Chapter 4.

Enzymes
 are biological catalysts that speed up the rate of chemical
reactions
 provide a fantastic speed, specificity, and regulatory control to
reactions in the body
Chemical Nature of Enzymes (Properties)
 Enzymes are proteins (except ribozymes)
 reusable(not consumed or permanently changed) by the reaction
 act repeatedly to increase the rate of reactions
 are often very “specific” – promote only 1 particular reaction
 acts up on reactants called substrates
 They have enormous power of catalysis. They increase rate of
reaction to 105 to 1010 folds.
 Enzymes do not alter the equilibrium constant (Keq). 1
How enzymes work?
 For a chemical reaction A → B to occur, energy is required.
 When enough energy is supplied, A undergoes to transition
state which is an unstable state. So, it gets converted to product
B which is more stable.
 The amount of energy needed to convert a substance from
ground state to transition state is called activation energy
 Enzymes accelerates the rate of reaction by decreasing the
energy of activation

2
 Enzyme Specificity
 Enzymes have varying degrees of specificity for substrates
 Enzymes may recognize and catalyze:
- a single substrate
- a group of similar substrates
- a particular type of bond
- a particular site on a substrate
- a particular stereoisomer

3
 Enzyme Specificity examples….continue
 Substrate Specificity
 Enzymes are specific towards their substrates
Example,

 Reaction Specificity
 A given enzyme catalyze only one specific reaction. example,
lipases only hydrolyze lipids, Proteases hydrolyzes proteins, do
not catalyze any other types of reactions.
 Optical Specificity/ Sterospecificity
 enzymes are able to recognize optical isomers of the substrate.
For example, enzymes of amino acid metabolism act only on L-
isomers (L-amino acid) but not D-isomers (D-amino acids). 4
 Group/ broad Specificity
 Some lytic (hydrolases) enzymes act on specific groups. e.g.
Proteases are specific for peptide groups, glycosidases are
specific to glycosidic bonds.
 Absolute Group Specificity
 Certain lytic enzymes exhibit high order group specificity. For
example, chymotrypsin is protein splitting enzyme i.e., it
hydrolyzes peptide bonds. But it preferentially hydrolyzes
peptide bonds in which carboxyl group is contributed by
aromatic amino acids phenylalanine, tyrosine and tryptophan.
 Likewise, trypsin another peptide bond hydrolyzing enzyme
preferentially hydrolyzes peptide bonds in which carboxyl
group is contributed by basic amino acids.

5
Enzyme Classification and Nomenclature
1. Recommended names
 Enzymes were assigned various trivial names as they were
discovered, which are convenient to use.
 The name of an enzyme has two parts.
 The first part indicates name of its substrate and second part
ending in ‘ase’
For example; Sucrase, Protease, Lipase
2. Systematic Name
 International Union of Biochemistry classified all enzymes
into six major classes based on the type of reaction they
catalyze and reaction mechanism
 Each enzyme has code (EC) number.
 EC is a four-digit number. The first digit indicates major
class, second digit indicates sub class, third digit denotes 6
sub-sub class and final digit indicates specific enzyme.
 Example, the formal systematic name of the enzyme catalyzing
the reaction;
ATP + D-glucose → ADP + D-glucose 6-phosphate is: “ATP: D-
hexose-6-phospho-transferase”, which indicates that it catalyzes
the transfer of a phosphoryl group from ATP to glucose.
• Its Enzyme Commission number (E.C. number) is 2.7.1.1. Here,
 a member of class 2 (transferase)

 sub-class 7 ( transferof a phosphoryl group

phosphotransferase)
 sub-sub class 1 (alcohol is the phosphoryl acceptor).

 The 4th digit 1- indicates D-glucose as the phosphoryl

group acceptor.. The term “hexose-6” indicates that the

alcohol phosphorylated is that of carbon six of a hexose
 For many enzymes, a trivial name is more commonly used in
this case hexokinase
 The six major classes of enzymes
1. Oxidoreductases: They catalyze oxidation and reduction
reactions. Eg. Pyruvate Dehydrogenase (PDH)
2. Tranferases: They catalyze transfer of groups. e.g..
Aminotransferases (ALT, AST…), PFK etc.
3. Hydrolases: They catalyze hydrolysis of peptide, ester,
glycosyl etc. bonds. Eg. Digestive enzymes (Amylases etc.).
4. Lyases: They catalyze removal of groups from substrates by
mechanisms other than hydrolysis forming double bonds. e.g..
Aldolase, Thiolases,
T Fumerase
5. Isomerases: They catalyze inter conversion of optical,
functional and geometrical isomers. e.g.. Phosphohexose
isomerase.
6. Ligases: They catalyze linking together of two compounds.
The linking is coupled to the breaking of phosphate from ATP
Eg. DNA Ligase 8
9
10
Enzyme-substrate interaction

 Substrate interact with enzyme at its active site

 The active site is a specialized region of the enzyme where
the enzyme interacts with the substrate.
 It is generally a pocket or cleft that is specialized to
recognize specific substrates and catalyze chemical
transformations.
 Substrates bind in active site by weak non-covalent
interactions (hydrogen bonding, hydrophobic interactions,
ionic interactions)
 The interactions hold the substrate in the proper orientation
for most effective catalysis
11
 Two models for enzyme/substrate interactions:

1. Lock and Key Model:

(Fisher’s template theory)
 Proposed by Emil Fischer
in 1894
 Substrate (key) fits into a
perfectly shaped space in
the enzyme (lock)
 similarity between shape
of enzyme and substrate
 Implies a very RIGID
inflexible active site
12
 In the induced-fit model
(Koshlan’s induced fit theory)

 Proposed by Daniel E. Koshland

in 1958
 account the flexibility of proteins
 A substrate fits into a general
shape in the enzyme, causing the
enzyme to change shape
(conformation)
 This conformation change
converts the enzyme into a new
structure in which the substrate
and catalytic groups on the
enzyme are properly arranged to
accelerate the reaction. 13
 Mechanisms of Enzyme Activity: The Chemistry of Catalysis
 The mechanism of enzyme action deals with molecular events
associated with conversion of a substrate to product in an
enzymatic reaction.
1. General Acid-Base Catalysis:
 Enzyme functional groups in the active site can serve as acids or
bases to assist in proton transfer reactions and facilitate bond
cleavage.
 The functional groups of the amino acid residues (e.g., glutamate,
aspartate, histidine, serine, tyrosine, cysteine, lysine and arginine)
at the active site of the enzyme participate in the catalytic process
as proton donors (acids) or proton acceptors (bases) in order to
stabilize developing charges in the transition state .
 Acid catalysis occurs when the partial and temporary proton transfer from an acid lowers
the activation energy required to reach and stabilize the transition state and increases
reaction rate. Oppositely, base catalysis occurs when the partial and temporary proton
abstraction by a base lowers the activation energy to reach and stabilize the transition
14
state and increases reaction rate.
 2. Metal Ion Catalysis /Electrostatic catalysis
 Is utilized by about one third of all known enzymes.
 The required metal is either a tightly bound prosthetic group as
in metalloenzymes, e.g., Fe2+, Zn2+, Co3+ and Mn2+. Or, the
element may work through a loose association with the enzyme
as a cofactor in metal-activated enzymes, e.g., Na+, K+, Mg2+ and
Ca2+.
 Metal ion hold substrate properly oriented by coordinate covalent
bonds, enhance a reaction by polarizing the scissile bond or by
stabilizing a negatively charged intermediate
 Participate in redox reactions by reversible electron transfer
between metal ions and substrate
3. Covalent Catalysis
 Occurs when a nucleophile (electron rich) functional group on
an enzyme reacts to form a covalent bond with substrate
15
 By the transient covalent bonding of enzyme and substrate
creates covalent reaction intermediate that helps reducing the
energy of later transition states of the reaction and accelerate
reaction rate.
 At a later stage in the reaction, covalent bonds are broken to
regenerate the enzyme
4. Proximity and Orientation Catalysis
 Proximity is the maximization of the concentration of
substrate(s) at the active site for the action of the enzyme
catalytic groups.
 The effect is similar to an effect induced by increasing substrate
concentration. Since enzymes have very high affinity (low Km)
for their substrates, they sequester substrates into their active
sites (by transient covalent and non covalent bonds).
 This property enables enzymes to convert substrate into its
product
16

 The Substrates are oriented in optimal position to react.

Molecular components of Enzymes

17
 Enzyme Cofactors
 Cofactors are non-protein molecules required for activity of
some enzymes
 are two types of cofactors:
 Organic cofactors(co-enzymes) such as NAD+, FAD+, CoA
 Inorganic cofactors(metal Ions) such as Fe 2+, Mg2+, Mn2+, or
Zn2+
 A coenzyme or metal ion that is very tightly or even covalently
bound to the enzyme protein is called a prosthetic group.
 A complete, catalytically active enzyme together with its bound
coenzyme and/or metal ions is called a holoenzyme.
 The protein part of such an enzyme is called the apoenzyme or
apoprotein.
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 Isoenzymes (Isozymes)
 These are enzymes that catalyze the same reaction but

originated at different sites and show different physical and

chemical characteristics such as amino acid composition,
specificity, electrophoretic mobilities, tissue distribution, and
substrate affinity.

Eg. Hexokinase and Glucokinase

Lactate Dehydrogenase (LDH) isoenzymes; namely

LDH-1, LDH-2, LDH-3, LDH-4 and LDH-5

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 Enzyme Kinetics

 Factors Affecting Enzyme Action

 The rate of an enzyme catalyzed reaction is often called its
velocity.
 The velocity of all enzymes is dependent on;
 Substrate concentration

 enzyme concentration

 Temperature

 pH (Hydrogen ion concentration)

 Inhibitors

 Activators(cofactors)

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 1. Effect of substrate
 The rate of an enzyme-catalyzed reaction increases with
substrate concentration until a maximal velocity (Vmax) is reached
 At low substrate concentrations a doubling of [S] will lead to a
doubling of the initial velocity (V0).
 At higher substrate concentrations the enzyme becomes saturated
and further increases in [S] lead to very small changes in V0.
 This occurs because, at saturating substrate concentrations
effectively all of the enzyme molecules have bound substrate. The
overall enzyme rate is now dependent on the rate at which the
product can dissociate from the enzyme,
 The shape of the resulting graph when V0 is plotted against [S] is
called a hyperbolic curve
 The simplest of these equations, the Michaelis-Menten equation,
relates the initial velocity (V0) to the concentration of substrate
21

[S] and the two parameters Km and Vmax..

Where, Km “Michaelis constant”= ½Vmax, reflects the affinity of the enzyme
for that substrate . Small Km= high affinity of the enzyme for substrate, Large
Km= low affinity of enzyme for substrate because a high concentration of substrate
is needed to half-saturate the enzyme
Vmax= is the reaction rate when the enzymes are saturated, and is independent of
the Substrate concentration.
Vo= - is the number of substrate molecules converted to product per unit time.
[S]- substrate concentration
•Km is a characteristic constant of E

[S] =Km ， v = Vmax/2

22

[S] << Km ， v ∝ [S]

 2. Enzyme concentration
 At saturating substrate concentration, a doubling of the enzyme
concentration will lead to a doubling of V0.
 The rate of enzyme catalyzed reaction is directly proportional to
the concentration of enzyme (V ≈ [E] )
 [S] is held constant/not limiting.
 gives a straight line graph when V0 is plotted against [E].

23
 3. Temperature
 Temperature affects the rate of reactions in two ways.
• First, arise in temperature increases the thermal energy of the
substrate molecules. This raises the proportion of substrate
molecules with sufficient energy to overcome energy of
activation, and hence increases the rate of the reaction.
 A second effect comes into play at higher temperatures.
Increasing the thermal energy of the molecules increase the
chances of breaking the multiple weak, non-covalent interactions
 Ultimately this will lead to the denaturation (unfolding) of the
enzyme (human E start at >40oc)., decreasing enzyme activity

24
 4. pH
 Each enzyme has an optimum pH
 Slight deviations in the pH from the optimum lead to a decrease
in the reaction rate.
 Larger deviations in pH lead to denaturation of the enzyme
due to changes in the ionization of amino acid residues and
the disruption of non covalent interactions.
 Many enzymes have a pH optimum of around 6-8,
 but there is great diversity in the pH optima of enzymes, due to
the different environments.

25
Enzyme inhibition

5. Inhibitors
 Any substance that can diminish the velocity of an enzyme-
catalyzed reaction is called an inhibitor
 Enzyme inhibition can be of two main types:
 irreversible or

 reversible.

 Reversible inhibition can be subdivided into

 competitive

 noncompetitive (Mixed inhibition)

 Uncompetitive inhibition

26
1. Irreversible Inhibitors

 Enzyme is covalently modified after interaction with inhibitor

 One bound with inhibitor the enzyme is no longer act as a
catalyst – loses enzymatic activity
 Original activity cannot be regenerated by increasing
substrate concentration or removing the remaining free
inhibitor
 Also called suicide inhibitors
e.g. nerve gas completely inactivate acetyl cholinesterase (an
enzyme that catalyzes the hydrolysis of acetylcholine to
acetic acid and choline)
Iodoacetamide, heavy metal ions (Hg++), oxidising agents.

27
2. Reversible Inhibitors
 Inhibitor bind to enzyme by non-covalent interaction and are
subsequently released
a. Competitive inhibitors
 Shape and structure of inhibitor is very similar to substrate
 Inhibitor mimic substrate (or transition state) and fits into the
active site. Physically blocks substrate’s access into the active site
 Effects of competition can be overcome by increasing [S].

28
Example 1. Allopurinol
Allopurinol is a drug used in the treatment of gout.
Gout is due to excessive production of uric acid. Xanthine
oxidase is an enzyme involved in the formation of uric acid from
hypoxanthine.
 Allopurinol is a structural analog of hypoxanthine and hence it
is an antimetabolite of hypoxanthine.
Example 2: Anticancer drugs
Competitive inhibitors used in the treatment of cancer are
aminopterin and amethopterin (methotrexate).
They are structural analog of folic acid. They are competitive
inhibitors for the enzyme dihydrofolate reductase.
They are used in the treatment of leukaemia, a type of cancer. 29
b. Non-competitive inhibitors
 Inhibitor binds to a site other than the active site
 Binding causes a change in the structure of the enzyme so that it
cannot catalyze a reaction
 The effects of non-competitive inhibition cannot be overcome by
increasing [S]
 These inhibitors bind reversibly either to the free enzyme or the
ES complex to form the inactive complexes EI and ESI
 The E-I complex formation does not affect the binding of
substrates but E-I-S complexes do not proceed to form products

30
 This kind of inhibition is common in allosteric enzymes which
have allosteric sites other than the substrate binding site
Example: the inhibition of L-threonine dehydratase by L-isoleucine
 Reversible non-competitive inhibitors are rare.
 Most of the known non-competitive inhibitors are irreversible.
They are referred as enzyme poisons.
1.Iodoacetate- blocks the formation of 1,3-bisphosphoglycerate
from glyceraldehyde-3-phosphate by inhibiting enzyme
glyceraldehyde-3-phosphate dehydrogenase
2. Fluoride- blocks the action of enolase, which converts 2-
phosphoglycerate to phosphoenolpyruvate.
3. Heavy metals like Hg2+, Ag+, Pb2+ and Arsenite are also enzyme
poisons. They interact with –SH group of enzyme and inactivate
it
31
C. Uncompetitive reversible inhibition
• In this case the uncompetitive inhibitor binds only to ES complex
at locations other than the catalytic site (active site)
• Substrate binding modifies enzyme structure making inhibitor
binding site available
• The E-I-S complexes do not proceed to form products.
• The E-I-S complexes do not backward to the substrates and
enzymes.
• This inhibition has the effects on reducing both Vmax and Km.
• Inhibitor has no structural similarity to the substrate and does not
bind the free enzyme but binds the enzyme after complexation
with the substrate that exposes the inhibitor binding site (ESI).
• Its binding, although away from the active site, causes structural
distortion of the active site of the complexed enzyme that
inactivates catalysis.
32
 E + ES E +
S P
+
I

Uncompetitive inhibition
ESI

Summary of reversible inhibition

33
Regulation of enzyme activity
 Maintenance of an ordered state in a timely fashion and without
wasting resource
 Regulation means to make an enzyme more or less active
 Regulation is necessary to control the rates of reactions
1. Feedback/end product regulation
 an enzyme early on in the pathway is inhibited by an end-
product of the metabolic pathway in which it is involved.

2. Reversible covalent modification

 The activity of many enzymes is altered by the reversible
making and breaking of a covalent bond between the enzyme
and a small non-protein group (Phosphorylation
34

dephosphorylation,Methylation-demethylation,glycosylation etc
For example, phosphorylation of glycogen phosphorylase (an
enzyme that degrades glycogen) increases activity, whereas the
addition of phosphate to glycogen synthase (an enzyme that
synthesizes glycogen) decreases activity
3. Proteolytic cleavage
 Zymogens (inactive precursor to an enzyme)
 activated by cleavage of a specific peptide bond

Example: Trypsin and Chymotrypsin

 Initially synthesized as trypsinogen and
chymotrypsinogen which are both inactive
 Formed in the pancreas where they would do damage if

active
 In the small intestine, where their digestive properties

are needed, they are activated by cleavage of specific

peptide bonds by entropeptidase 35
 Importance of Enzymes

 Enzymes play an important role in Metabolism, Diagnosis,

and Therapeutics.
 All biochemical reactions are enzyme catalyzed in the living
organism.
 Level of enzyme in blood are of diagnostic importance e.g. it
is a good indicator in disease such as myocardial infarction.
 Enzyme can be used therapeutically such as digestive
enzymes.
 Enzymes have industrial application

36
 Clinical Application of Enzymes
1. Use of enzymes in therapy
Coagulation Factors (II, VII, VIII, IX, X)- to Prevent
bleeding
 DNase- to remove DNA clogging in lumen of tubes in cystic
fibrosis
Asparaginase: To remove Asn from extracellular fluid and
starve neoplastic cells
Patients who have had a heart attack or stroke are frequently
treated by intravenous administration of tissue plasminogen
activator (tPA) or streptokinase, enzymes that break down fibrin
clots that clog blood vessels
2. Evaluate the status of a given organ (liver, heart,) by serum
enzyme
3.To use enzymes as reagents to quantify the amount of an analyte
eg. Glucose oxidase as Glucose titer, uricase used for uric 37acid
analysis
 Plasma enzymes
Plasma enzymes are of two sources
A. Plasma-derived /Plasma specific/ plasma functional enzymes
 Their field of activity is plasma components and their activity is
higher in plasma than in cells, e.g. coagulation and lipoprotein-
metabolizing enzymes.
B. Cell-derived /Non-plasma specific /plasma non-functional
enzymes
 A very low plasma level normally exists due to normal wear
and tear and diffusion through undamaged cell membranes.
 Gross damage to the cells or abnormal membrane permeability,
overproduction of the enzymes or abnormal high cellular
proliferation and/or wear and tear may allow their leakage in
abnormally high amount into plasma.
 Enzymes that have been shown to have diagnostic value are
present in plasma. 38
39
 Enzymes of different origin are present in plasma and are used
for diagnosis.
 Because of normal cell turn over they present in small quantity ,
however, increased quantity is due cells injuries.
1. Acid phosphatase: Tumor marker in prostate carcinoma
2.Alanine aminotransferase(ALT): indicator of hepatocellular
damage.
3. Alkaline phosphatase: increase in cholestatic liver disease and
is marker of osteoblast activity in bone disease.
4. Amylase/Lipase: indicator of cell damage in acute pancreatitis.
5. Aspartate amino transferase (AST): indicator of hepatocellular
damage or as a marker of muscle damage such as MI
6. Creatine Kinase(CK): marker of muscle damage and acute MI.
7. γ-glutamyl transpeptidase: sensitive marker of liver cell
damage
40
8. Lactate dehydrogenase: marker of muscle cell damage.
Application of enzymes in diagnosis of diseases

41
 Assignment
1. Write the Diagnostic function of enzymes and give
some examples
2. Drive a Michael-Menten equation
3. What are Isozymes? Give some examples with their
locaton and function.

42