Chapter 3 enzyme 2015814

04/06/2023 24LOCK-AND-KEY THEORY  The lock-and-key theory:  explains the high specificity of enzyme  Enzyme surface accommodates substrates having specific shapes and sizes  Only spe

CHAPTER 3

ENZYME

04/06/2023 2

CONTENTS

Nomenclature and Classification

Mechanism of enzyme action

04/06/2023 3

ENZYME NOMENCLATURE

Traditional name: some of the earliest enzymes to be

discovered:

 Name: ending in – “in” to indicate they were proteins

 Ex: the digestive enzymes: pepsin, trypsin, chymotrypsin

 urease catalyzes hydrolysis of urea

 DNA polymerase catalyzes the polymerization of

nucleotides to form DNA

 Common name

04/06/2023 5

EXAMPLES

04/06/2023 6

ENZYME NOMENCLATURE

 EC (Enzyme Commission) systematic name:

 [International Union of Biochemistry and Molecular

Biology: IUBMB]

 Systematic name indicating:

 The substance acted on

 The functional group acted on

 The type of reaction catalyzed

 All EC names end in –ase

(2) : the class name (transferase)

(7): the sub class (phosphotransferase)

 (1): the sub sub class (a phosphotransferase with a hydroxyl group as acceptor)

 (1): the sub sub sub class (D-hexose as the

phosphoryl group acceptor)

04/06/2023 8

EC CLASSIFICATION OF ENZYMES

Enzymes: grouped into 6 major classes (based on the rxns which they catalyze)

04/06/2023 9

ENZYME CLASSIFICATION

04/06/2023 10

ENZYME CLASSIFICATION

Oxidoreductases, Transferases and Hydrolases

Lyases, Isomerases and Ligases

04/06/2023 by TDLV 13

OXIDOREDUCTASE

 Ex: dehydrogenases (Lactate Dehydrogenase

(LDH)): accept and donate electrons (H + ), using

cofactors such as NAD+/NADH as an electron

donor or acceptor

LDH

04/06/2023 by TDLV 14

Lyases

 Lyases: cleaving bonds

 Ex: aldolases: fructose diphosphate aldolase: in glycolysis)

 Thiolases: β-ketoacyl-CoA thiolase: breakdown of fatty acids

04/06/2023 by TDLV 15

Isomerases

Isomerases: catalyzing the rearrangement of atoms in a molecule

04/06/2023 by TDLV 16

Ligases

 Ligases: catalyzing synthesis of bonds

04/06/2023 17

ENZYME NOMENCLATURE

 Ex: EC number: 2.7.1.1.:

(2) : the class name (transferase)

(7): the sub class (phosphotransferase)

 (1): a phosphotransferase with a hydroxyl group

as acceptor

 (1): D-hexose as the phosphoryl group acceptor

04/06/2023 19

CLASSWORK

? Match the following general enzyme names to the

reactions that they catalyze

Enzyme Reaction catalyzed

Decarboxylase Hydrolysis of cellulose

Peptidase Hydrolysis of pectin

Pectinase Hydrolysis of peptide linkages

Lipase Removal of carboxyl groups from compounds

Cellulase Hydrolysis of lipid

04/06/2023 20

STRUCTURE OF AN ENZYME

 Some enzymes are proteins

 Some enzyme are RNAs

 Some contains both a protein (apoprotein/apoenzyme) and a nonprotein

 Nonprotein: either a coenzyme (vitamins) or a cofactor (inorganic ions)

 Some enzymes require both a coenzyme and one cofactor for activity

04/06/2023 21

COFACTOR

Table1: Some common cofactors (hard to separate)

04/06/2023 22

COENZYME

Table2: Some common coenzymes

04/06/2023 23

ACTIVE SITE OF AN ENZYME

 At the active site:

 The chemical transformation of the substrate

caused by functional groups on the enzyme

04/06/2023 24

LOCK-AND-KEY THEORY

 The lock-and-key theory:

 explains the high specificity of enzyme

 Enzyme surface accommodates substrates having

specific shapes and sizes

 Only specific substances “fit” in an active site to

form an ES complex

04/06/2023 25

INDUCED-FIT THEORY

 Limitation of lock-and-key theory:

 requires enzymes conformations to be rigid

 More flexible theory: induced-fit theory

 proposes enzymes have flexible conformations that

may adapt to incoming substrates

 The active site adopts a shape that is complementary

to the substrate only after the substrate is bound

04/06/2023 26

Fig : lock-and-key theory Fig : induced-fit theory

04/06/2023 27

MECHANISM OF ENZYME ACTION

Step 1: form enzyme-substrate complex

 Enzyme and substrate combine to form substrate complex

enzyme-E + S ES

+

04/06/2023 28

MECHANISM OF ENZYME ACTION

Step 2: form enzyme-product complex

 Enzyme-product complex formed

E S EP

04/06/2023 29

MECHANISM OF ENZYME ACTION

Step 3: enzyme & product separate

 Enzyme-product complex formed

EP E + P

04/06/2023 30

MECHANISM OF ENZYME ACTION

04/06/2023 31

MECHANISM OF ENZYME ACTION

04/06/2023 by TDLV 32

ENZYME CATALYSIS MECHANISM

1 Acid-base catalysis: hydrolysis of ester/ peptide

bonds, phosphate group reactions, addition to

carbonyl groups, etc

2 Covalent catalysis: formation of a

catalyst-substrate covalent bond

Serine proteases: acyl-serine intermediate Cysteine proteases: acyl-cystein intermediate Protein kinases and phosphatases: phospho-amino acid intermediates

04/06/2023 by TDLV 33

Catalysis mechanism

 General acid-base catalysis:

– donating a proton (act as a

general acid), or

– accepting a proton (, act as a

general base)

• Protein functional groups can

function as general acid/base

catalysts:

– e.g His imidazole, -amino group,

-carboxyl group, thiol of Cys, R

group carboxyls of Glu, Asp,

aromatic OH of Tyr, etc

04/06/2023 by TDLV 34

 Covalent catalysis: a transient covalent bond is formed between the enzyme and the substrate

– side chains of His, Cys, Asp, Lys and Ser can participate in covalent

catalysis by acting as nucleophiles

• Electrophilic catalysis: covalent intermediate btw the cationic

electrophile of the enzyme and an electron rich portion of the substrate molecule

– The a.acid side chains do not provide this so enzyme electrophilc

catalysis require electron deficient organic cofactors or metal ions

04/06/2023 by TDLV 36

Mechanisms of catalysis

04/06/2023 by TDLV 37

3 Metal ion catalysis: Ionic interactions between an

enzyme-bound metal and a substrate can help orient the

substrate for reaction or stabilize charged reaction transition states  weak bonding interactions

Metals can also mediate oxidation-reduction reactions by reversible changes in the metal ion’s oxidation state

04/06/2023 by TDLV 38

Mechanisms of catalysis

Metal ion catalysis:

• Metal ions often used for one or more of the following:

– binding substrates in the proper orientation (e.g

Cytochromes)

– mediating oxidation-reduction reactions

– electrostatically stabilizing or shielding negative charges

that would otherwise repel the attack from an electrophile

(electrostatic catalysis)

– Simply stabilize the catalytically active form of the enzyme

04/06/2023 by TDLV 39

Mechanisms of catalysis

Metal ion catalysis:

• Metalloenzymes contain tightly bound metal ions: (usually

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

• Metal-activated enzymes contain loosely bound metal ions:

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

• Its role is largely to bind the (-)ly charged groups in NZ 

more active form

• Mg +2 (intracellular NZs) and Ca +2 (extracellular NZs)

• Ca +2  extracellular NZs, e.g Salivary and pancreatic

-amylase: maintain the structure required for activity

• Mg +2  intracellular NZs, most kinases

04/06/2023 by TDLV 41

Mechanisms of catalysis

Fe, Cu, Zn and Mo

• Found in trace amounts

• Cu +2 & Zn +2  superoxide dismutase: Zn +2 ions

have a structural role, Cu +2 ions are involved in

reaction

• The iron-molybdenum cofactor (FeMoco) of

nitrogenase: metallocluster biological nitrogen

fixation

04/06/2023 by TDLV 42

Mechanisms of catalysis

4 Catalysis by alignment

• "Strain“: binding of the substrate to the enzyme caused the

substrate to become distorted toward the transition state

• Transition state stabilization: the transition state makes

better contacts with the enzyme than the substrate does.

04/06/2023 43

ENZYME ACTIVITY

 Enzyme activity: the catalytic ability of an

enzyme to increase the rate of a reaction

 Turnover number (TON): the number of

molecules of substrate acted on by one molecule

of the enzyme per minute

 TON~1000 (substrate molecules per minute)

04/06/2023 44

ENZYME ACTIVITY

 Enzyme activity: the total units of enzyme activity in a

solution

 Specific activity: the number of enzyme activity units

per milligram of total protein

 Specific activity: a measure of enzyme purity

 the higher the specific activity, the purer the enzyme

04/06/2023 45

ENZYME ACTIVITY

 1.0 unit of enzyme activity (1IU: International Unit):

 the amount of enzyme causing transformation of 1.0

μmol of substrate per minute at 25⁰C under optimal conditions of measurement

 for some enzymes: IU may be defined differently

 IUs measure how much enzyme is present in a solution

04/06/2023 46

 Enzyme assays:

 experiments performed to measure enzyme activity

 often done by monitoring the rate at which a

characteristic color of a product forms or the color

of a substrate decreases  colorimetric method

 For reactions involving H+ ions, monitoring the rate

of change in pH over time

04/06/2023 47

REGULATION OF ENZYME ACTIVITY

Enzymes are often regulated by the cell

Cells use several methods to control when & how well enzymes work

– activation of zymogens

– allosteric regulation

– genetic control

 are synthesized and stored as inactive precursors

 when the enzyme is needed the zymogen is

released and activated (cleavage of one or more

peptide bonds)

 digestive enzymes: pepsin, trypsin, chymotrypsin

04/06/2023 49

EXAMPLES OF ZYMOGENS

04/06/2023 50

ALLOSTERIC REGULATION

 Modulators: compounds alter enzymes by changing

the 3D conformation of the enzyme

 activators: increase enzyme activity

 inhibitors: decrease enzyme activity: non-competitive

inhibitors

• Allosteric enzymes: enzymes with quaternary

structures with binding sites for modulators

04/06/2023 51

ALLOSTERIC REGULATION

• Feedback inhibition: when the end product of a

sequence of enzyme-catalyzed reactions inhibits

an earlier step in the process

 allows concentration of the product to be maintained within very narrow limits

04/06/2023 52

GENETIC CONTROL

 Synthesis of enzymes is under genetic control

by nucleic acids

 Increasing the number of enzymes molecules

present through genetic mechanisms  increase

production of needed products

• Enzyme induction: enzymes are synthesized in

response to cell need

 allows an organism to adapt to environmental

changes

04/06/2023 53

GENETIC CONTROL

 Galactosidase: enzyme in Escherichia coli

 Hydrolyze lactose to D-galactose and D-glucose

– In the absence of lactose in the growth medium

 there are very few β-galactosidase molecules

– In the presence of a lactose-containing medium

 thousands of molecules of enzyme are produced

– If lactose is removed  production of the enzyme

decreases

Enzyme Activity

Enzyme activity: total units of activity in a given

volume of solution.

Specific activity: the number of units of activity per

amount of total protein (mass)  follow the increasing purity of an enzyme through several procedural steps.

Molecular activity: Used to compare activities of

different enzymes.

Also called the turn-over number (TON = kcat)

Enzyme Activity

Classical units:

Unit of enzyme activity:

Enzyme Activity

New international units:

Unit of enzyme activity:

mol substrate transformed/sec = katal

04/06/2023 by TDLV 57

ENZYME ACTIVITY

 One unit of enzyme activity : the amount of the

enzyme that catalyzes the conversion of 1 μmol

of substrate or formation of one μmol product per

minute at a specific condition (pH, temperature,

substrate concentration much greater than the value of Km)

04/06/2023 by TDLV 58

REGULATION OF ENZYME ACTIVITY

Enzymes are often regulated by the

cell

Cells use several methods to control

when & how well enzymes work

04/06/2023 60

THE EFFECT OF TEMPERATURE

 Increasing temperature  reaction rate increases

• Because enzymes are proteins, beyond a certain

temperature, the enzyme denatures

• Every enzyme has an optimum temperature (at which

the enzyme activity is highest)

 above or below the optimum temperature  the rate

is lower

04/06/2023 61

THE EFFECT OF pH VALUE

 Every enzyme has an optimum pH value (at which

the enzyme activity is highest)

 above or below the optimum pH value  the rate is lower

04/06/2023 62

THE EFFECT OF pH VALUE

04/06/2023 63

THE EFFECT OF pH VALUE

04/06/2023 64

THE EFFECT OF ENZYME AMOUNT

• Enzyme amount increases  reaction rate increases (at constant substrate concentration)

• Linear relationship between reaction rate and

enzyme amount

Enzyme amount: too high

 no effect on rxn rate (substrate concentration becomes the limiting

factor)

04/06/2023 65

THE EFFECT OF SUBSTRATE CONCENTRATION

• Increasing [S] increases reaction rate

• The rate reaches a maximum (Vmax), and remains constant after that

• The maximum rate is reached when the enzyme is saturated with substrate

- a single substrate (absolute)

- a group of similar substrates

- a particular type of bond

- Stereochemical specificity: Only work with the proper D- or L- form

04/06/2023 by TDLV 67

ENZYME SPECIFICITY

04/06/2023 by TDLV 68

Enzyme kinetics

• The concentration-rate curve is described by the

Michaelis-Menten equation

04/06/2023 by TDLV 70

Michaelis – Menten equation

04/06/2023 by TDLV 71

04/06/2023 by TDLV 72

• Km is the Michaelis constant

= a measure of affinity of substrate for the enzyme - the lower the Km is, the greater the affinity is

• Because Vmax is never reached, Km can only be

estimated from the concentration-rate curve

• Vmax and Km are key paramemeters in defining how an enzyme functions.

04/06/2023 by TDLV 74

04/06/2023 by TDLV 75

04/06/2023 by TDLV 76

04/06/2023 by TDLV 77

Calculation of kinetic parameters

Kinetic parameters of enzyme-catalysed reaction (Vmax, Km)

Determined graphically by measuring velocity (ΔC/Δt)

of enzyme-catalyzed reaction at different

concentrations of substrate (Vo versus [substrate])

04/06/2023 by TDLV 78

04/06/2023 by TDLV 79

04/06/2023 by TDLV 80

04/06/2023 by TDLV 81

3 PARTS OF S CONCENTRATION RANGE

• when [S]= K M , the equation reduces to

• when [S] >> KM, the equation reduces to

• when [S] << KM, the equation reduces to

04/06/2023 by TDLV 82

Kcat: turnover number (TON)

minutes

Vo = Vmax[S] can be transformed by

Km + [S] taking reciprocals.

 plot the curve as a straight line in the form

y = mx + c

Lineweaver Burk plot

Plotting 1 against 1 gives a straight line Vo [S]

Using these transformations , Km and Vmax can be determined easily and accurately.

04/06/2023 by TDLV 87

LINEWEAVER – BURK DOUBLE RECIPROCAL PLOTS

04/06/2023 by TDLV 88

Kcat

Kcat: more general rate constant: to describe

the limiting rate of any enzyme-catalyzed

reaction at saturation.

If the reaction has several steps and one is

clearly rate-limiting kcat is equivalent to the rate constant for that limiting step

04/06/2023 by TDLV 89

Kcat

In the Michaelis-Menten equation,

kcat=Vmax/[Et], Vmax=kcat[Et]

Equation Michaelis-Menten becomes

kcat is a first-order rate constant  has units of

It is equivalent to the number of substrate molecules

converted to product in a given unit of time on a single enzyme molecule when the enzyme is saturated with

substrate

04/06/2023 by TDLV 90

Kcat

04/06/2023 by TDLV 91

kcat/Km

kcat/Km: the criterion of substrate specificity, catalytic

efficiency and "kinetic perfection”

kcat/Km used as a measure of 2 things:

1 enzyme's substrate preference

2 enzyme's catalytic efficiency

1 enzyme's preference for different substrates (substrate specificity)

– The higher the kcat/Km, the better the enzyme works on that substrate

– e.g., chymotrypsin: protease that clearly "prefers" to

cleave after bulky hydrophobic and aromatic side chains