LIFE SCIENCES (FBI 1224)

CHAPTER 2:
BIOCATALYSIS
Dr Nur Afiqah Mohamad
LEARNING OUTCOME
At the end of the lecture, the students should be
able to explain:
 The properties of enzymes

 Mechanism of enzyme action

 The hypotheses related to the mechanism of

action of enzymes
 Factors affecting enzyme function

 Negative feedback
The living cell is a miniature chemical factory where thousands of
reactions occur.
METABOLISM
 All biochemical reactions that occur in living
cells.
 Biochemical reactions are divided into anabolic
and catabolic reactions.
 Anabolic reaction : complex molecules
synthesised from simpler molecules usually with
the expense of energy.
 Often involve condensation.

 Example : synthesis of starch from glucose

METABOLISM
 Catabolic reaction : Complex molecules are
broken down into simpler molecules. Usually
energy is released.
 Often involve oxidation and hydrolysis.

 Example : cellular respiration, glucose oxidised to

form water, carbon dioxide and energy (ATP),
hydrolysis of starch, proteins, lipids
METABOLISM
 Chemical reactions of life

 Forming bond between molecules

 dehydration
 synthesis

 anabolic reactions

 Breaking bonds between molecules

 hydrolysis
 digestion

 catabolic reactions
EXAMPLES
 dehydration (synthesis)

enzyme

 hydrolysis (digestion)

enzyme
CHEMICAL REACTIONS & ENERGY
 Some chemical
reactions release
energy
 exergonic
 digesting polymers
 hydrolysis =
catabolism
CHEMICAL REACTIONS & ENERGY
 Some chemical
reactions require
input of energy
 endergonic
 building polymers
 dehydration
synthesis =
anabolism
ACTIVATION ENERGY
Breaking down large molecules
requires an initial input of energy
 activation energy
 large biomolecules are stable
 must absorb energy to break bonds

cellulose energy CO2 + H2O + heat

ACTIVATION ENERGY
 the
minimum energy that must be
input to a chemical system,
containing potential reactants, in
order for a chemical reaction to
occur.
TOO MUCH ACTIVATION ENERGY FOR LIFE
 Activation energy
 amount of energy needed to destabilize the bonds of a
molecule. It can be provided in the form of heat from
surroundings, but high temperature can kill cells.

glucose Not a match!

That’s too much
energy to expose
living cells to!
CATALYSTS
 So what’s a cell got to do to reduce activation
energy?
 get help! … chemical help…

ENZYMES!

∆G
REDUCING ACTIVATION ENERGY
 Catalysts
 reducing the amount of energy to start a reaction
 lowering the activation energy of the reaction

uncatalyzed reaction
Pheeew…
that takes a lot
catalyzed reaction less energy!

NEW activation energy

reactant

product
ENZYMES
 Biological catalysts
 globular proteins
 facilitate chemical reactions
 increase rate of reaction without being consumed
 reduce activation energy

 required for most biological reactions

 highly specific
 thousands of different enzymes in cells
 control reactions
of life
 ENZYMES VOCABULARY
substrate
 reactant which binds to enzyme
 enzyme-substrate complex: temporary association

product
 end result of reaction
active site
 enzyme’s catalytic site; substrate fits into active site

active site
substrate products

enzyme
PROPERTIES OF ENZYMES
 Reaction specific
 each enzyme works with a specific substrate
 chemical fit between active site & substrate

 Not consumed in reaction

 single enzyme molecule can catalyze thousands or more
reactions per second
 enzymes unaffected by the reaction

 Affected by cellular conditions

 any condition that affects protein structure
 Required in small amounts
 Lowers the activation energy
 Does not alter the nature or properties of the end products
NAMING CONVENTIONS
 Enzymes named for reaction they catalyze
 sucrase breaks down sucrose
 proteases break down proteins
 lipases break
down lipids
 DNA polymerase builds DNA
 adds nucleotides
to DNA strand
 pepsin breaks down
proteins (polypeptides)
TWO MODELS TO EXPLAIN HOW ENZYMES
WORK

Lock and Key

Model

Induced Fit Model

LOCK AND KEY HYPOTHESIS
What is the ‘lock and key’ hypothesis?
 It is the old view of enzyme specificity, that
there was an exact match between the active
site and the substrate.
 Substrate fits into the active site rather like a
key fits into a lock. The substrate is the “key”
that fits exactly the enzyme “lock”.

21
LOCK AND KEY MODEL
 Simplistic model of enzyme action
 substrate fits into 3-D structure of enzyme’
active site
 H bonds between substrate & enzyme
 like “key fits into lock”
INDUCED FIT HYPOTHESIS
What is induced fit hypothesis?
 shape of the active site adjusts to fit the
substrate.
INDUCED FIT MODEL

 More accurate model of enzyme action

 3-D structure of enzyme fits substrate
 substrate binding cause enzyme to change shape leading
to a tighter fit
 “conformational change”
 The active site in the enzyme is not rigid, but flexible.
 Shape of active site is not exactly complementary to

substrate
 Binding of substrate induces the enzyme to change its shape

slightly leading to a better fit between its active site and the
substrate. There is also a slight alteration to the shape of teh
substrate.
FACTORS AFFECTING ENZYME FUNCTION

1. Substrate concentration
2. Enzyme concentration
3. Temperature
4. pH
5. Salinity
6. Cofactors
7. Inhibitors

catalase
1. SUBSTRATE CONCENTRATION
 FACTORS AFFECTING ENZYME FUNCTION
 Substrate concentration
 as ↑ substrate = ↑ reaction rate
 more substrate = more frequently collide with enzyme
 reaction rate levels off
 all enzymes have active site engaged
 enzyme is saturated

 maximum rate of reaction

reaction rate

substrate concentration
2. ENZYME CONCENTRATION
What’s
happening here?!
reaction rate

enzyme concentration
 FACTORS AFFECTING ENZYME FUNCTION
 Enzyme concentration
 as ↑ enzyme = ↑ reaction rate
 more enzymes = more frequently collide with substrate
 reaction rate levels off
 substrate becomes limiting factor
 not all enzyme molecules can find substrate
reaction rate

enzyme concentration
3. TEMPERATURE
AT TEMPERATURES ABOVE THE OPTIMUM
 Molecules vibrate and twist so rapidly that some
of their hydrogen bonds and hydrophobic
interactions break.
FACTORS AFFECTING ENZYME FUNCTION
 Temperature
 Optimum T°
 greatest number of molecular collisions
 human enzymes = 35°- 40°C

 body temp = 37°C

 Heat: increase beyond optimum T°

 increased energy level of molecules disrupts bonds in enzyme &
between enzyme & substrate
 H, ionic = weak bonds

 denaturation = lose 3D shape (3° structure)

 Cold: decrease T°
 molecules move slower
 decrease collisions between enzyme & substrate
 EFFECT OF TEMPERATURE ON THE RATE OF REACTION

Rate of reaction
3 The optimum temperature is reached.
Enzyme is most active.
(enzyme activity)

4 Beyond the optimum

2 As the temperature rises, temperature, enzyme
enzyme activity increases as activity decreases.
indicated by the increase in
the rate of reaction it
catalyses. Usually the
enzyme is twice as active 5 At point D, the enzyme
for every 10°C rise in has lost its ability to
1 An enzyme
temperature until the catalyse the reaction.
is less active
optimum temperature is
at very low
reached.
temperatures.

0 optimum temperature D Temperature

ENZYMES AND TEMPERATURE
 Different enzymes function in different organisms
in different environments

hot spring
human enzyme bacteria enzyme
reaction rate

37°C 70°C
temperature
(158°F)
 4. PH
• Most enzymes work best when the pH value is between 6 and 8
• Optimum pH for an enzyme is the pH at which the maximum
rate of reaction occurs.

pepsin trypsin

pepsin
reaction rate

trypsin

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14
pH
FACTORS AFFECTING ENZYME FUNCTION
 pH
 changes in pH
 disrupts ionic bonds, disrupts 3D shape
 disrupts attractions between charged amino acids

 affect 2° & 3° structure

 denatures protein

 can also cause changes to the charges found at the active site
and on the substrate (affecting formation of enzyme-substrate
complex)
 optimal pH?
 most human enzymes = pH 6-8
 depends on localized conditions

 pepsin (stomach) = pH 2-3

 trypsin (small intestines) = pH 8

0 1 2 3 4 5 6 7 8 9 10 11
5. SALINITY
reaction rate

salt concentration
FACTORS AFFECTING ENZYME FUNCTION
Salt concentration
 Its chemical formula is NaCl, or sodium (Na) chloride
(Cl).
 changes in salinity
 adds or removes cations (+) & anions (–)
 disrupts bonds, disrupts 3D shape

 disrupts attractions between charged amino acids

 affect 2° & 3° structure

 denatures protein

 enzymes intolerant of extreme salinity

 Dead Sea is called dead for a reason!
6. COFACTORS
 Non protein components required for the efficient
functioning of enzymes.
 Inorganic or organic.

 Can be grouped into coenzymes, prosthetic

groups and metal ions.
 Coenzymes
 Loosly associated with the enzyme and can be readily
detached.
 Small organic molecules
 Weaken the bonds of substrate, allowing it to react
with an enzyme.
 Eg : NAD (nicotinamide adenine dinucleotide), ATP
(adenosine triphosphate).
 In animals some types of coenzymes are often
produced from vitamins in the diet.
 NAD from vitamin B complex.
 Prosthetic groups
 Nonprotein portions tightly bound/permanently
attached to the active site
 Organic substances
 Example: haem is the prosthetic group of the enzyme
catalase.
 Metal ions
 Some enzymes are tightly bound to metal ions,
example, Zn2+ in carboxypeptidase and Ca2+ in
thrombokinase.
 Metal ions may be bonded through covalent bonds.
 Help draw electrons away from substrate molecules
making bonds less stable and easier to break.
 Binding of metal ions also can change the shape of
substrates and enzymes allowing enzyme-substrate
formation.
 Other examples: magnesium, iron, potassium
7. INHIBITORS
 Small molecules which reduce the rate of an
enzyme-controlled reaction.
 Irreversible
 Inhibitor reacts with active site
 Enzyme is permanently damaged

 Reversible
 No covalent bonding to active site
 i. Competitive
 ii. Non-competitive inhibition
IRREVERSIBLE INHIBITORS
IRREVERSIBLE INHIBITORS
 Bind tighly and permanently to enzymes with
strong covalent bonds and destroy their function.
 Small concentrations of heavy metal ions eg:
mercury, silver and arsenic combine.
 Some type of insecticides eg: malathion
REVERSIBLE INHIBITORS
When an inhibitor binds loosely and temporarily with the
enzyme by weak hydrogen bonds.
A. COMPETITIVE INHIBITOR
 Inhibitor & substrate “compete” for active site (similar shapes)
 penicillin
blocks enzyme bacteria use to build cell walls
 disulfiram (Antabuse)
treats chronic alcoholism
 blocks enzyme that
breaks down alcohol
 severe hangover & vomiting
5-10 minutes after drinking
 Overcome by increasing substrate
concentration
 saturate solution with substrate
so it out-competes inhibitor
for active site on enzyme
B. NON-COMPETITIVE INHIBITOR
 Inhibitor binds to site other than active site
 allosteric inhibitor binds to allosteric site
 causes enzyme to change shape
 conformational change
 active site is no longer functional binding site

 keeps enzyme inactive

 some anti-cancer drugs
inhibit enzymes involved in DNA synthesis
 stop DNA production
 stop division of more cancer cells
EFFECT OF CONCENTRATION OF A COMPETITIVE
INHIBITOR ON THE RATE OF REACTION
ALLOSTERIC ENZYMES
 Certain enzymes are subject to allosteric
regulation.
 Allosteric enzymes are large and complex, having
quatenary structures.
 Allostery means different shape.
ALLOSTERIC INHIBITION
 Allosteric means other site.
SWITCHING OFF
 These enzymes have two receptor sites.
 One site fits the substrate like other enzymes.

 The other site fits an inhibitor molecule.

ALLOSTERIC INHIBITION
 A type of reversible inhibition which allows the
rate of enzyme catalysed reactions to be
controlled.
 The end product of the reaction can act as a non-
competitive inhibitor and bind at a site on the
enzyme.
NEGATIVE FEEDBACK
 In feedback inhibition, the end product of a
metabolic pathway shuts down the pathway.
END POINT INHIBITION
 threonine
FEEDBACK INHIBITION
 Example
 synthesis of amino acid,
isoleucine from amino acid,
threonine
 isoleucine becomes the
allosteric inhibitor of the first
step in the pathway
 as product accumulates it
collides with enzyme more often
than substrate does

isoleucin
e
WHAT IS THE VALUE OF NEGATIVE
FEEDBACK INHIBITION TO A CELL?