with substrate.
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kcat = Vmax / [Etotal]

kcat has a unit of reciprocal time (s-1)

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The ratio Kcat / Km is a measure of catalyticefficiency

k1 k2
E+S ES E+P
Km (≈ k-1/k1) determines k-1 K2 (= Kcat) determines
how fast ES is formed how fast P is formed
from E and S from ES

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Compare the catalytic efficiency of catalase and fumarase
Break
Inhibition of Enzyme Activity

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Different types of enzyme inhibition

Competitive
inhibitor

Noncompetitive
Reversible inhibitor
Enzyme
inhibition Uncompetitive
inhibitor
Irreversible

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 Competitive Inhibition

• The inhibitor competes with the substrate for the active site.
• Competitive inhibitors are not catalyzed by the enzyme.
• The inhibitor does not inactivate the enzyme.
• Competitive inhibition can be “diluted” by adding more substrate.
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Adapted from Biochemistry, 5th Edition by Richard Harvey and Denise Ferrier (2011)
Many competitive inhibitors are
structurally similar to the substrates

Statin drugs are structural analogs of

the natural substrate. The drugs inhibit
de novo cholesterol synthesis and
thereby lower plasma cholesterol
levels.

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 Competitive inhibition

• Effect on Vmax: unchanged

• Effect on Km: increased (lower substrate affinity)
• Effect on the Lineweaver-Burk plot: refer to the graph
Adapted from Biochemistry, 5th Edition by Richard Harvey and Denise Ferrier (2011)
 Noncompetitive inhibition

The inhibitor can bind

to E or ES complex,
and thus inactivate
the enzyme

Inhibitor binds at a site distinct from the

substrate active site
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Adapted from Biochemistry, 5th Edition by Richard Harvey and Denise Ferrier (2011)
 Noncompetitive inhibition

• Effect on Vmax: decreased

• Effect on Km: the same
• Effect on the Lineweaver-Burk plot: refer to the graph
Adapted from Biochemistry, 5th Edition by Richard Harvey and Denise Ferrier (2011)
 Irreversible inhibition

Inhibition of acetylcholinesterase
(an enzyme that cleaves the
neurotransmitter acetylcholine) by
some insecticides => neurotoxic
effect

• Irreversible inhibitor binds strongly to the active site, usually by

forming covalent bonds with the functional groups of the active site
(note: noncovalent binding is also possible).

• Suicide inactivator: irreversible inhibitor that undergoes the first few

chemical steps of the normal enzymatic reactions, but cannot be
converted to a “product” (i.e. permanently inactivate the enzyme)
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Regulation of enzyme activity

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Many enzymatic reactions are tightly regulated

Glycolysis
pathway

Uncontrolled velocity is
dangerous!

What will it happen if enzymes are not regulated? 47

 Allosteric regulation

• Allosteric enzymes are usually

larger and have two or more
subunits (catalytic vs
regulatory)

• Allosteric enzyme undergoes

conformational change in
response to binding of modulator
(*not to be confused with
noncompetitive inhibitors, which
do not necessarily have
conformational changes)

• The modulator can be inhibitory

or stimulatory

• Allosteric modulators are

Subunit interactions in an allosteric generally small metabolites or
enzyme, and interactions with inhibitors cofactors (e.g. ATP, CTP)
and activators 48
Aspartate transcarbamoylase, an allosteric enzyme
The holoenzyme is composed of two catalytic trimers and
three regulatory dimers

Note the change of

quaternary structure

ATP: positive regulator

CTP: negative regulator
(one of the end products of
ATCase is a highly regulated the pathway)
enzyme that catalyses the first
committed step in pyrimidine
biosynthesis (cytosine/thymine) 49
Some enzymes are regulated by
reversible covalent modification

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 Regulation of enzyme activity by covalent
modification

• Phosphoryl groups affect structure and catalytic activity of

enzymes

• Kinase: catalyze addition of a phosphoryl group (usually from ATP)

• Phosphatase: catalyze removal of phosphoryl group

• The covalent modification can be reversed (reversible)

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 Glycogen
phosphorylase

(Glucose)n + Pi (Glucose)n-1 + Glucose 1-phosphate

Glycogen

Glycogen Glycogen
Inactive form phosphorylase phosphorylase Active form

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Some enzymes are regulated by
proteolytic cleavage of an
enzyme precursor

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Digestive enzymes are synthesized and stored as
inactive precursors – zymogens or proenzymes

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Adapted from Biochemistry, 4th edition by Zubay (1998)
 Activity of enzyme is regulated

Acute pancreatitis is a life-

threating disorder.

Pancreatitis-induced necrosis is
caused by activation of pancreatic
enzymes within the ductile system
of the pancreas.
During an episode of acute pancreatitis, trypsinogen comes
into contact with lysosomal enzymes (specifically cathepsin),
which activate trypsinogen to trypsin.

Serum contains natural inhibitor for

trypsin (antitrypsin)

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Google images Adapted from Biochemistry, 4th edition by Zubay (1998)
Enzymes in health and
diseases

56
Many drugs are enzyme inhibitors
Penicillin (found from fungus) specifically
inhibits transpeptidase that are needed for the
cross-linking of peptidoglycans during the final
step in cell wall biosynthesis in bacteria.

Penicillin has a strained four-membered lactam ring

that mimics the transition state of the normal
substrate.

When penicillin binds to the active site of the enzyme,

its lactam ring opens, forming a covalent bond with a
serine residue at the active site.

Penicillin is a potent irreversible inhibitor of bacterial

cell-wall synthesis, making the bacterium osmotically
fragile and unable to survive in the body.

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Adapted from Biochemistry, 4th edition by Zubay (1998)
HIV proteases are targets of enzyme inhibition

58
https://doi.org/10.2147/HIV.S300055 Adapted from Biochemistry, 4th edition by Zubay (1998)
 HIV protease inhibitor

The inhibitors mimic the substrate at transition-state

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Lehninger Principles of Biochemistry, David L. Nelson & Michael M. Cox, 7th Ed. (2017) Adapted from Biochemistry, 4th edition by Zubay (1998)
 Irreversible enzyme
inhibition can be lethal

60
Adapted from Biochemistry, 4th edition by Zubay (1998)
Organophosphorus are potent neurotoxins !

Diisopropylphospho-
fluoridate (DIPF)

Acetylcholine is a
neurotransmitter

61
Adapted from Biochemistry, 4th edition by Zubay (1998)
Enzymes are importance
markers for medical
diagnosis and treatment

62
Adapted from Biochemistry, 4th edition by Zubay (1998)
 63
Adapted from Biochemistry, 4th edition by Zubay (1998)
Industrial applications of Enzymes

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Adapted from Biochemistry, 4th edition by Zubay (1998)
 Actilyse treats acute ischemic stroke
and acute myocardial infarction (by
venus injection)

Actilyse (Alteplase or t-PA) is a thrombolytic medication.

T-PA is a serine protease that binds to fibrin (a major component of the

blood clot) in a thrombus and initiates fibrinolysis
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Adapted from Biochemistry, 4th edition by Zubay (1998)
 References

• Medical Biochemistry, by John W. Baynes & Marek H. Dominiczak,

5th Ed. (2019) – Chapter 6

• Lehninger Principles of Biochemistry, by David L. Nelson & Michael

M. Cox, 7th Ed. (2017) - Chapter 6

• Lippincott’s Illustrated Reviews: Biochemistry 5th Edition, by Richard

Harvey & Denise Ferrier (2011)

• Biochemistry 5th Edition, by JM Berg, JL Tymoczko & L Stryer (2002)

• Online resources (e.g. AK Lecture)

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