Module 7: Overview

Classes of enzymes
A) Enzyme substrate complex
Enzyme substrate complex
Reaction Activation energy

Enzyme catalysis
B) Enzyme catalysis and Chymotrypsin
Catalytic mechanism of Chymotrypsin
Catalytic Triad

C) Neuraminidase and Enzyme inhibition Competitive inhibitors as drugs

Why is Relenza such a good inhibitor?

Hypothesis of an ES complex
D) Enzyme kinetics and enzyme regulation Michaelis-Menten Kinetics
Kinetics of Enzyme Inhibition

1
Why do we want to study enzyme function?

The following 3 chemicals are enzyme inhibitors – they inhibit cyclooxygenases

They have a world-wide market of $billions (11.4 in 2014)!
O

1 – this drug inhibits COX-1

OH It has good anti-coagulant properties
Many side effects
O Covalent inhibitor!

H
N
2 – this drug inhibits COX-2 (and 3?)
It has no effect on blood clotting
It is hepatoxic, mechanism not well understood
O
HO

OH
3 – the exact mechanism is unknown
It is a non-specific inhibitor of COX-2(?)
O
This molecule is chiral (what does that mean?)
Molecular recognition is at the heart of drug development

 So what we are going to look at is how we do

the biochemistry of enzymes so that we
understand the chemistry of catalysis, and then
you should be able to apply that knowledge to
understanding inhibition which is how many
drugs work

March 2021 Molecule of the Month Cisplatin

bound to DNA
https://pdb101.rcsb.org/motm/255

Cisplatin increased the cure rate for testicular

cancer from 5% to 80%
Enzymes and substrates

Recall from our last section:

• Proteins bind ligand molecules ex: myoblobin binds O2

No change to O2 or the protein – simple binding reaction

Compare with enzymes:
• Enzymes also bind “ligands” but these are now called substrates.

• Substrates are chemically modified by enzymes – substrate is

changed into the product.

The basic equation for an enzyme reaction is:

E(Enzyme) + S(Substrate) → E(Enzyme) + P(Product)

What are enzymes, and how do they work?

Concept of flexible active site stated earlier by Linus Pauling

(1946):

 Hypothesized that an enzyme is a flexible template that is

most complementary to substrates at the transition state
rather than at the ground state

 As reaction proceeds, enzyme conforms better to the

transition-state structure

 Transition-state stabilization results in rate enhancement

Lock-and-key versus induced-fit
The enzyme-substrate complex

kcat1

E+S ES EP E+P

kcat-1

E = enzyme; S = substrate; P = product

ES and EP are intermediate complexes
Reaction rate depends on activation energy

•For any reaction, including exergonic reactions, there is a barrier

– the activation energy, an energy quantity of the transition state.

•transition state represents a high-energy distortion of bonds that

reactants must go through to become a product! A transient
distortion that often has very unfavorable positions.
We use a free energy diagram to describe a reaction

Reaction coordinate diagram. The free energy of the system is plotted against the progress of
the reaction S → P. A diagram of this kind is a description of the energy changes during the
reaction, and the horizontal axis
(reaction coordinate) reflects the progressive chemical changes (e.g., bond breakage or
formation) as S is converted to P. The activation energies, ΔG‡, for the S → P and P → S
reactions are indicated. ΔG′° is the overall standard free energy change in the direction S → P
Transition States and Reaction Rates

k
S P
k-1
An example of a transition state

Chapter 8, Figure 8.2c, Free energy diagrams for the simple reaction AB
How do we lower the activation energy?

- We could add heat – but biological systems operate at a narrow temp range
(except for extremophiles)

- We could find some way to lower the ΔGo‡

- Transition state theory says that the catalyst binds the transition state to achieve
this. Our catalyst is an enzyme.

- In this case the enzyme actually distorts the substrate into a shape resembling
the transition state – this “activates” the substrate for a chemical transformation.
Enzymes stabilize the transition state to achieve rate
enhancement (catalysis)
Rate Equations and Constants

k k
S P S1 + S2 P
k-1 k-1
Second-order reaction
First-order reaction
[P]
Equilibrium constant Keq = [P] Keq =
[S1] [S2]
[S]
Reaction equationV = k[S] V = k[S1][S2]

k has units of s-1 k has units of M-1s-1

In order to understand catalysis we need to consider two kinds of reactions.

1st order where the rate is proportional to concentration of the single starting material

2nd order where the rate is dependant on the concentrations of two reactants
Sample Enzymatic Rate Enhancements
Intermediate states are important for overcoming the energy barrier

The [ES] complex is

not the transition
state – but an
intermediate on the
way to the transition
state.

k1 k2
E + S  [ES]  [EP]  E + P
How to lower the activation energy?

Covalent Mechanism: Formation of transient covalent

intermediates between substrate functional groups and enzyme
functional groups in active site – alternate states of lower energy!

Non-covalent Mechanism: weak, favorable non-covalent

bonds between enzyme and substrate (and transient state).
Each interaction releases a small amount of free energy
called binding energy ΔGB.
Many interactions add to a large –ve ΔGB. This lowers the
activation energy of reactions!
ΔGB = weak interactions during binding specificity + weak
interactions that stabilize the transient states!!
How to lower the activation energy? (2)

Through binding substrate, transition state, product:

• increases local concentration
• orients and restricts movement of functional groups (entropy
reduction)
• specificity of reaction
• microenvironment – desolvation for example
How to lower the activation energy? (2)
Summary: Reaction coordinate diagram for a simple enzyme-catalyzed reaction

Can you identify and explain

each step?
Role of binding energy in catalysis

Can you identify and explain

each step?

Role of binding energy in catalysis. To lower the activation energy for a reaction, the
system must acquire an amount of energy equivalent to the amount by which ΔG‡ is
lowered. Much of this energy comes from binding energy, ΔGB, contributed by formation
of weak noncovalent. interactions between substrate and enzyme in the transition state.
Sample exam question

Which of the following statements about enzymes is true:

(A) Enzymes are optimized for binding their substrate in
its most stable conformation
(B) Enzymes can change the equilibrium of a reaction
(C) Enzymes can lower the activation energy of a reaction
(D) Enzymes can raise the activation energy of a reaction
(E) Enzymes are forever changed by their reactions

24
Lecture 7D
Enzyme kinetics
Outline
Initial rate of reaction (V0), the Michaelis constant, and Vmax
Concept of steady state kinetics of a enzymatic reaction
Assumptions of steady state kinetics lead to the Michaelis-Menten equation
Kinetics of Enzyme Inhibition
Allosteric regulation

Learning Objective
Understand the basic quantitative models that help determine enzyme kinetics
Compare enzyme specificities quantitatively. Interpret v/[s] and lineweaver plots.
Describe and differentiate between different types of reversible enzyme inhibition using
quantitative measures
25
What is Enzyme Kinetics?

Kinetics is the study of the rate at which compounds react

•Rate of enzymatic reaction is affected by

• Enzyme concentration
• Substrate concentration
• Effectors
• Temperature
• pH
Why Study Enzyme Kinetics?

It is a quantitative description of biocatalysis

•Determine the order of binding of substrates

•Elucidate acid-base catalysis

•Understand catalytic mechanism

•Find effective inhibitors – like potential drugs

•Understand regulation of activity

Experimental observations led to the hypothesis of an ES complex
This kinetic pattern led scientists to propose in 1903 that the combination
of an enzyme with its substrate molecule to form an ES complex is a
necessary step in enzymatic catalysis. This idea was expanded into a
general theory of enzyme action, particularly by Leonor Michaelis and
Maud Menten in 1913. They postulated that the enzyme first combines
reversibly with its substrate to form an enzyme-substrate complex in a
relatively fast reversible step:

The ES complex then breaks down in a slower second step to

yield the free enzyme and the reaction product P:

Because the slower second reaction must limit the rate of the
overall reaction, the overall rate must be proportional to the
concentration of
the species that reacts in the second step—that is, ES.
ES complex formation and Vmax

k1
E+S ES Fast and reversible
k-1
k2 = (kcat)
ES E+P Slower, rate-limiting
k-2 [ES] is going to determine overall rate

At low [S], there is little ES – most enzyme is unoccupied

At low [S], rate is proportional to [S] –more S, forces ES formation!

At high [S], enzyme is mostly in ES – thus [ES] becomes main

parameter – further increase in [S] has little effect – can’t make
more ES! Thus Vmax is achieved.
 The basic kinetic equation for this course

Enzyme Substrate Enzyme- Enzyme- Enzyme Product

Substrate Product
E S complex complex
E P
ES EP

Reaction rate V = kcat[ES]

A theoretical enzyme kinetic profile: [ES] is essentially constant – so we call this
steady state

The steady state in enzyme kinetics. The figure

shows how the concentrations of substrate [S],
free enzyme [E], enzyme–substrate complex [ES],
and product [P] vary with time for a simple
enzyme catalyzed reaction described by E + S H
ES ¡ E + P. After a very brief initial period, [ES]
reaches a steady state in which ES is consumed
approximately as rapidly as it is formed, so
d[ES]/dt ≈ 0.
The concentrations of E and ES are greatly
exaggerated for clarity.
Note that [E] + [ES] = [E]t, or total enzyme
concentration, and that [ES] actually falls very
slowly as substrate is consumed, while [E]
accordingly rises.
Steady-state Kinetics

•Studies the initial rate, which we call V0

•At this stage, [Substrate] can be assumed to be nearly

constant
•[Product] is very small

•ES breakdown to E + P is rate limiting step - kcat

•Enzyme-Substrate complex is nearly constant

•Overall rate proportional to [ES] rate = kcat[ES]
difficult to measure [ES]
k1 kcat so, use:
E+S ES E+P 1. observed rate
k-1 2. initial [S]
3. total [E] 4. [P]
Michaelis-Menten kinetics

𝑘1 𝑘𝑐𝑎𝑡
E+S 𝑘 −1
ES E+P
 Km is the concentration of substrate that produces half the
maximum reaction rate (Vmax)

 Km is a dissociation constant of ES, so the smaller the Km

the stronger the interaction between E and S

 We are assuming the EP breakdown is fast – but that is not

always true.

[ E nz ] [¿ ] (k-1 + kcat)
K M= =
[ ES ] k1
Enzymes display saturation kinetics

rate = kcat[ES]
Vmax = kcat[E]t
Significance of Km and Vmax

Vmax and KM are the two parameters which define the kinetic behavior of an enzyme
as a function of [S].

Vmax is a rate of reaction. It will have units of: moles/min, moles/s, umol/min etc.
Vmax depends on the structure the enzyme itself and the concentration of enzyme
present.

KM is the concentration substrate required to approach half the maximum reaction

velocity. if [S]>>Km then Vo will be close to Vmax.

KM is a concentration. It will have units of: (M/L),or (uM/L),etc.

KM depends only on the structure of the enzyme and is independent of enzyme

concentration.

36
Interpreting kcat and kcat/Km

kcat
= the specificity constant for the enzyme when [S] << Km
Km - it is used to compare catalytic efficiencies of different enzymes.

k is referred to as the turn over number, or the number of molecules of

cat
substrate converted per molecule of enzyme.

Under [S] << Km the ratio of kcat/Km provides a direct measure of enzyme
efficiency for that enzyme and different substrates.
Vmax and KM can be determined from a double reciprocal
plot (Lineweaver-Burk)
Kinetics terms you should know

Km = the equilibrium constant for the ES complex,

and is also dependant on the rate of catalysis.

kcat = the limiting rate for any enzyme reaction at

saturation (turn over number). This is Vmax/[Et].
This is the number of substrate molecules turned
to product per unit of time.
kcat
= the specificity constant for the enzyme when [S] << Km
Km - it is used to compare catalytic efficiencies of different enzymes.

Simplified Michaelis-Menton mechanism when kcat is rate limiting

k1 kcat
E+S ES E+P
k-1
Sample kcat/Km question (Mastering q68 Tutorial)

The Michaelis-Menten parameters, kcat and KM , for selected enzymes are given in the table below. Use these
parameters to determine the catalytic efficiency of each enzyme. Based on your calculations arrange the enzymes in
increasing order of their efficiencies.

Note catalytic efficiency is the same as ‘specificity’, so we calculate the specificity constant Kcat/Km for each enzyme:

e.g. urease = kcat=1.0×104 s−1 divided by KM=2.5×10−2 M = 4.00×10^5M −1s−1

Ranked from highest to lowest : Crotonase > Acetylcholinestaerase > Catalase > Triosephosphate > Urease

Therefore Crotonase is the most specific (efficient) enzyme. Note Km is also a dissociation constant for the ES complex, so
Crotonase has also the lowest Km (binds the substrate with greatest affinity).

40
Sample Vmax/Km question (Mastering q68 Tutorial cont’d)

Given this data, enzyme _____obeys the Michaelis-Menten equation. Analyzing the shape of this plot,
the estimated value of Vmax is _____mmol/s and Km is ______mmol/L.

You can plot these values but inspecting the numbers

Is usually sufficient. Here enzyme B plateaus and resembles
MM kinetics, so we take the [S] at 50% of Vmax (read from
Table) Km= 8mmol/L; Vmax is plateau point = 16 mmol/s

41
An enzyme is discovered that catalyzes the chemical reaction

A team of motivated researchers sets out to study the enzyme, which

they call
happyase. They find that the kcat for happyase is 600 s-1 and carry out
several
additional experiments.
When [Et] = 20 nM and [SAD] = 40 μM, the reaction velocity, V0, is 9.6 μM
s-1. Calculate Km for the substrate SAD.

We know kcat, [Et], [S],

and V0. We want to solve
for Km.
Kinetics of Enzyme Inhibition

•Many drugs are enzyme inhibitors. The development of some drugs has been
advanced by kinetic studies, as well as structural studies of the target enzymes.

•We will consider only one class of enzyme inhibitors:

• reversible inhibitors (noncovalently bound)

• irreversible inhibitors (covalently bound) are also common but we don’t

have time to cover them in this course.
Reversible Inhibition: Competitive Inhibition

Alpha value: If Km was 100 nM and changes to 300 nM then α = 3

Think of this as the degree of inhibition.
Graphical view of reversible inhibition : competitive inhibition

Apparent KM is increased
Vmax is unchanged
Reversible Inhibition: Uncompetitive
Inhibition

There is a protein conformation change upon inhibitor binding which prevents catalysis.
Graphical view of reversible inhibition: uncompetitive inhibition
Mixed inhibition : a special case
Graphical view of reversible inhibition: mixed inhibition
Make sure you know how they are different!

or increased

50
Sample Vmax question (Mastering q68 Tutorial)

An enzyme that follows Michaelis-Menten kinetics has a KM value of 7.00 μM and a kcat value of 206 s−1 . At an
initial enzyme concentration of 0.0100 μM , the initial reaction velocity was found to be 1.07×10−6 μM/s . What was
the initial concentration of the substrate, [S] , used in the reaction ?

Use the most convenient form (the one on the left)

kcat = (206 s−1 ) and [E]t = (0.0100 μM )

Km = 7.00uM, v=1.07x10-6 uM/s

Plug in and solve for [S]

[S]i =3.64×10−6μM

51
Sample Ki question (Mastering 8.16)
Enalaprilat is a competitive inhibitor of the angiotensin-converting
enzyme (ACE), which cleaves the blood-pressure regulating peptide angiotensin I.
ACE has a Km = 12 uM for angiotensin I, which is present
in plasma at a concentration of 75 uM. When enalaprilat is present at
2.4 nM, the activity of ACE in plasma is 10% of its uninhibited activity.
What is the value of Ki for enalaprilat?

The inhibited reaction will have an apparent KM = KM, and the observed velocity will be 10% of the
uninhibited reaction under the conditions given in the problem. These two ideas can be expressed
mathematically in this way:
Vmax[S] 0.1Vmax[S]
vinhibited  =
 K M  [S] K M  [S]

this can be rearranged to give (divide by Vmax[S] on both sides):

 K M  [S]
10 
K M  [S]
solve for  using the given values of [S] and KM:
[I ]
 66.25 1
KI
See text pg 287 for alpha = (1+ [I]/KI)
solve for KI
2.410 9 M
KI  3.710 11M
66.25
53
In most enzymes, the binding energy used to form the ES complex is just one
of several contributors to the overall catalytic mechanism. Once a substrate is
bound to an enzyme, properly positioned catalytic functional groups aid in
the cleavage and formation of bonds by a variety of mechanisms, including
general acid-base catalysis, covalent catalysis, and metal ion catalysis. These
are distinct from mechanisms based on binding energy because they
generally involve transient covalent interaction with a substrate or group
transfer to or from a substrate.
Enzymatic catalysis proceeds by one or more of:

•General acid/base catalysis (GABC)

•Covalent catalysis
•Electrostatic stabilization
• 1/3 of enzymes are metalloenzymes
•Proximity effects
• The closer reactants are the faster the
rate
•Preferential stabilization of the transition
state
•Protein conformational changes also play an
important role in catalysis

This is why enzymes are often MUCH bigger than their substrate!
General acid-base catalysis

Amino acids in general acid-base catalysis. Many

organic reactions that are used to model biochemical
processes are promoted by proton donors (general
acids) or proton acceptors (general bases). The active
sites of some enzymes contain amino acid functional
groups, such as those shown here, that can
participate in the catalytic process as proton donors
or proton acceptors
Enzymes use acid-base chemistry for many reactions! This is why we care about amino acid pKa

What are the possible amino acids

in this diagram?

What are their normal side chain

pKa’s?
As you already know, some chemistry cannot be performed directly by the amino acid side
chains – so we use molecules called co-enzymes.
Metal ions in enzymes – help with a variety of reactions.
Covalent Catalysis: Enzyme Example

H2 O
A-B A + B
H2 O
A-B + X: A-X + B A + X: + B

• Amino acid functional (R-) group can react to form

transient covalent species with a reactant

• Sometimes this is a coenzyme (e.g. prosthetic) functional

group

• At the end of reaction, functional group is reformed

Covalent Catalysis: In Enzymes

• Proteases and peptidases

• chymotrypsin, elastase, subtilisin
• reactive serine nucleophile x-CH2-O-
• Some aldehyde dehydrogenases
• glyceraldehyde-3phosphate dehydrogenase
• reactive thiolate nucleophile x-CH2-S-

• Aldolases and decarboxylases

• amine nucleophile x-CH2-NH2:
• Dehalogenases and some glycosidases
• carboxylate nucleophile x-CH2-COO-
Chymotrypsin is a serine endopeptidase
produced by the acinar cells of the pancreas.

Chymotrypsin becomes activated after

proteolysis of chymotrypsinogen by trypsin.

While trypsin hydrolyzes at lysine and arginine,

chymotrypsin selectively cleaves peptide bonds
formed by aromatic residues (tyrosine,
phenylalanine, and tryptophan).

How is this specificity achieved, and how does

catalysis take place?
Specificity of chymotrypsin. Chymotrypsin cleaves proteins on the
carboxyl side of aromatic or large hydrophobic amino acids
(shaded orange). The likely bonds cleaved by chymotrypsin are
indicated in red.
Chymotrypsin – a surface view
Inhibitors as research tools – DIPF protease inhibitor

An unusually reactive serine residue in chymotrypsin.

Chymotrypsin
is inactivated by treatment with diisopropylphosphofluoridate
(DIPF),
which reacts only with serine 195 among 28 possible serine
residues.
(a) A representation of primary
structure, showing disulfide bonds and
the amino acid residues crucial to
catalysis. The protein consists of three
polypeptide chains linked by disulfide
bonds.
The active-site amino acid
residues are grouped together in the
three-dimensional structure. (b) A
depiction of the enzyme emphasizing
its surface. The hydrophobic pocket in
which the aromatic amino acid side
chain of the substrate is bound is
shown in
yellow. Key active-site residues,
including Ser195, His57, and Asp102,
are red. (c) The
polypeptide backbone as a ribbon
structure. Disulfide bonds are yellow;
the three chains are colored as in part
(a).
Specificity pocket of chymotrypsin. Notice that
this pocket is lined with hydrophobic residues
and is deep, favoring the binding of residues
with long hydrophobic side chains such as
phenylalanine (shown in green). The active site
serine residue (serine 195) is positioned to
cleave the peptide backbone between the
residue bound in the pocket and the next
residue in the sequence. The key amino acids
that constitute the binding site are identified.
The active site of chymotrypsin
 The catalytic machinery for chymotrypsin
Asp102

His57

Normal pKa for His = 6

In Chymotrypsin the
pKa for His57 = >12

What does that mean

for the reaction? Ser195

Gly193
The catalytic triad
The catalytic triad, shown on the left, converts serine 195
into a potent nucleophile, as illustrated on the right.

The side chain of serine 195 is hydrogen bonded to the imidazole ring of histidine 57.
The —NH group of this imidazole ring is, in turn, hydrogen bonded to the carboxylate
group of aspartate 102. This constellation of residues is referred to as the catalytic
triad.
The mechanism of chymotrypsin – formation of a covalent intermediate
After substrate binding (step 1), the reaction
begins with the
oxygen atom of the side chain of serine 195
making a nucleophilic attack on
the carbonyl carbon atom of the target peptide
bond (step 2). There are now
four atoms bonded to the carbonyl carbon,
arranged as a tetrahedron,
instead of three atoms in a planar
arrangement. This inherently unstable
tetrahedral intermediate bears a formal
negative charge on the oxygen atom
derived from the carbonyl group. This charge is
stabilized by interactions with NH groups from
the protein in a site termed the oxyanion hole
 Formation of a tetrahedral intermediate (oxyanion hole)

www.youtube.com/watch?v=gJNMryCX3YY
How the oxyanion hole stabilizes a transition state

https://www.youtube.com/
watch?v=K21VDO6CtJk

74
Covalent catalysis involves the formation of a covalent intermediate;
The Acyl-Enzyme intermediate

Watch this for the complete sequence

https://www.youtube.com/
watch?v=OY1WsqlcUdo
The second step is hydrolysis using water: a second covalent intermediate forms
The second step is hydrolysis using water: a second covalent intermediate forms

https://www.youtube.com/
watch?v=gJNMryCX3YY

77
Reaction summary : 8 steps
What you need to know about Chymotrypsin cleavage of peptides:

• What is the nucleophile?

• What other amino acids could fill that role?

• How is the nucleophile activated?

• What is the role of His57?
• What is the role of Asp102?

• What is the acyl-enzyme intermediate?

• How is product released from this intermediate?

DO NOT SIMPLY MEMORIZE THE STEPS – make sure you know the chemical
logic of this mechanism
Sample exam question
Chymotrypsin uses a catalytic triad for catalysis: Asp102-His57-Ser195.

(i) In a mutant version of chymotrypsin, Asp102 is changed to Asn102. In this mutant with Asn102,
the activity
Vmax of Chymotrypsin is decreased 5000-fold (compared to the normal protein).
Explain how this could this happen. If a diagram helps draw one. (2 marks)

(ii) At pH 5 Chymotrypsin has very little enzyme activity. Why is that the case? (2 marks)

(iii) Chymotrypsin uses a two-step mechanism. What is the chemical nature of the common
intermediate in both step? (2 marks)

(iv) Why is Chymotrypsin specific for cleavage near aromatic amino acid? (2 marks)

Answer:
1. Asn102 cannot orient the His57 in the same way as Asp102 – because the critical h-bond is missing.
Without this h-bond His57 cannot be a strong base to activate Ser195. (2 marks)
2. His57 is protonated at pH 5 and so cannot act as base to activate Ser 195. (2 marks)
3. Both steps proceed through a covalent intermediate called the “acyl-enzyme intermediate” (2
marks).
4. There is a hydrophobic pocket that the aromatic amino acids fit into to create specificity. (2 marks)

80
Catalytic triads are found in other hydrolytic enzymes

Structural similarity of
trypsin and chymotrypsin. An overlay of
the structure of chymotrypsin (red) on that
of trypsin (blue) is shown. Notice the high
degree of similarity. Only a-carbon-atom
positions are shown.
What we’ve done…

General acid-base catalysis becomes crucial in the active site of an

enzyme, where water may not be available as a proton donor or acceptor

The Ser195 in chymotrypsin is converted into a strong nucleophile by means of its position
in a catalytic triad with His57 and Asp102

The chymotrypsin reaction proceeds in eight steps:

(1) substrate binding, (2) nucleophilic attack of serine on the peptide carbonyl group,
(3) collapse of the tetrahedral intermediate, (4) release of the amine component,
(5) water binding, (6) nucleophilic attack of water on the acyl-enzyme intermediate,
(7) collapse of the tetrahedral intermediate; (8) release of the carboxylic acid component.

The catalytic triad is a constellation of residues that is found in other enzyme (hydrolases, transferases)
reaction active sites

82
Competitive enzyme inhibitors as drugs

An example of this is a drug used to treat Influenza virus.

You should be able to recognize the “flu” drug as a mimic

of the transition state – and a good competitive inhibitor.

Influenza A virus neuraminidase (aka sialidase)

The influenza virus: a continuing health
risk
Haemagglutinin

H5N1

Neuraminidase

Sialic acid, Neu5Ac, Neuraminic acid

Epidemics & pandemics
The functional role of viral neuraminidase
Action of a neuraminidase inhibitor
Mechanism of neuraminidase

O O O O O O
H H
O H O
OH H2O ROH OH O OH
HO CO2H HO HO CO2H
OH OH OH
O OR O CO2H O OH
AcHN AcHN AcHN
HO H HO O HO H
H H
O O O O O O
sialosyl-enzyme
intermediate
How is the binding of Relenza different from Sialic acid?

Sialic acid Relenza

So why is Relenza such a good inhibitor?

This inhibitor engages other

amino acids (E227) through
interaction with the guanidinyl
group at the C4 position.
Relenza looks more like the
transition state than substrate.
What we’ve done…

• The chemistry of amino acid chains drives biological reactions!

• pKas can be drastically altered by the environment the amino acids are in.
Like what we see for His57 in chymotrypsin.

• The availability of inhibitors as research tools facilitates our understanding of

how enzyme architecture contributes to activity.
• Normally we can not trap substrates as they turn over, so we rely on
inhibitors to provide the snapshot of the active site.

• Protein structure aided design of drugs needs a well understood enzyme

mechanism, and requires a high resolution crystal structure with a bound ligand.

• Understanding the mechanism of an enzyme and having a crystal structure are

powerful tools in drug design.
• Filling a negatively charged pocket in neuraminidase with a positively
charged group provides a 106 fold increase in inhibition. This is an
incredibly large increase for a simply chemical change.
Regulation of enzyme activity

In cells the activity of most enzymes is highly regulated in order to

keep homeostasis.

Regulation at committed steps of pathways

 feedback inhibition/activation at control points – don’t waste energy
making something you have enough of
 often mediated by allosteric enzymes – Enzymes where effectors
bind at sites other than the active site

Covalent modification
 reversible (e.g., many cell signaling processes, like phosphorylation)
 irreversible (e.g., zymogen activation; blood clotting – proteases
activate other enzymes).
Regulation of enzyme activity : Serines,
Threonines and Tyrosines!!
Feedback inhibition

Reversible covalent modification

 (phosphorylation, can be both
 activating or deactivating)
Allosteric regulation of aspartate transcarbamoylase

CTP, UTP, TTP

ACTase is regulated by CTP – the end product of the pathway

ACTase is negatively regulated by CTP and positively regulated by ATP

Why would this be the case?
Allosteric regulation of aspartate
transcarbamoylase

Idealized curve – look familiar?