BIOCHEMISTRY

Enzymes and Coenzymes

CHEMISTRY & BIOCHEMISTRY
Contents
• General Characteristics of Enzymes
• Enzyme Structure
• Enzyme Nomenclature
• Enzyme Function
• Enzyme Specificity
• Factors Affecting Enzyme Activity
• Enzyme Inhibition
• Regulation of Enzyme Activity
• Medical Uses of Enzymes
General Characteristics of Enzymes
• ENZYME
– Usually a protein, acting as catalyst in specific
biochemical reaction

• Each cell in the human body contains

1,000s of different enzymes
– Every reaction in the cell requires its own
specific enzyme

• Most enzymes are globular proteins

– A few enzymes are made of RNA
• Catalyze biochemical reactions involving
nucleic acids

• Enzymes undergo all the reactions of

proteins
– Enzymes denaturation due to pH or temperature
change
• A person suffering high fever runs the risk of
denaturing certain enzymes
Enzyme Structure
• SIMPLE ENZYMES
Composed only of protein

• CONJUGATED ENZYMES
Composed of:
– Apoenzyme
• Conjugate enzyme without
its cofactor

• Protein part of a • The apoenzyme can’t catalyze its reaction

conjugated enzyme without its cofactor.
– The combination of the apoenzyme with the cofactor
makes the conjugated enzyme functional.
– Coenzyme (Cofactor)
• Holoenzyme = apoenzyme + cofactor
• Non-protein part of a – The biochemically active conjugated enzyme.
conjugated enzyme
Coenzymes and cofactors
• Coenzymes provide additional chemically reactive functional groups
besides those present in the amino acids of the apoenzymes
– Are either small organic molecules or inorganic ions

• Metal ions often act as additional cofactors (Zn2+, Mg2+, Mn2+ & Fe2+)
– A metal ion cofactor can be bound directly to the enzyme or to a coenzyme

• COENZYME
– A small organic molecule, acting as a cofactor in a conjugated enzyme
• Coenzymes are derived from vitamins or vitamin derivatives
– Many vitamins act as coenzymes, esp. B-vitamins
Enzyme definitions

Term Definition
Enzyme Protein only enzyme that facilitates a chemical reaction
(simple)
Coenzyme Compound derived from a vitamin (e.g. NAD+) that
assists an enzyme in facilitating a chemical reaction
Cofactor Metal ion (e.g. Mg2+) that that assists an enzyme in
facilitating a chemical reaction
Apoenzyme Protein only part of an enzyme (e.g. isocitrate
dehydrogenase) that requires an additional coenzyme to
facilitate a chemical reaction (not functional alone)
Holoenzyme Combination of the apoenzyme and coenzyme which together
facilitating a chemical reaction (functional)
Enzyme Nomenclature • Suffix of an enzyme –ase
– Lactase, amylase, lipase or protease
• Denotes an enzyme
• Enzymes are named according
to the • Some digestive enzymes have the suffix –in
– Pepsin, trypsin & chymotrypsin
type of reaction they • These enzymes were the first ones to be studied
catalyze and/or their substrate
• Prefix denotes the type of reaction the
enzyme catalyzes
– Oxidase: redox reaction
• Substrate = the reactant upon – Hydrolase: Addition of water to break one
which the specific enzyme acts component into two parts

– Enzyme physically binds to the • Substrate identity is often used together

substrate with the reaction type
– Pyruvate carboxylase, lactate dehydrogenase

Enzyme Substrate Enzyme/substrate complex

6Enzyme
MajorClass C lasses of Enz ymes
Reaction Catalyzed Examples in Metabolism
Oxidoreductase Redox reaction (reduction & Examples are dehydrogenases
oxidation) catalyse reactions in which a
substrate is oxidised or reduced
Transferase Transfer of a functional group Transaminases which catalyze
from 1 molecule to another the transfer of amino group or
kinases which catalyze the
transfer of phosphate groups.
6 Major Classes
Hydrolase Hydrolysis reaction Lipases catalyze the hydrolysis of Enzymes
of lipids, and proteases catalyze
the hydrolysis of proteins
Based on the type of
Addition / removal of atoms to / Decarboxylases catalyze the
reaction they catalyze
Lyase
from double bond removal of carboxyl groups

Isomerase Isomerization reaction Isomerases may catalyze the

conversion of an aldose to a
ketose, and mutases transfer
functional group from one atom
to another within a substrate. The table explains
Synthesis reaction Synthetases link two smaller
the functions of
Ligase
(Joining of 2 molecules into one, molecules are form a larger one. enzymes and how
forming a new chemical bond, they are classified
coupled with ATP hydrolysis) and named.
Enzyme Active Site
• Active site
– The specific portion of an enzyme (location)
where the substrate binds while it undergoes
a chemical reaction

• The active site is a 3-D ‘crevice-like’ cavity

formed by secondary & tertiary structures
of the protein part of the enzyme
– Crevice formed from the folding of the protein
• Aka binding cleft

– An enzyme can have more than only one

active site

– The amino acids R-groups (side chain) in the

active site are important for determining the
specificity of the substrate
Enzyme – Substrate Complex
• When the substrate binds to the enzyme active site an
Enzyme-Substrate Complex is formed temporarily
– Allows the substrate to undergo its chemical reaction much faster
Lock & Key Model of Enzyme Action
• The active site is fixed, with a rigid shape (LOCK)

• The substrate (KEY) must fit exactly into the rigid enzyme (LOCK)

• Complementary shape & geometry between enzyme and substrate

– Key (substrate) fits into the lock (enzyme)

• Upon completion of the chemical reaction, the products are released from the active site, so the next
substrate molecule can bind
Induced Fit Model of Enzyme Action
• Many enzymes are flexible & constantly change their shape
– The shape of the active site changes to accept & accommodate the
substrate
• Conformation change in the enzyme’s active site to allow the substrate to
bind

• Analogy: a glove (enzyme) changes shape when a hand (substrate) is

inserted into it
Enzyme Specificity
• Absolute Specificity
– An enzyme will catalyze a particular reaction for only one substrate
– Most restrictive of all specificities
• Not common

– Catalase has absolute specificity for hydrogen peroxide (H2O2)

– Urease catalyzes only the hydrolysis of urea

• Group Specificity
– The enzyme will act only on similar substrates that have a specific functional
group

• Carboxypeptidase cleaves amino acids one at a time from the carboxyl end of the
peptide chain

• Hexokinase adds a phosphate group to hexoses

Enzyme Specificity
• Linkage Specificity
– The enzyme will act on a particular type of chemical bond, irrespective of the
rest of the molecular structure
– The most general of the enzyme specificities

• Phosphatases hydrolyze phosphate–ester bonds in all types of phosphate esters

• Chymotrypsin catalyzes the hydrolysis of peptide bonds

• Stereochemical Specificity
– The enzyme can distinguish between stereoisomers
– Chirality is inherent in an active site (as amino acids are chiral compounds)

• L-Amino-acid oxidase catalyzes reactions of L-amino acids but not of D-amino

acids
Factors Affecting Enzyme Activity
Enzyme activity
• Measure of the rate at which an enzyme converts substrate to products
in a biochemical reaction

4 factors affect enzyme activity:

• Temperature

• pH

• Substrate concentration: [substrate]

• Enzyme concentration: [enzyme]

 Temperature (t)
• With increased t the EKIN increases
– More collisions
– Increased reaction rate

• Optimum temperature (tOPT) is the t,

at which the enzyme exhibits
maximum activity
– The tOPT for human enzymes = 370C

• When the t increases beyond tOPT

– Changes in the enzyme’s tertiary
structure occur, inactivating & denaturing
it (e.g. fever)

• Little activity is observed at low t

 pH
• Optimum pH (pHOPT) is the pH, at which the
enzyme exhibits maximum activity

• Most enzymes are active over a very narrow

pH range
– Protein & amino acids are properly maintained

– Small changes in pH (low or high) can result in

enzyme denaturation & loss of function

• Each enzyme has its characteristic pHOPT,

which usually falls within physiological pH
range 7.0 - 7.5

• Digestive enzymes are exceptions:

– Pepsin (in stomach) – pHOPT = 2.0

– Trypsin (in SI) – pHOPT = 8.0

 Substrate Concentration
• If [enzyme] is kept constant & the [substrate] is
increased
– The reaction rate increases
until a saturation point is
met
• At saturation the reaction rate stays the same even if
the [substrate] is increased

– At saturation point substrate molecules are bound to

all available active sites of the enzyme molecules

• Reaction takes place at the active site

– If they are all active sites are occupied the reaction is
going at its maximum rate
• Each enzyme molecule is working at its maximum
capacity

– The incoming substrate molecules must “wait their turn”

Enzyme Concentration
• If the [substrate] is kept constant & the
[enzyme] is increased
– The reaction rate increases

– The greater the [enzyme], the greater the

reaction rate

• RULE:
– The rate of an enzyme-catalyzed reaction is
always directly proportional to the amount
of the enzyme present

• In a living cell:
– The [substrate] is much higher than the
[enzyme]
• Enzymes are not consumed in the reaction

• Enzymes can be reused many times

 Key concept: function of an
enzyme
What is the function of an enzyme in a chemical reaction?

What happens to the enzymes when the body

temperature rises from 37ᵒC to 42ᵒC?

If an enzyme has broken down and is non-functional,

what would happen to the chemical reaction
normally facilitated by the enzyme? Explain.
G
Enzyme Inhibition
• ENZYME INHIBITOR
– A substance that slows down or stops the normal catalytic function of
an enzyme by binding to the enzyme

• Three types of inhibition:

– Reversible competitive inhibition

– Reversible non-competitive inhibition

– Irreversible inhibition
Reversible Competitive Inhibition
• A competitive inhibitor resembles the
substrate
– Inhibitor competes with the substrate for binding to
the active site of the enzyme

– If an inhibitor is bound to the active site:

• Prevents the substrate molecules to access
the active site
– Decreasing / stopping enzyme activity

• The binding of the competitive inhibitor to the

active site is a reversible process
– Add much more substrate to outcompete the
competitive inhibitor

• Many drugs are competitive inhibitors:

– Anti-histamines inhibit histidine decarboxylase,
which converts histidine to histamine
 Reversible Noncompetitive Inhibition
• A non-competitive inhibitor decreases
enzyme activity by binding to a site on the
enzyme other than the active site

– The non-competitive inhibitor alters the tertiary

structure of the enzyme & the active site

• Decreasing enzyme activity

• Substrate cannot fit into active site

– Process can be reversed only by lowering the

[non-competitive inhibitor]

• Example:
– Heavy metals Pb2+ & Hg2+ bind to –SH of
Cysteine, away from active site
• Disrupt the secondary & tertiary structure
Irreversible Inhibition
• An irreversible inhibitor inactivates an enzyme
by binding to its active site by a strong covalent bond

– Permanently deactivates the enzyme

– Irreversible inhibitors do not resemble substrates

• Addition of excess substrate

doesn’t reverse this process
– Cannot be reversed

• Chemical warfare (nerve gases)

• Organophosphate insecticides
Allosteric Enzymes
• Allosteric enzymes have a quaternary
structure
– Are composed of 2 or more protein chains
– Possess 2 or more binding sites • Binding of a regulator molecule
to its regulatory site causes
• 2 types of binding sites: changes in 3-D structure of the
enzyme & the active site
– One binding site for the substrate
– Binding of a Positive regulator
• Active site up-regulates enzyme activity
• Enhances active site, more able
– Second binding site for a regulator molecule to accept substrate
• Regulatory site
– Binding of a Negative regulator
(non-competitive inhibitor)
• Active & regulatory binding sites are down-regulates enzyme activity
distinct from each other in shape & location • Compromises active site, less
able to accept substrate
Feedback Control Example:
The degradation of glucose
• A process in which activation or inhibition of one of the earlier reaction through a metabolic pathway
steps in a reaction sequence is controlled by a product of this reaction can be regulated in several
sequence. ways
The enzyme PFK is
allosterically inhibited by
– One of the mechanisms in which allosteric enzymes are regulated
the product ATP

– Most biochemical processes proceed in several steps & each step is Glycolysis (makes ATP) is
catalyzed by a different enzyme slowed when cellular ATP
• The product of each step is the substrate for the next step / enzyme. is in excess
Observe animation of feedback
control

Reaction 1: converts Reaction 2: converts Reaction 3: converts

reagent A into product B reagent B into product C reagent C into product D
 The different
effects
Positiveof&
Negative
regulators
on an
Allosteric
enzyme
 •
Proteolytic Enzymes Most digestive & blood-clotting enzymes
are proteolytic
– Blood clotting enzymes break down
& Zymogens proteins within the blood so that they can
form the clot
• 2nd mechanism of allosteric enzyme regulation • Platelets interspersed with tangled
protein (collagen and thrombin)
– Production of an enzyme in an inactive form
• Activation of a zymogen requires the
– Activated when required (right time & place) removal of a peptide fragment from the
• Activated aka “turned on”
zymogen structure
– Changing the 3-D shape & affecting the
• Proteolytic enzymes catalyze breaking of active site
peptide bond in proteins • E.g. Pepsiongen (zymogen)
– To prevent these enzymes from destroying the >>> Pepsin (active proteolytic enzyme)
tissues, that produced them, they are released in
an inactive form = ZYMOGENS
Activation of a
Zymogen
Covalent Modification of Enzymes
• Covalent modifications are the 3rd mechanism of enzyme activity regulation
– Aprocess of altering enzyme activity by covalently modifying the structure of the enzyme
• Adding / removing a group to / from the enzyme

• Most common covalent modification = addition & removal of phosphate group:

– Phosphate group is often derived from an ATPmolecule

• Addition of phosphate = phosphorylation is catalyzed by a Kinase enzyme

• Removal of phosphate = dephosphorylation is catalyzed by a Phosphatase enzyme

– Glycogen synthase: involved in synthesis of glycogen

• Deactivated by phosphorylation

– Glycogen phosphorylase: involved in breakdown of glycogen

• Activated by phosphorylation.
Vitamins as Coenzymes
• Many enzymes require B vitamins as coenzymes
– Allow the enzyme to function

• Coenzymes serve as temporary carriers of atoms or functional groups

– Coenzymes provide chemical reactivity that the apoenzyme lacks

– Important in metabolism reactions to release energy from foods

• E.g. redox reactions where they facilitate oxidation or reduction

• B vitamins don’t remain permanently bonded to the apoenzyme

– After the catalytic action the vitamin is released & can be repeatedly used by various enzymes

– This recycling reduces the need for large amounts of B vitamins

 Key concept: sites with enzymes,
coenzymes
Why is an enzymes active site important to the
function of the enzyme?

Why is the enzymes regulatory binding site important

for controlling the activity of the enzyme?

Why are coenzymes (derived from vitamins)

important to the function of some enzymes?
G
Drugs Inhibiting Enzyme Activity
• Many prescription drugs inhibit enzymes

• ACE Inhibitors
– Inhibit Angiotensin-Converting Enzyme
• Lowers blood pressure

• Sulfa drugs
– Antibiotics acting as competitive inhibitors of bacterial enzymes
• Involved in conversion of PABA to Folic acid
– Deficiency of folic acid retards bacterial growth, eventually killing them

• Penicillin's
– β-lactam antibiotics inhibit transpeptidase
• Transpeptidase enzyme strengthens the cell wall
– Forms peptide cross links between polysaccharides strands in bacterial cell walls

– Without transpeptidase enzyme (inhibited by Penicillin) >>> weakened cell wall, bacteria dies
Medical Uses of Enzymes
• Enzymes can be used in diagnosis & treatment of certain diseases

• Lactate dehydrogenase (LDH) is normally not found in high levels in

blood, as it is produced in cells
– Increased levels of LDH in the blood indicate
myocardial infarction (MI) (Heart attack)

– Tissue plasminogen activator (TPA) activates the enzyme plasminogen that

dissolves blood clots
• Used in the treatment of MI

• There is no direct test to measure urea in the blood

– Urease converts urea into ammonia, which is easily
measured & is used as urea indicator
• Blood Urea Nitrogen (BUN) is used to measure kidney function

– High urea levels in the blood indicate kidney malfunction

Isoenzymes
• Isoenzyme catalyze the same reaction
in different tissues in the body
– e.g. lactate dehydrogenase (LDH) consists of 5 isoenzymes
• Each isoenzyme of LDH has the same function
– Converts lactate to pyruvate

• LDH1 isoenzyme is more prevalent in heart muscle

• LDH5 form is found in skeletal muscle & liver

• Isoenzymes can be used to identify the damaged or diseased organ or tissue

• It is a marker for a particular location

• If LDH1 isoenzyme was found in the blood >>> indicates heat muscle damage