Introduction to Enzymes:

Basic concepts of enzyme action

Notes Authored by: Professor Victor Gault

BMS102 Biochemistry
Weeks 2 and 3: 2018

Enzymes: Powerful Catalysts

• An enzyme is a biological catalyst
• Without enzymes to speed up biochemical reactions
life would not exist
• Nearly all enzymes are proteins
• Allow reactions to occur under mild (physiological
conditions)
• Highly specific
• Subject to regulation
Example:

Carbonic anhydrase (one of the fastest enzymes known)

CO2 + H20 → H2CO3 107 times faster

Each enzyme molecule (carbonic anhydrase) can hydrate about 106 molecules of CO2
per second

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 Enzymes: Highly Specific
• Specific both in reactions they catalyse and their choice
of reactants (or substrates)
• Catalyse a single reaction OR a series of closely
related chemical reactions
Example: proteolytic enzyme(s) – hydrolyse peptide bond(s)

Arg H

Trypsin Thrombin

Enzyme specificity due to precise interaction of substrate

with enzyme (precision due to 3D structure of enzyme)

Enzymes: Six Major Classes

Class 1: OXIDOREDUCTASES: catalyse oxidation-
reduction reactions
These enzymes transfer electrons between molecules
Example: Lactate Dehydrogenase

Note: requires the coenzyme nicotinamide adenine dinucleotide (NAD)

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 Enzymes: Six Major Classes
Class 2: Transferases: catalyse transfer of functional
groups between molecules
Aminotransferases (a class of transferases) shuffle amine
groups between donor and acceptor molecules
Example: Alanine Transaminase

Enzymes: Six Major Classes

Class 3: Hydrolyases: catalyse hydrolysis by cleaving
molecules by the addition of water
Trypsin is an example (seen earlier) and another is:
Example: Pyrophosphatase

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 Enzymes: Six Major Classes
Class 4: Lyases: catalyse addition of atoms or functional
groups to a double bond OR removes them to form a
double bond
Example: Fumarase

Enzymes: Six Major Classes

Class 5: Isomerases: catalyse movement of functional
groups within a molecule
Generally simplest enzymatic reactions as only 1 substrate
and 1 product
Example: Alanine racemase

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 Enzymes: Six Major Classes
Class 6: Ligases: catalyse bond formation or joining two
molecules AT EXPENSE OF ATP.
An example is DNA ligase or another one is:
Example: Glutamine synthetase

Enzymes: Many Require Co-Factors

• Whilst amino acid functional group provides diversity,
catalytic activity of many enzymes require co-factors
• Enzyme without its co-factor is termed an apoenzyme
• Complete catalytically active enzyme is a holoenzyme
• Co-factors:
(1) small organic molecules derived from vitamins and
called coenzymes and
(2) metals
• Tightly bound coenzymes are called prosthetic (helper)
groups
APOENZYME (inactive) + cofactor

HOLOENZYME (active)

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 Enzymes: Examples of Co-Factors

Co-factor Enzyme

Coenzyme
Thiamine pyrophosphate (TPP)
Flavin adenine nucleotide (NAD)
Nicotinamide adenine dinucleotide (NAD)
Coenzyme A (CoA)
Pyruvate dehydrogenase
Monoamine oxidase
Lactate dehydrogenase
Acetyl CoA carboxylase

Metal
Zn2+ Carbonic anhydrase
Ni2+ Urease
Se Glutathione peroxidase
K+ Acetoacetyl CoA thiolase

Enzymes: Thermodynamic Free Energy

• Enzymes speed up the rate of chemical reactions but properties of the
reaction (ie. it takes place at all) depends on free-energy differences.
• Free energy (G) is measure of useful energy (i.e capable of doing work).
• The free-energy change of a reaction (∆G) informs us as to whether a
reaction can take place spontaneously:
(1) Reaction can take place spontaneously only if ∆G is negative
(exergonic)
(2) Reaction cannot take place spontaneously if ∆G is positive
(endergonic)
(3) At equilibrium, no net change in concentration of products and
reactants and ∆G is zero
(4) ∆G of a reaction is independent of the path (or molecular
mechanism) of the transformation
(5) ∆G provides no information about the rate of a chemical reaction

Negative ∆G indicates a reaction can take spontaneously - it does not

signify the rate that a reaction will proceed (free energy of activation)

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Enzymes: Reaction Rate and Equilibrium
• Enzymes cannot change the Laws of Thermodynamics – they cannot
alter the equilibrium of a chemical reaction

aA bB

[B]b [Product]
Keq = =
[A]a [Reactant]
Keq is equilibrium constant - reflection of
difference of ∆G between reactants &
products Same equilibrium point is reached but more
quickly in the presence of the enzyme

• Reaction has reached equilibrium – reactant still being converted to

product, but product is being converted back into reactant at a rate
whereby the concentration of product stays the same

Enzymes accelerate attainment of equilibrium but do not shift their

positions

Enzymes: Transition State

• ∆G (reactants and products) accounts for equilibrium of a reaction
• How do enzymes accelerate how quickly this equilibrium is attained?

S X‡ P

X‡ is called a Transition State – it has higher ∆G than does S or P

X‡
X‡ is an unstable arrangement of
atoms – chemical bonds are in the
process of being broken or formed

Difference in free energy between

transition state and substrate is the
free energy of activation or
activation energy (∆G ‡)

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 Enzymes: Activation Energy
• Enzymes function to lower the ∆G‡ OR facilitate the
formation of the transition state

P + Q have energy below A + B

Reaction proceeds from A +B to P + Q

• Substrate and enzyme combine to create a pathway whose transition-

state energy is lower than when enzyme is not present
• More molecules have required energy to reach transition state and thus
more product is formed faster (BUT NOT ANY MORE PRODUCT!)
At the heart of enzyme catalysis is stabilisation of the
transition state

Enzymes

Enzymes do not

Change the equilibrium constant

Change ∆G reaction [∆G = ∆G Products - ∆G Reactants ]

Enzymes do

Lower the ∆G‡ for the reaction

Stabilise the Transition State

Make the forward reaction much more favourable

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 Enzyme-Substrate Complex
Reactants that enter into an enzymatic reaction are
termed SUBSTRATES Enzyme
+
Transition State Product

E + S ES ES* EP E+ P
Enzyme Enzyme-Substrate Enzyme-Product
+ Complex Complex
Substrate
Active
Site

Rate of reaction (Velocity)

Rate of reaction (Velocity)

Uncatalysed Catalysed

Substrate Concentration Substrate Concentration

Active Site
• Region of an enzyme that binds substrates (and co-factors, if any)
• It is the interaction of the enzyme and the substrate at the active site that
promotes formation of the transition state
• Active site region that most directly lowers ∆G‡ of the reaction
• The Active Site:
(1) Is a 3D cleft or crevice (different parts of aa sequence. In
Lysozyme aa residues 35, 52, 62, 63, 101 and 108)
(2) Small part of the total volume of enzyme (‘extra’ aa used in
regulatory role or form channels to bring substrates to active sites
(3) Unique microenvironment (nonpolar environment enhances
binding of substrates and catalysis – water usually excluded)
(4) Substrates bound to enzymes by multiple weak attractions
(non-covalent weak reversible interactions – H-bonds, vdWaals)
(5) Binding specificity depends on precisely defined
arrangements of atoms in the active site (due to short-range
forces close contact is required – substrate must have matching
shape to active site)

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 Substrate Binding
Substrate bound in the active
site by multiple weak bonds
(mainly non-covalent)

Electrostatic interactions
Hydrogen bonds
Van der waals
Hydrophobic interactions

Lock and Key Theory

• Emil Fischer (1894) – enzyme is a rigid template (LOCK) and the
substrate is a matching KEY
• Active site of the enzyme is complementary in shape to the substrate

This theory hinged on the idea

that the active site residues are
completely immobile

We now know that the active

site in enzymes is flexible

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 Induced-Fit Theory
• Daniel Koshland Jr (1958) – flexible nature of protein structure taken
into account
• The active sites of some enzymes assume a shape that is
complementary to that of the substrate only AFTER substrate has been
bound

This leads to conforming

the shape of the active
site to the shape of the
substrate in its transition
state conformation
Dynamic recognition OR
induced-fit

Binding Energy
• When weak interactions between complementary enzyme and substrate
are formed free energy is released – binding energy
• This full complement of interactions or binding is only formed when the
substrate is in the transition state
• Maximal binding energy released only when the enzyme facilitates
formation of the transition state
• Experimentally how do we know that transition state is important in
catalysis?

Transition-state analogues: potent inhibitors of enzymes

Planar TS where alpha-C is trigonal – not tetrahedral

Pyrrole-2-carboxylate also has trigonal geometry: it
can bind to the racemase enzyme 160 times tighter
than proline. It is a potent inhibitor
TS analogues can be used as useful enzyme
inhibitors

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 Enzyme Kinetics and
Regulation

Enzyme Kinetics
• Kinetics – study of the rates of chemical reactions (enzyme kinetics is
the study of the rates of enzyme-catalysed reactions)
• What is the ‘rate’ or ‘velocity’ of a chemical reaction?

A P
• Velocity (V) is the quantity of reactant, A, that disappears in a specified
unit of time, t. (equal also to velocity of the appearance of P)

V = -d[A]/dt = d[P]/dt
d is decrease in substrate concentration or increase in product
concentration (can follow spectrophotometrically)

• Velocity is directly related to concentration of A by a constant, k, called

the rate constant

V = k[A]

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 Enzyme Kinetics
• Reactions in which V is directly proportional to reactant concentration
are called first-order reactions
• First-order rate constants have the unit of s-1
• Many reactions do not simply involve one reactant but two (second-
order reactions)

A + B P

V = k[A][B]

• Second-order rate constants have the unit of M-1s-1

NB: Some second-order reactions can appear to be first-order reactions.
If [B] exceeds that of [A] and if [A] is present in low concentrations, reaction
rate will be first-order with respect to A and will not appear to depend on the
concentration of B: These reactions are termed pseudo-first-order-
reactions: quite a lot of these in biochemistry!!!

Michaelis-Menten Model
• Initial velocity of catalysis is
defined as number of moles of
product formed per unit time
shortly after a reaction has begun
– varies with [S] as shown

• Initial velocity rises linearly as [S]

increases and then begins to
level off (plateau)

k1 k2
E+S ES E+P
k-1 k-2

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 Progress curve for an
enzyme-catalyzed reaction

• The initial velocity (vo) is

the slope of the initial
linear portion of the
curve
• Rate of the reaction
doubles when twice as
much enzyme is used

Michaelis-Menten Model
• We can simplify by ignoring reverse reaction of product forming
substrate
• V0 is defined then as the rate of increase in product with time when [P] is
low – ie. at times close to the start of the reaction (hence, V0)
• So……V0 is determined for each substrate concentration by measuring
the rate of product formation at early times before P accumulates.

k1 k2
E+S ES E+P
k-1

• Velocity at the beginning of an enzyme-

catalyzed reaction is Vo (initial velocity)
• k1 and k-1 represent rapid noncovalent
association /dissociation of substrate from
enzyme active site
• k2 = rate constant for formation of product
from ES

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 Michaelis-Menten Model
• In 1913, Leonor Michaelis and Maud Menten proposed a simple model
to account for these enzyme characteristics
• Critical feature in their model is that a specific ES complex is a
necessary intermediate in catalysis
• The Michaelis-Menten equation describes variation of enzyme activity as
a function of substrate concentration:

[S]
V0 = Vmax
[S] + KM
Km is the Michaelis constant – unique to each enzyme and independent of
enzyme concentration
Vmax directly dependent on enzyme concentration and can only be attained
when all of the enzyme is bound to substrate

Michaelis-Menten Model

• Vmax reached when enzyme is saturated with substrate (high [S])

• High [S] - reaction rate is independent of [S]
• Low [S] reaction is first order with respect to S
• The shape of a V0 versus [S] curve is a rectangular hyperbola,
indicating saturation of the enzyme active site as [S] increases
• Km is equal to substrate concentration at which the reaction velocity
is half its maximal value

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 Km
• Km equal to substrate concentration at which half the active sites are
filled (half Vmax)
• Km provides a useful measure of substrate concentration required for
significant catalysis to take place
• The lower the value of Km the tighter the substrate binding
• Km can be a measure of the affinity of enzyme for substrate
• Km values depend on environmental conditions (pH, temperature etc.)

Enzyme Substrate Km (µM)

Chymotrypsin Acetyl-L-tryptophanamide 5000

β-galactosidase Lactose 4000
Pencillinase Benzylpencillin 50
Lysozyme Hexa-N-acetylglucosamine 6

Km: Clinical Relevance?

• Alcohol dehydrogenase in liver converts ethanol to acetaldehyde

• Aldehyde dehydrogenase then converts acetaldehyde into acetate
• There is a low km mitochondrial form of aldehyde dehydrogenase and a
high km cytoplasmic form of aldehyde dehydrogenase
• In some people, mitochondrial form is less active – thus less
acetaldehyde is converted to acetate
• Facial flushing, tachycardia etc…..

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 Vmax and kcat
• Vmax (or maximal velocity) essentially tells us about the turnover
number of an enzyme
• Number of substrate molecules that an enzyme can convert into product
per unit time when the enzyme is fully saturated with substrate
• Turnover number is equal to the rate constant k2 – termed kcat

Vmax = k2[E]T

k2 = Vmax/[E]T

SO

kcat = Vmax/[E]T

Examples: Carbonic anhydrase (600,000 s-1); acetylcholinesterase (25,000 s-1);

chymotrypsin (100 s-1); Lysozyme (0.5 s-1)

Kcat / Km

kcat/Km is a measure of catalytic efficiency

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 Measuring Km and Vmax

Reciprocal of both sides of michaelis-

menton equation
Plot 1/V0 versus 1/[S]. This is a double-
reciprocal plot or Lineweaver-Burk
plot

Straight line with a y-

intercept of 1/Vmax, slope
of km/Vmax and x-intercept
of -1/km

Self-Check Problem
The initial rate for an enzyme-catalysed reaction has been determined at a
number of substrate concentrations as follows:

[S] (µM) V (µM min-1)

3 10.4
5 14.5
10 22.5
30 33.8
90 40.5

Estimate Vmax and Km from a direct graph of V versus [S]. Do you think you
get clear answers?

Now use a Lineweaver-Burk plot to analyse the same data. Does this work
better?

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The initial rate for an enzyme-catalysed reaction has been determined at a
number of substrate concentrations as follows:

[S] (µM) V (µM min-1)

3 (0.33) 10.4 (0.096)
5 (0.20) 14.5 (0.069)
10 (0.10) 22.5 (0.044)
30 (0.033) 33.8 (0.030)
90 (0.011) 40.5 (0.025)

Estimate Vmax and Km from a direct graph of V versus [S]. Do you think you
get clear answers? Not really that good!!!!

Now use a Lineweaver-Burk plot to analyse the same data. Does this work
better? Vmax = 47.6 µM min-1 and Km = 11 µM

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 Regulation
• Most biochemical reactions start with 2 substrates and yield 2 products:

A + B P + Q

• Multiple-substrate reactions can be either sequential reactions or

double-displacement reactions (or ping-pong)
• Sequential: all substrates must bind to enzyme before any product
released. These can be either ordered or random
• Double-displacement reactions (ping-pong): one or more products
are released before all substrates bind enzyme (they will have a
substituted enzyme intermediate – enzyme temporarily modified).
Substrates/products bounce on and off an enzyme (like a ping-pong ball)

First substrate (A) binds and first catalytic step takes place resulting in a substituted enzyme. The first
product (P) then leaves. The second substrate (B) binds to the substituted enzyme and the second catalytic
step takes place forming final product (Q).

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 Allosteric Regulation
• Enzymes which follow Michaelis-Menten kinetics are not regulated in the
cell – i.e. if substrate is present then the enzyme catalyses

• Enzymes that regulate the flux of biochemical compounds in metabolic

pathways are called allosteric enzymes

Allosteric Enzymes: Hypothetical Examples

E1 E2 E3 E4
A B C D E

• 4 reactions catalysed by 4 different enzymes;

• End product E only required in limited concentrations and can’t be stored
• Initial reactant A is valuable and should be conserved unless E is needed
• B, C and D have no biological role whatsoever except as intermediates
• Reaction of A → B is the committed step
• After this reaction, B is committed to conversion into E
• When E is present in sufficient amounts, E reversibly binds to E1 (the
committed step) and inhibits reaction – Feedback inhibition

(-)
E1 E2 E3 E4
A B C D E
Feedback inhibitors do NOT bind at the active site but at a distinct
regulatory site on the allosteric enzyme

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 (-)
E1 E2 E3 E4
A B C D E
(+)
E7 E8
(+)
I J
E7 E6
F G H
(-)

Many biochemical pathways must communicate with each other. How can
they be coordinated so that appropriate concentration of J is produced?

Allosteric enzymes can recognise inhibitor molecules as well as

stimulatory molecules

Allosteric Enzymes
• Have a second regulatory site (allosteric site) distinct from the
active site
• Bind to allosteric site and regulate enzyme activity via
conformational changes (alterations in quaternary structure)
• Always catalyse the committed step of metabolic pathways
• Allos ‘other’ and stereos ‘structure’
• Do not conform to Michaelis-Menten kinetics
• Activators can lower Km, inhibitors can raise Km

Allosteric enzymes display a sigmoidal

dependence (S-shape) of reaction rate
(velocity) on substrate concentration.
Michaelis-Menten enzymes display
hyperbolic curve

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 Allosteric Enzyme: Phosphofructokinase
• One of the most important regulatory allosteric enzymes in glycolysis and is
composed of 4 subunits
• Catalyses ‘committed step’ in glycolysis: fructose-6-phosphate and ATP to
fructose 1,6-biphosphate and ADP
• ATP acts as an allosteric inhibitor and fructose 1,6-biphosphate acts as an
allosteric activator

Allosteric Regulation - Clinical Relevance?

Phosphoribosylpyrophosphate
synthetase (PRS)
Ribose 5-P + ATP 5-phosphoribosyl-1-pyrophosphate + AMP
(-)

Nucleotide synthesis

PRS enzyme becomes insensitive to feedback

inhibition – regulatory allosteric site on enzyme
becomes mutated
Uric acid and urate salts are quite insoluble –
humans are predisposed to crystalline uric acid
deposition (referred to as gout)
Gout arises from overproduction of purine
nucleotides (elevated uric acid synthesis) and/or
impaired uric acid excretion from kidneys

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 Enzyme Mechanisms and
Inhibitors

Enzymes: Catalytic Strategies

All enzymes operate by facilitating formation of the transition state
What mechanism(s) do enzymes use to form the transition state?

1. Covalent catalysis
2. Acid-base catalysis
3. Metal ion catalysis
4. Stabilisation of transition state

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 Covalent Catalysis
Active site contains a reactive group (usually powerful nucleophile)
that becomes temporarily covalently modified during catalysis.

Biologically important nucleophilic groups:

-
Hydroxyl group R-OH R-O: + H+

Sulfhydryl group R-SH R-S: - + H+

Amino group R-NH3+ R-NH2 + H+

R R

Imidazole group HN + NH HN N: + H+

Acid-base Catalysis
A molecule other than water plays the role of a proton donor or acceptor
(e.g. chymotrypsin uses a histidine residue as a base catalyst to enhance
nucleophilic power of serine).
What side-chains can act donate or accept protons?
Amino Acid pK a β-COOH
Often used in the hydrolysis of ester/ COO - O
peptide bonds, phosphate group Aspartic acid 3.90 H C CH
2
C
reactions, addition to carbonyl NH 3+ O-
γ-COOH
groups, etc. COO - O
Glutamic acid 4.07 H C CH CH C
2 2
An enzyme avoids unstable charged NH 3+ O-

intermediates in reaction (which COO - imidazole

N
would have high free energies) by Histidine 6.04 H C CH
2
NH 3+ N
having groups appropriately located
to: COO - sulfhydryl
Cysteine 8.33 H C CH SH
2
NH 3+
donate a proton (act as a general
phenol
acid), or COO -
Tyrosine 10.13 H C CH OH
2
NH 3+
accept a proton (abstract a proton, ε-amino
COO -
act as a general base) +
Lysine 10.79 H C CH 2 CH 2 CH2 NH 3
NH 3+

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 Metal Ion Catalysis
Metal ions can function catalytically in several ways:

Metal ions are often used for one or more of the following:
•binding substrates in the proper orientation and thus increasing
binding energy
•May generate a nucleophile by increasing acidity of a nearby
molecule (e.g. water)
•electrostatically stabilizing or shielding negative charges by
acting as an electrophilic catalyst

Metalloenzymes contain tightly bound metal ions: (usually Fe+2,

Fe+3, Cu+2, Zn+2, or Mn+2)

Metal-activated enzymes contain loosely bound metal ions:

(usually Na+, K+, Mg+2, or Ca+2)

Transition State Stabilisation

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 Enzyme Activity and Temperature
Increase temperature - rate of most chemical reactions normally increases
Why? Brownian motion of molecules increases – interactions between
substrate and enzyme more likely.
Comes a point were optimum is reached – afterwards loss in activity. Why?

Remember: enzyme is a protein – it

becomes denatured (weak bonds can no
longer hold it in its 3D shape that is
required for biological activity)

Enzyme Activity and pH

The activity of most enzymes displays a bell-shaped curve as a function of
changing pH
Optimal pH (pH at which enzyme operates at maximal activity) – depends
on environment. Why?
Functional ionisable side groups of amino acids (acidic, basic groups etc.)
Optimum pH for most enzymes is between 5 and 9

NB: pH of the environment (e.g. in the

body) can act as a regulator device (e.g.
phosphofructokinase)

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 Enzyme Activity and Inhibitors
Binding of specific small molecules and ions can inhibit the activity of many
enzymes. Why?
Inhibition can be either reversible or irreversible
Reversible inhibition is largely distinguished by a rapid dissociation of the
enzyme-inhibitor complex (unlike irreversible)
3 main types of reversible inhibition:
1.Competitive inhibition
2.Uncompetitive inhibition
3.Noncompetitive inhibition

Competitive Inhibition

• Competitive inhibitor usually resembles the substrate and binds only to free
enzyme active site - not enzyme-substrate complex
• Enzyme can bind substrate or inhibitor but NOT BOTH – thus competitive inhibitor
reduces proportion of enzyme molecules bound to substrate – decrease catalysis
• Kinetically: competitive inhibitor has no effect on Vmax but increases Km
• Examples: sulfanilamide (antibiotic); ibuprofen; statins

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 Uncompetitive Inhibition

• Uncompetitive inhibitor binds only to the enzyme-substrate complex and NOT to

free enzyme
• This type of inhibition cannot be overcome by addition of more substrate
• Kinetically: uncompetitive inhibitor decreases Vmax and decreases Km (note lines
in plot are parallel)
• Examples: glyphosate (herbicide)

Noncompetitive Inhibition
• Noncompetitive inhibitor can bind simultaneously with the substrate to the enzyme
at different binding sites
• Can bind to both free enzyme and the enzyme-substrate complex
• This type of inhibition cannot be overcome by addition of more substrate
• Kinetically: noncompetitive inhibitor decreases Vmax and Km does not change
• Examples: Doxycycline (antibiotic)

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 Irreversible Inhibition
• Irreversible inhibitors form stable covalent bonds with the enzyme (e.g.
alkylation or acylation of an active site side chain)
• There are many naturally-occurring and synthetic irreversible inhibitors
• Incubation of inhibitor with enzyme results in loss of activity

Example
• Diisopropyl fluorophosphate (DFP) is an organic phosphate that
inactivates serine proteases
• DFP reacts with the active site serine (Ser-195) of chymotrypsin to form
DFP-chymotrypsin
• Such organophosphorous inhibitors are used as insecticides or for
enzyme research
• These inhibitors are toxic because they inhibit acetylcholinesterase (a
serine protease that hydrolyzes the neurotransmitter acetylcholine)

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