biocatalysts · 1897 –eduard buchner –ability of yeast extracts that lacked any living yeast...

ENZYMES BIOCATALYSTS

2009/2010

INGRID ŽITŇANOVÁ

ENZYMOLOGY

HISTORY

17th -18th century – digestion of meat caused by stomach secretions

- conversion of starch to glucose by saliva

19th century – L. Pasteur – fermentation of sugar to alcohol by yeasts

- vital force in yeasts required for this fermentation

1897 – Eduard Buchner – ability of yeast extracts that lacked any

living yeast cells to ferment sugar – in 1907 – Nobel Prize for

chemistry - discovery of cell free fermentation

1926 – James B. Sumner isolated the first enzyme – urease and

prooved its protein character

en zyme – in yeasts

Louis Pasteur

Eduard Buchner

http://en.wikipedia.org/wiki/File:Eduardbuchner.jpg

• Enzymes are biocatalysts

Increase the rate of a reaction

Not consumed by the reaction

Enzymes are often very “specific” – promote only 1 particular

reaction

In the single cell - more than 3000 enzymes

BIOCATALYSTS VS. INORGANIC

CATALYSTS

Enzymes:

1) More efficient - higher reaction rate

2) Milder reaction conditions (20-40C, pressure 0.1 MPa, pH = 7)

3) Higher specificity of the reaction

4) Ability to be regulated at different levels (inhibitors, activators)

5) They are non-toxic

6) Enzymes – organic compounds, chemic. catalysts – inorg. compounds

Catalyst rate enhancement

Inorganic catalysts 102 -104 fold

Enzymes up to1020 fold

Catalyst time for reaction

With enzyme 1 second

Without enzyme 3 x 1012 years

How much is 1020 fold?

• Glucose oxidation in solution and in human

organism

Example of enzymes effectiveness

• The oxidation of a fatty acid to carbon dioxide and

water

in the body: reaction takes place smoothly and rapidly within a

narrow range of pH and temperature

in the test tubes: extreme pH, high temperature and corrosive

chemicals

ENZYME STRUCTURE

• Enzymes are proteins (chain of amino acids)

• Enzyme will twist and fold into a specific shape due

to how the amino acids are attracted to each other

• Enzyme shape attracts specific

molecules - substrates – molecules

that bind to the enzyme

Carbonic anhydrase

in lungs: H2CO3 CO2 + H2O

Enzymes DO NOT change the equilibrium constant

of a reaction (accelerate the rate of the forward and

reverse reactions equally)

Carbonic anhydrase

in tissues: CO2 + H2O H2CO3

Carbonic anhydrase

CO2 + H2O H2CO3

HOLOENZYME

Inorganic elements serving as enzyme cofactors

Cytochrome oxidase

Cytochrome oxidase, catalase, peroxidase

Pyruvate kinase

Hexokinase, pyruvate kinase

Arginase,

Dinitrogenase

Urease

Glutathione peroxidase

Carboanhydrase, alcohol dehydrogenase

Cu2+, Zn2+, Mn2+ Superoxide dismutase

Cofactors

serve several apoenzymes:

NAD+ (nicotinamide adenine dinucleotide) - a coenzyme for a great

number of dehydrogenases: alcohol dehydrogenase,

malate dehydrogenase

lactate dehydrogenase reactions

Role of organic cofactors:

transport of chem. groups from 1 reactant to another

Classification of cofactors according to

the type of a transferred molecule

1) Transfer of H atoms

NAD+ (nicotine amide adenine dinucleotide) - transport of H-

FAD (flavine adenine dinucleotide) – transport of 2H

FMN (flavine mononucleotide), lipoic acid - transport of 2H

3) Transfer of groups of atoms

adenosine phosphates (ATP, ADP) - phosphate group

coenzyme A – acyl groups

thiamine diphosphate - aldehydes

pyridoxal phosphate – amine groups

biocytin – CO2

tetrahydrofolate (coenzyme F) – one-carbon groups

2) Transfer of electrons

coenzyme Q, porfyrin derivatives

Vitamins are often converted to coenzymes

Vitamin Coenzyme Function

Thiamin diphosphate decarboxylation

Flavin mononucleotide (FMN) carries hydrogen

Nicotinamide adenine dinucleotide carries hydrogen H-

(NAD+), (NADH)

Biocytin CO2 fixation

Coenzyme A acyl group carrier

B1

B2

B3

pantothenic acid

B5

H

ACTIVE SITE

Active siteSubstrate

ACTIVE SITE

ACTIVE SITE = pocket in the enzyme where substrates

bind and catalytic reaction occur

CATALYTIC SITE(where the reaction proceeds)

BINDING SITE(where a substrate binds)

Some enzymes contain more active sites (2-4), they can

bind more substrate molecules

Aminoacids of the active site can be located at different

regions of a polypeptide chain

hydrogen

bonding

binding pocketionic interaction

ionic interaction

hydrophobic

interaction

2. non-covalent interactions

between substrate and

the active site:

- hydrogen bonding

- ionic interactions

- hydrophobic interactions

Binding sites (substrate, cofactor)

Catalytic site

ENZYME SUBSTRATE

ACTIVE SITE

Substrates bind in active site by following interactions:

hydrogen bonds

hydrophobic interactions

ionic interactions

covalent bonds (occasionally)

The interactions hold the substrate in the proper orientation for most

effective catalysis

The ENERGY derived from these interactions = “Binding energy“

1/ E + S E-S Formation of E-S complex

2/ E-S E-S* Activation of the complex

3/ E-S* E-P Conversion of substrate to a product

4/ E-P E + P Separation of product from enzyme

ES* = enzyme/transition state complex

Stages of enzyme reaction

E + S ES

First step of enzyme catalysis

FORMATION OF THE ENZYME-SUBSTRATE

COMPLEX

ES transition state complex

Second step

FORMATION OF THE TRANSITION

STATE COMPLEXNote change

Transition State:

a. Old bonds break and

new ones form.

b. Substance is neither

substrate nor product

c. Unstable short lived

species with an equal

probability of going

forward or backward.

Third step

FORMATION OF THE ENZYME-PRODUCT

COMPLEX

ES* EP

Fourth step

RELEASE OF THE PRODUCT

EP E + P

Mechanisms of substrate conversion

• Enzyme binds

2 substrates,

that they are in

close vicinity

• Charges in the

active site

induce changes

in the charges in

S molecule

• Deformation of S

facilitates its

conversion to a

product

• Activation energy is the energy required to start a

reaction.

MECHANISM OF ENZYME ACTION

• Enzymes decrease the activation energy of a reaction

by formation of active enzyme - substrate complex

Uncatalyzed reaction

Catalyzed reaction

Substrate

Product

En

erg

y

Transition state

• The lower the free energy of activation, the more

molecules have sufficient energy to pass through the

transition state, and, thus, the faster the rate of the

reaction.

Enzyme activity

The katal (symbol: kat) - the SI unit of catalytic activity

1 kat = mol . s-1

One katal is the catalytic activity that changes one mole

of substrate per second at optimal pH.

Enzyme

Substrate Product

SPECIFIC ACTIVITY – katal/kg (μkat/mg) protein

MOLAR ACTIVITY – katal/mol protein

1 U = μmol . min-1

1 kat = mol/s = 60 mol/min= 60.106 μmol.min-1 = 6.107 U

1 U = μmol.min-1 = 10-6 mol/60 s = 16.7 . 10-9 kat

Enzyme has SPECIFICITY – it can discriminate among

possible substrate molecules:

ENZYME SPECIFICITY

EnzymeEnzyme

SubstrateSSS

Enzymes are very specific

and only work with certain substrates

SUBSTRATE SPECIFICITY(apoenzyme responsible)

1) Strictly specific enzymes - only react with a single

substrate (DNA polymerase, urease)

2) Less specific enzymes

a. Group specific - recognize a functional group (-OH, -NH2...)

(alcoholdehydrogenase)

b. Linkage specific – particular type of chemical bond regardless

. of the rest of the molecular structure (peptidase, esterase)

SPECIFICITY OF EFFECT (cofactor responsible)

Each enzyme can catalyze only a certain type of a

chemical reaction

SPECIFICITY OF EFFECT (cofactor responsible)

OXIDOREDUCTASES – oxidation/reduction reactions - transfer of H and O atoms or electrons from one substance to another (alcoholdehydrogenase)

TRANSFERASES – transfer of a functional group - methyl-, acyl-, amino- or

phosphate group (hexokinase)

HYDROLASES – catalyze hydrolysis of various bonds (carboxypeptidase A)

LYASES – cleave bonds by means other than hydrolysis and oxidation

(pyruvate decarboxylase)

ISOMERASES – intramolecular changes of „S“ (maleate isomerase)

LIGASES – join two molecules with covalent bonds with the

use of energy from ATP (pyruvate carboxylase)

MODELS FOR ENZYME/SUBSTRATE

INTERACTIONS

1) Lock and Key Model (Emil Fischer 1894)

This model assumed that only a substrate of a proper shape

could fit with the enzyme

Substrate

Active siteES complex

Enzyme

Enzyme

Substrate

A. Substrate (key) fits into a perfectly shaped space in the enzyme

(lock)

B. Highly stereospecific

C. Site is preformed and rigid

2) Induced Fit Model (Daniel Koshland 1958)

This model assumes continuous changes in active site

structure as a substrate binds

Enzyme

Substrate

Active site

ES complex

Takes into account the flexibility of proteins

A substrate fits into a general shape in the enzyme, causing the enzyme to change shape (conformation)

Change in protein configuration leads to a near perfect fit of

substrate with enzyme

Induced fit model

• Uncatalyzed reactions often are extremely slow.

Principles of Catalysis

• They are slow because of the heigh activation energy

• Creating an EC complex reduces bonds in the substrate and

makes the substrate easier to convert to the product – it lowers

the activation energy.

Enzyme Nomenclature

1. Trivial names

2. Systematic nomenclature

Enzyme Nomenclature

1. Trivial names

everyday use (pepsin, trypsin)

Usually named by suffix –ase to: - the name of a substrate (urease)

- the catalytic reaction (glucose

oxidase)

Some examples:

Alcohol dehydrogenase - oxidation of alcohols

DNA polymerase - polymerization of nucleotides

Protease - hydrolysis of proteins

Methyltransferase - methyl group transfer

2. Systematic names

Introduced in 1961 (enzyme commision of IUB)

Systematic names:

a) characterizing catalytic reaction

b) recommended – commonly used

c) international – code number

L-lactate + NAD+ pyruvate + NADH + H+

2. Systematic names

a) Characterizing the reaction:

L-lactate : NAD+ - oxidoreductase

name of substrates + name of the reaction catalyzed + suffix

(separated by the colon)

–ase

b) Recommended name: Lactate dehydrogenase

c) Code number : EC 1.1.1.1

EC 1.x.x.x oxidoreductases

EC 2.x.x.x transferases

EC 3.x.x.x hydrolases

EC 4.x.x.x lyases

EC 5.x.x.x isomerases

EC 6.x.x.x ligases (synthetases)

ENZYME NOMENCLATURE

b) Recommended name: Lactate dehydrogenase

L-lactate + NAD+ pyruvate + NADH + H+

c) Code number : EC 1. 1. 1. 1

oxidoreductase

acting on the CH-OH group

NAD+ as acceptor

alcohol dehydrogenase

ISOZYMES – ISOENZYMES

• catalyze the same reaction

• have different primary structure

•are produced by different genes (= true isozymes), or produced

by different posttranslational modification (= isoforms)

• have different physical and chemical properties

• can be localized in different organs and cell compartments

pyruvate

Lactate dehydrogenase

LDH1 – LDH5

• Slightly different amino acid sequence

• Detection of specific LDH isozymes in the blood - diagnostics

of tissue damage such as occurs during myocardial infarction

lactate

Lactate dehydrogenase – composed of M a H subunits

5 isomers of lactate dehydrogenase

M4

M3H

M2H2

MH3

H4

M4 M3H M2H2 MH3 H4

Liver

Muscle

White cells

Brain

Red cells

Kidney

Heart

Separation by electrophoresisLDH-1

LDH-2

LDH-3

LDH-4

LDH-5

LDH1

LDH2

LDH5

Control serum

LDH1

LDH2

LDH3

LDH3

LDH5

ENZYME COMPLEXES

Formation of a binary complex between 2 enzymes of a certain

reaction chain

Aldolase + glycerolphosphate dehydrogenases

Multienzyme complexes

pyruvate dehydrogenase system

complex of fatty acid synthesis (7 enzymes)

Multienzyme complex of pyruvate

dehydrogenase

MULTIENZYME COMPLEX

Regulation of enzyme activity

A) Without the change in the quantity of enzyme

molecules 1) Physico-chemical factors

2) Presence of inhibitors and activators

3) Allosteric regulation of enzyme activity

4) Regulation by modification of enzyme molecule

5) Compartmentalization of enzymes

B) With the change of the number of enzyme

molecules1) Induction and repression

2) Regulated degradation of proteins

1. Physico-chemical factors

Substrate concentration

Enzyme concentration

Temperature

pH

Ionic strength

Substrate Concentration

• for isosteric enzymes

• for single-substrate reactions

Saturation curve

Km

½ Vmax

• fixed amount of enzyme

MICHAELIS and MENTEN equation

v - reaction rate

vmax - maximal reaction rate

[S] - substrate concentration (mol/L)

Km - Michaelis constant (mol/L)

vmax [S]

v =

Km + [S]

The MICHAELIS´ CONSTANT (Km) – is the substrate

concentration at which the reaction rate is half of maximal,

and is an inverse measure of the substrate's affinity for the

enzyme

Maud Menten

Leonor Michaelis

Lineweaver – Bürk equation

(reciprocal transformation of M-M equation)

1 Km + [S] Km 1 [S] Km 1 1

v vmax [S] vmax [S] vmax [S] vmax [S] vmax

=== + +. .

Vmax [S]

v =

Km + [S]

• For single substrate reactions

-1/Km

1/vmax

1/v

1 Km 1 1

v vmax [S] vmax

= +.

Lineweaver – Burk plot

1/S

y a x b

y = ax + b

Multi-substrate reactions

1) Ternary-complex mechanism

(sequential)

2) Ping-pong mechanism• Formation of binary complexes – E - S1

- E – S2

ordered

random

• Substrates bind to the enzyme at the same time to produce a

ternary complex

Ternary

complex

1. Ternary complex mechanism

Ternary complex mechanism

Ternary

complex

+ + +

Intermediate

transaminase

Ping- pong mechanism




Temperature

pH

Ionic strength

E

No enzyme

enzyme

Concentration of Enzyme

• Substrate is present in a large excess

Co

nc.

of

pro

du

ct




Temperature

pH

Ionic strength

Optimal

temperature

TEMPERATURE

• Disruption of hydrogen bonds

• Disruption of the shape of the enzyme

Denaturation:

enzyme stability curve

kinetic energy curve

• Optimal t of most enzymes – similar or little higher than the t of

cells in which they occur

Shrimp

(cold water)

Bacteria

(hot springs)Human

Temperature




Temperature

pH

Ionic strength

pH

Alters the state of ionization of

charged amino acids in enzyme

Enz- + SH+ EnzSH

Effect of pH

Deviation from optimal pH - protein unwinding

- dissociation to subunits

- conversion to more compact form

LOSS of

activitySH+ + OH- S + H2O .......... high pH

Enz- + H+ EnzH ............... low pH




Temperature

pH

Ionic strength



molecules 1) Physico-chemical factors






molecules1) Induction and repression


ENZYME INHIBITION

Nonspecific

Denaturation

Acids and bases

Temperature

Alcohol

Heavy metals

Reducing agents

Specific

Competitive

Noncompetitive

Uncompetitive

ReversibleIrreversible

Specific

DIPFP, IAA

Irreversible inhibitors

bind at the active site, or at a different site

cannot be removed by dialysis

often contain reactive functional groups forming covalent

adducts with AA side chains

inhibition cannot be reversed

Examples of irreversible inhibition

DIPFP (Diisopropyl fluorophosphate)- inhibits enzymes with

serine (acetyl cholinesterase) in the active site

IODOACETAMIDE- inhibits enzymes with cysteine in the

active site

ASPIRIN - suppresses the production of prostaglandins and

thromboxanes due to its irreversible inactivation of the

cyclooxygenase

Irreversible inhibition - DIPFF

Diisopropyl fluorophosphate

Diisopropylfluorophosphate

• neurotoxin

• inhibitor of acetylcholinesterase (prolonged muscle

contraction - death)

Acetylcholine esterase

NH2

NH2

Iodoacetamide

Irreversible inhibitions

Iodoacetamide

• proteins cannot form disulfide bonds

• toxic, carcinogen, reproductive damage

I

ARACHIDONIC ACID

Cyclooxygenase

ASPIRIN (Acetylsalicylic acid)

Inflammation,

Temperature

Irreversible inhibition - ASPIRIN

PROSTAGLANDINS

Active cyclooxygenase

Salicylic acid

Inactive cyclooxygenase

(Aspirin)

OH O- CO – CH3

ENZYME INHIBITION

Nonspecific

Denaturation

Acids and bases

Temperature

Alcohol

Heavy metals

Reducing agents

Specific

Competitive

Noncompetitive

Uncompetitive

ReversibleIrreversible

Specific

DIPFP, IAA

Reversible

1) COMPETITIVE INHIBITION

• Inhibitor structurally similar to the substrate

• The inhibitor competes with the substrate for the enzyme

active site

• Increasing concentration of substrate will outcompete the

inhibitor for binding to the enzyme active site

• Reversible inhibition

COMPETITIVE INHIBITION

Active center

ENZYME

Inhibitor

Complex Enzyme-Inhibitor

ENZYME

SubstrateS

S

I I

S

S

ENZYME ENZYMEI

IS

S

S S

S

S

S

S

KmI

1/2vmax

vmax = vImax Km < KI

m

Km

1/vmax

1/[S]

1/vI

-1

Km

-1

KmI

Lineweaver – Burk plot

Competitive inhibition

COO¯ COO¯

CH2 - 2H CH

+ FAD + FADH2

CH2 SDH CH

COO¯ COO¯

Succinate Fumarate

COO¯ COO¯

CH2 CO

COO¯ CH2

COO¯

Malonate Oxalacetate

COMPETITIVE INHIBITION

XANTHINE URIC ACID

Xanthine oxidase

ALLOPURINOLGOUT

CH3-CH2-OH CH3-C H CH3-C

ethanol acetaldehyde acetate

CH3-OH H-C H H-C

methanol formaldehyde formiate

CH2-OH CHO COOH

CH2-OH CH2-OH COOH

ethylene glycol glycol aldehyde oxalic acid

Ethanol – antidotum in methanol and

ethylene glycol poisoning

O O

O-

Alcohol dehydrogenase

OO

O-



Noncompetitive inhibition

Substrate

EnzymeInhibitor

site

Active

site

Enzyme binds substrate Enzyme releases products

Inhibitor

Inhibitor binds and

alters enzyme´s shape

Binding of substrate is

reduced

Inhibition:

Reaction:

• Inhibitor binds to the enzyme at a different place then

the substrate

• Inhibitor – structurally different from the substrate

(Hg2+, Zn2+, Cu2+,Pb2+, CN¯, CO)

No inhibitor

With inhibitor

Km = Kmv

max> v

max

II

1/v

1/[S]01/Km

1/V

I1

Noncompetitive inhibition

1/V

No inhibitor

• Noncompetitive inhibitors do not influence binding of S into the

active site of enzyme but they reduce the rate of its conversion to a

product. Therefore Km is unchanged and vmax is reduced.

• Because EIS decomposes more slowly than ES, the rate of

enzymatic reaction slows down

• Inhibitor binds only to the complex enzyme – substrate.

E + S [ES] [ES]I

I S

KmI < Km vI

max < vmax

Uncompetitive inhibition

Figure 4 – Illustrations

Uncompetitive inhibition

Uncompetitive inhibitors:

• Anticancer drugs

• Lithium

http://spotlite.nih.gov/assay/index.php/File:Manual_sect12_new_fig4.gif

http://spotlite.nih.gov/assay/index.php/File:Manual_sect12_new_fig4.gif

• vImax < v Km

I < Km

UNCOMPETITIVE INHIBITION

• multiple substrate mechanisms (ping-pong mechanism)

Normal

With inhibitor

With inhibitor Normal

Both the effective Vmax and effective Km are reduced with an inhibitor

V

1/V

Km Km -1/Km -1/Km

Enzyme Inhibitions (Mechanisms)

I

I

S

S

S I

IS

E

Different siteCompete

for an active site

Inhibitor

Substrate

E + S→ES→E + P

+I

↓

EI

←

↑

E + S→ES→E + P

+ +I I

↓ ↓

EI+S→EIS

←

↑ ↑

E + S→ES→E + P

+I

↓

EIS

←

↑

E

I

S

Juang RH (2004) BCbasics

I Competitive NoncompetitiveI I Uncompetitive

E



molecules

1) Physico-chemical factors






molecules

1) Induction and repression


Allosteric enzymes

Allosteric enzymes – change their conformational

ensemble upon binding of an effector

catalytic center – binds substrate

binds activator

allosteric center

binds inhibitor

subunits

Active siteallosteric

center

• Binding of the inhibitor to a site other than the active site changes

the shape of the active site – substrate cannot bind there

The allosteric inhibition

The allosteric activation

• Binding of the activator to a site other than the active site changes

the shape of the active site – substrate can bind there

Activator - stabilizes R (relaxed)

Inhibitor stabilizes T (tense)

Sigmoidal curve

Allosteric enzymes

Allosteric enzyme

Isosteric enzyme

do not obey Michaelis-Menten kinetics

Allosteric enzymes

display sigmoidal plots of the reaction velocity (v) versus

substrate concentration [S]

the binding of substrate to one active site can affect the properties

of other active sites in the same molecule

their activity may be altered by regulatory molecules that are

reversibly bound to specific sites other than the catalytic sites

Allosteric effectors of isocitrate

dehydrogenase

HH

Respiratory chain ATP

HO-C-COO-

CO2

Allosteric effectors of ICDH

ISOCITRATE

(+)NAD+ NADH + H+(-)

(+)ADP ATP(-)

(+)CITRATE

KREBS CYCLE

α-KETOGLUTARATE

ALLOSTERIC INHIBITION

A B C D E P

E1 E2 E3 E4 E5

Feed-back regulationFeed-back regulation

Feed-forward activation

• Metabolite B produced at the beginning of the metabolic pathway

can activate a downstream enzyme e.g.E4

Activation of allosteric enzymes

Cooperative model

(Concerted model)

Sequential model

• Both models postulate that enzyme subunits exist in one of

two conformations, tensed (T) or relaxed (R)

• Relaxed subunits bind substrate more readily than those in

the tense state.

S1 S2,S3

S4

Cooperative model

(MONOD 1965)

T (Tensed) R (Relaxed)

S1 S2,S3

S4

Nonactive form Active form

after binding a substrate a conformational change in one subunit is

necessarily conferred to all other subunits.

all subunits must exist in the same conformation

Sequential model

SEQUENTIAL MODEL

substrate-binding at one subunit only slightly alters the structure of

other subunits so that their binding sites are more receptive to

substrate

subunits need not exist in the same conformation

conformational changes are not propagated to all subunits

Ligand binding may result in an increased or a reduced affinity for the

ligand at the next binding site



molecules







molecules



4) Regulation by modification of enzyme

molecule

a) Limited proteolysis

b) Covalent modifications


Inactive form of enzyme PROENZYME (ZYMOGEN) is

cleaved by proteases to the active enzyme

PROENZYME ACTIVE ENZYMEtrypsinogen trypsin (- pentapeptide)

pepsinogen pepsin (-1/5 molecule)

Enzymes produced by cells in the active form could damage own

protein structures (digestive enzymes)

Nonactive Active

substratesubstrate

Hydrolytic enzymes

PEPSIN

Pepsinogen Pepsin (peptide)

H+ (44 Aminoacids)

ENTEROPEPTIDASE

Trypsinogen Trypsin (6 AA)

TRYPSIN

Chymotrypsinogen Chymotrypsin + dipeptide

Similar mechanisms:

Proinsulin insulin pro-thrombin thrombin

Fibrinogen fibrin

4) Regulation by modification of enzyme

molecule


b) Covalent modifications

b) Covalent modification of enzyme molecule

• Covalent attachment of a modifying group to a specific functional

group on the enzyme

A/ PHOSPHORYLATION, DEPHOSPHORYLATION

reversible modification, binding of a phosphate group to a

molecule by a specific kinase (in mammals)

B/ ADENYLATION – reversible binding of a nucleotide (e.g. AMP)

(in bacteria)

C/ ADP-RIBOZYLATION - reversible binding of ADP-ribosyl.

Donor of the ADP-ribosyl group is the coenzyme NAD+;

Phosphorylation,

Dephosphorylation

• Kinases - phosphorylate proteins

• Phosphatases - dephosphorylate

Phosphorylation

• on serine, threonine, tyrosine,

• conformational change of the structure

• on nonpolar part of proteins – increase

of polarity – change of conformation

Advantages of

phosphorylation/dephosphorylation:

It is rapid (takes a few seconds)

It does not require new proteins to be made or

degraded

It is easily reversible



molecules







molecules



Regulation of enzyme activity by changing

the number of enzyme molecules

1) Induction of enzyme synthesis

Constitutive enzymes – present at constant

concentrations (Krebs cycle)

Inducible enzymes – de novo synthesis of the enzyme

according to the need of a cell

2) Repression of enzyme synthesis – inhibition of

gene expression (actinomycins –inhibit transcription

streptomycin – inhibit translation)

- lactose

lactase

lactase

lactose



molecules







molecules



Degradation of proteins in

eukaryotic cells

a) lysosomes - degradation of intracellular proteins

with a long half-life, extracellular proteins

associated with cell membrane

b) proteasomes – degradation of intracellular

proteins with a short half-life

Lysosomes

//upload.wikimedia.org/wikipedia/commons/1/1a/Biological_cell.svg

//upload.wikimedia.org/wikipedia/commons/1/1a/Biological_cell.svg

PROTEASOME

• Protein complex with proteolytic activity

• Located in the nucleus and the cytoplasm

• Proteins degraded in proteasome: transcription

factors, cyclins, proteins encoded by viruses...

Function:

Degradation of unneeded or damaged proteins by

proteolysis

19S regulatory subunit

19S regulatory subunit

20S catalytic subunit

http://upload.wikimedia.org/wikipedia/commons/5/54/Proteaosome_1fnt_side.png

Regulation of enzyme activity by degradation

Regulated by proteases – hydrolysis of peptide bonds

Proteins Peptides shorter peptides, aminoacids

proteases peptidases

endopeptidases – cleave intramolecular peptide bonds

Peptidases (trypsin, pepsin)

exopeptidases – cleave off a terminal amino acid

(carboxypeptidase A)

SPECIFICITY OF PROTEASES

• Ability to cleave peptide bonds next to a specific amino acid

Chymotrypsin – active site – hydrophobic

- preferentially cleaves peptide bonds next to aromatic

amino acids

Trypsin –in active center – negative charge

- cleaves peptide bonds from amino acids with positively

charged side chain

Chymotrypsin

Trypsin

1) INTRACELLULAR ENZYMES

• Stay in a cell in which they were synthesized

• Many occur only in some organs or cell organels

• In healthy organism – minimal concentrations in blood

ENZYMES

2) EXTRACELLULAR ENZYMES

• Secreted from cells of their origin

(e.g. in animals into digestive juice, blood...)

biocatalysts · 1897 –eduard buchner –ability of yeast extracts that lacked any living yeast...

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