Enzymes BIOCATALYSTS VS

ENZYMESENZYMOLOGY BIOCATALYSTS

2009/2010

INGRID ŽITŇANOVÁ HISTORY

17th -18th century – digestion of meat caused by stomach secretions - conversion of starch to glucose by saliva Louis Pasteur 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 Eduard Buchner en zyme – in yeasts • 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 (biocatalysts):

1) More efficient - higher reaction rate

2) Milder reaction conditions (20 - 40°C, 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

How much is 1020 fold?

Catalyst time for reaction

With enzyme 1 second Without enzyme 3 x 1012 years 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 ➢ Enzymes DO NOT change the equilibrium constant of a reaction (accelerate the rate of the forward and reverse reactions equally)

Carbonic anhydrase CO2 + H2O H2CO3

Carbonic anhydrase in tissues: CO + H O 2 2 H2CO3 Carbonic anhydrase in lungs: H2CO3 CO2 + H2O ENZYMES

SIMPLE COMPLEX (HOLOENZYME)

APOENZYMES COFACTOR (Nonprotein) (Protein)

ORGANIC INORGANIC

COENZYME PROSTHETIC GROUP (loosely bound) (tightly bound) 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

Role of organic cofactors: transport of chem. groups from 1 reactant to another

◼ cofactors can serve several apoenzymes:

NAD+ (nicotinamide adenine dinucleotide) - a cofactor for a great number of dehydrogenases: alcohol dehydrogenase,

malate dehydrogenase

lactate dehydrogenase reactions 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 2) Transfer of electrons coenzyme Q, porfyrin derivatives

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 Vitamins are often converted to coenzymes

Vitamin Coenzyme Function

Thiamin diphosphate decarboxylation B1 Flavin mononucleotide (FMN) carries hydrogen B2 Nicotinamide adenine dinucleotide carries hydrogen H- B3 (NAD+), (NADH) B 5 acyl group carrier pantothenic acid Coenzyme A

H (B7) Biocytin CO2 fixation

B9-folic acid Tetrahydrofolate carries one carbon units

B12-cobalamine Methylcobalamine, adenosylcobalamine ACTIVE SITE

Active site Substrate ACTIVE SITE

BINDING SITE CATALYTIC SITE (where a substrate binds) (where the reaction proceeds)

▪ ACTIVE SITE = pocket in the enzyme where substrates bind and catalytic reaction occur ▪ 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 Aminoacids of the active site can be located at different regions of a polypeptide chain ➢ 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“ binding pocket hydrophobic ionic interaction hydrogen interaction bonding ionic interaction

2. non-covalent interactions between substrate and Interactionsthe active site:between enzyme and substrate - hydrogen bonding - ionic interactions - hydrophobic interactions Stages of enzyme reaction

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 First step of enzyme catalysis FORMATION OF THE ENZYME-SUBSTRATE COMPLEX

E + S ES Second step FORMATION OF THE TRANSITION STATE COMPLEX Note change

ES transition state complex 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 • Charges in the • Deformation of S 2 substrates, active site facilitates its that they are in induce changes conversion to a close vicinity in the charges in product S molecule MECHANISM OF ENZYME ACTION

• Enzymes decrease the activation energy of a reaction by formation of the active enzyme - substrate complex

• Activation energy is the energy required to start a reaction.

Transition state

Uncatalyzed reaction

Catalyzed reaction

Substrate Energy Product

• 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

Enzyme Substrate Product

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

One katal is the catalytic activity that changes one mole of substrate per second at optimal pH.

1 kat = mol . s-1 ◼ 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 SPECIFICITY

Enzyme has SPECIFICITY – it can discriminate among possible substrate molecules:

S S S Substrate

Enzyme Enzyme 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 - converts methanol, ethanol, ethylene glycol)

b. Linkage specific – particular type of chemical bond regardless

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

Substrate

Active site ES complex

Enzyme

➢ This model assumed that only a substrate of a proper shape could fit with the enzyme 1) Lock and Key Model (Emil Fischer 1894)

Substrate

Enzyme

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)

Substrate

Active site

Enzyme ES complex

➢ This model assumes continuous changes in the active site structure as a substrate binds Induced fit model

◼ 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 Principles of Catalysis

• Uncatalyzed reactions often are extremely slow.

• They are slow because of the heigh activation energy

• Enzymes lower the activation energy by creating an ES (enzyme-substrate) complex which reduces bond strength in the substrate and makes the substrate easier to convert to the product. 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 2. Systematic names

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

name of substrates + name of the reaction catalyzed + suffix –ase (separated by the colon) a) Characterizing the reaction: L-lactate : NAD+ - oxidoreductase b) Recommended name: Lactate dehydrogenase c) Code number : EC 1.1.1.1 CODE NUMBERS OF ENZYMES

◼ 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) L-lactate + NAD+ pyruvate + NADH + H+ b) Recommended name: Lactate dehydrogenase c) Code number : EC 1. 1. 1. 1

oxidoreductase NAD+ as acceptor

acting on the CH-OH group

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 Lactate dehydrogenase

pyruvate lactate

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 dehydrogenase – composed of M a H subunits

5 isomers of lactate dehydrogenase M4 M3H M2H2 MH3 H4

LDH-5 Liver

Muscle M4 LDH-4 White cells

M3H Brain LDH-3

Red cells M2H2 LDH-2 Kidney

MH3 Heart LDH-1 Separation by electrophoresis H4 Control serum

LDH1 LDH5

LDH2 LDH3 LDH2 LDH5 LDH3 LDH1 Regulation of enzyme activity

B) With the change of the number of enzyme molecules 1) Induction and repression 2) Regulated degradation of proteins 1. Physico-chemical factors

➢ Substrate concentration ➢ Temperature ➢ pH ➢ Ionic strength ➢ Redox potential Substrate Concentration

½ Vmax

Saturation curve

Km • for isosteric enzymes • fixed amount of enzyme • for single-substrate reactions MICHAELIS and MENTEN equation

vmax [S] v =

Km + [S]

Leonor Michaelis v - reaction rate

vmax - maximal reaction rate [S] - substrate concentration (mol/L)

Km - Michaelis constant (mol/L)

Maud Menten 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

Vmax [S] v =

Km + [S]

Lineweaver – Bürk equation (reciprocal transformation of Michaelis -Menten equation)

1 Km + [S] Km 1 [S] Km 1 1 = = . + = . +

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

• It is valid for single substrate reactions Lineweaver – Burk plot

1/v y = ax + b y a x b 1 K 1 1 = m . + v vmax [S] vmax 

1/vmax

-1/Km 1/S Multi-substrate reactions

ordered 1) Ternary-complex mechanism (sequential) random • Substrates bind to the enzyme at the same time to produce a ternary complex

2) Ping-pong mechanism • Formation of binary complexes – E - S1 - E – S2 1. Ternary complex mechanism

Ternary complex Ternary complex mechanism

Ternary complex Ping- pong mechanism

transaminase

Intermediate

+ + + 1. Physico-chemical factors

➢ Substrate concentration ➢ Temperature ➢ pH ➢ Ionic strength ➢ Redox potential TEMPERATURE

enzyme stability curve

Denaturation: • Disruption of hydrogen bonds • Disruption of the shape of the enzyme • Optimal temperature t of most enzymes – similar or little higher than the t of cells in which they occur

Bacteria Shrimp Human (hot springs) (cold water)

Temperature 1. Physico-chemical factors

➢ Substrate concentration ➢ Temperature ➢ pH ➢ Ionic strength ➢ Redox potential pH alters the state of ionization of charged amino acids in enzyme Effect of pH

Enz- + SH+ EnzSH

- + Enz + H EnzH ...... low pH LOSS of activity + - SH + OH S + H2O ...... high pH

Deviation from optimal pH - protein unwinding - dissociation to subunits - conversion to more compact form 1. Physico-chemical factors

➢ Substrate concentration ➢ Temperature ➢ pH ➢ Ionic strength ➢ Redox potential Ionic strength

• Concentration of salts influences enzyme activity because the salts affect the hydration of proteins and consequently their solubility and shape of molecules. • Solubility of proteins at low ionic strengths increases with the concentration of salt (so-called salting in). Increasing salt concentration increases the solubility. • At very high ionic strengths charges of protein molecules are shaded, leading to the existence of very weak electrostatic interactions between protein molecules, and thus solubility is reduced (salting out) . 1. Physico-chemical factors

➢ Substrate concentration ➢ Temperature ➢ pH ➢ Ionic strength ➢ Redox potential REDOX POTENTIAL

Redox potential (RP) – a measure of the tendency of a chemical to acquire electrons and thereby be reduced

The more positive the potential, the greater the species' affinity for electrons and tendency to be reduced

Redox potential affects: • some oxidizable groups especially –SH

• spacial arrangement of the whole enzyme molecule

• substrate binding (formation of –S-S- bonds) Regulation of enzyme activity

B) With the change of the number of enzyme molecules 1) Induction and repression 2) Regulated degradation of proteins ENZYME INHIBITION

Irreversible Reversible

Nonspecific Specific Specific

Denaturation DPFP, IAA Competitive Acids and bases Noncompetitive Temperature Uncompetitive Alcohol Heavy metals Reducing agents 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

❑ IAA (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 – binds to –OH group of serine in the active site of enzyme

Diisopropyl fluorophosphate • neurotoxin • inhibitor of acetylcholinesterase (prolonged muscle contraction - death) Acetylcholine esterase

If the enzyme is inhibited, acetylcholine accumulates and nerve impulses cannot be stopped, causing prolonged muscle contraction - paralysis occurs and death may result since the respiratory muscles are affected. NH2

NH2

Iodoacetamide

Irreversible inhibitions Iodoacetamide – reacts with –SH groups in the active site • proteins cannot form disulfide bonds

• toxic, carcinogen, reproductive damage Irreversible inhibition - ASPIRIN

Inflammation, Temperature

ARACHIDONIC ACID PROSTAGLANDINS

ASPIRIN (Acetylsalicylic acid)

Cyclooxygenase (Aspirin)

OH O- CO – CH3

Active cyclooxygenase Inactive cyclooxygenase Salicylic acid

Acetylation of the enzyme results in a steric block, preventing arachidonic acid from binding ENZYME INHIBITION

Irreversible Reversible

Nonspecific Specific Specific

Denaturation DIPFP, IAA Competitive Acids and bases Noncompetitive Temperature Uncompetitive Alcohol Heavy metals Reducing agents 1) COMPETITIVE INHIBITION

• Inhibitor is 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 Inhibitors work by preventing the formation of Enzyme-Substrate Complexes because they have a similar shape to the substrate molecule. I I vmax = v max Km < K m

1/2vmax

I Km Km Competitive inhibition

1/v I

1/vmax

-1 -1 1/[S] I Km Km Lineweaver – Burk plot 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 Competitive Inhibitors as Medicines XANTHINE URIC ACID

ALLOPURINOL GOUT Xanthine oxidase

Ethanol – antidotum in methanol and ethylene glycol poisoning

Alcohol dehydrogenase O O CH3-CH2-OH CH3-C H CH3-C ethanol acetaldehyde acetate O-

Alcohol dehydrogenase O O CH3-OH H-C H H-C - methanol formaldehyde formiate O

Alcohol dehydrogenase

CH2-OH CHO COOH

CH2-OH CH2-OH COOH ethylene glycol glycol aldehyde oxalic acid Noncompetitive inhibition

• Inhibitor binds to the enzyme at a different place then the substrate Substrate Reaction: Active site Inhibitor Enzyme • Inhibitor – structurallysite different from the substrate Enzyme binds substrate Enzyme releases products Inhibition:

Inhibitor

Inhibitor binds and Binding of substrate is alters enzyme´s shape reduced No inhibitor

With inhibitor

I I v > v Km = Km max max 1/v I1

No inhibitor

1/V 1/V

0 1/Km 1/[S]

Noncompetitive inhibition • 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 Uncompetitive inhibition

• Inhibitor binds only to the complex enzyme – substrate.

E + S [ES] [ES]I

I S I I Km < Km v max < vmax Uncompetitive inhibition

Figure 4 – Illustrations

Uncompetitive inhibitors: • Anticancer drugs • Lithium UNCOMPETITIVE INHIBITION

• multiple substrate mechanisms (ping-pong mechanism) I I • v max < v Km < Km

1/V Normal With inhibitor Normal

V With inhibitor

Km Km -1/Km -1/Km

Both the effective Vmax and effective Km are reduced with an inhibitor 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 molecules 1) Induction and repression 2) Regulated degradation of proteins Allosteric enzymes

• Allosteric enzymes – change their conformation upon binding of an effector (activator, inhibitor) The allosteric inhibition

• 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 activation

• Binding of the activator to a site other than the active site changes the shape of the active site – substrate can bind there Allosteric enzymes

Single subunit enzymes

Allosteric enzyme

Sigmoidal curve Allosteric enzymes

◼ do not obey Michaelis-Menten kinetics

◼ 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

Respiratory chain ATP

CO2

HO-C-COO-

HH Allosteric effectors of ICDH

ISOCITRATE

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

(+)ADP ATP(-)

(+)CITRATE

KREBS CYCLE

α-KETOGLUTARATE ALLOSTERIC REGULATION

E1 E2 E3 E4 E5 A B C D E P

Feed-backFeed regulation-back inhibition Feed-forward activation

• Metabolite B produced at the beginning of the metabolic pathway

can activate a downstream enzyme e.g.E4 Mechanism of activation of allosteric enzymes

Cooperative model Sequential model (Concerted 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. Cooperative (concerted)model (MONOD 1965)

S1 S ,S S1 S2,S32 3

S4 S4

T (Tensed) R (Relaxed) Nonactive form Active form

❖ all subunits must exist in the same conformation

❖ after binding a substrate a conformational change in one subunit is necessarily conferred to all other subunits. SEQUENTIAL MODEL (KOSHLAND 1966) T-conformation nonactive

k 1 k3 S S1 + + S + S k 2 3 2 k S 4 S

R –conformation - active

S S S S k7 S4+ S S k8 S SEQUENTIAL MODEL

❖ subunits need not exist in the same conformation

❖ conformational changes are not propagated to all subunits

❖ substrate-binding at one subunit only slightly alters the structure of other subunits so that their binding sites are more receptive to substrate

❖ Substrate binding may result in an increased or a reduced affinity for the ligand at the next binding site Regulation of enzyme activity

a) Limited proteolysis b) Covalent modifications a) Limited proteolysis Inactive form of enzyme PROENZYME (ZYMOGEN) is cleaved by proteases to the active enzyme

substrate substrate

Nonactive Active PROENZYME ACTIVE ENZYME trypsinogen trypsin (- pentapeptide) pepsinogen pepsin (-1/5 molecule)

Enzymes produced by cells in the active form could damage own protein structures (digestive enzymes)

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

a) Limited proteolysis 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 Regulation of enzyme activity

◼ Enzymes are often compartmentalized - stored in a particular organelle - they can find their substrates readily, don't damage the cell, and have the right microenvironment to work well

◼ digestive enzymes of the lysosome work best at a pH around 5 which is found in the acidic interior of the lysosome (but not in the cytosol, which has a pH of about 7.27).

Regulation of enzyme activity

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

- lactose

lactase lactose

lactase

- lactose

lactase Regulation of enzyme activity

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 PROTEASOME

• Protein complex with proteolytic activity 19S regulatory subunit • Located in the nucleus and the cytoplasm • Proteins degraded in proteasome: transcription factors, cyclins, proteins encoded by viruses...

20S catalytic subunit Function: Degradation of unneeded or damaged proteins by proteolysis 19S regulatory subunit Ubiquitin detachment and protein unfolding 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 Chymotrypsin

Trypsin ENZYMES

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

2) EXTRACELLULAR ENZYMES

• Secreted from cells of their origin (e.g. in animals into digestive juice, blood...) Enzyme Name Increased levels in disease

Alanine ALT aminotransferase Hepatopathy Aspartate AST aminotransferase Myocardial infarction Lactate LD dehydrogenase Myocardial infarction - LD1,2, hepatopathy - LD4,5 Myocardial infarction - CK-MB, skeletal muscle diseases - CK Creatine kinase CK- MM Alkaline ALP phosphatase Diseases of the bile duct and liver, bone diseases

ACP Acid phosphatase Prostate tumors

AMS Amyláza Tissue specificAkútna enzymes pankreatitída SOME ENZYME DEFECT DISORDERS

◼ Lactose intolerance – insufficient levels of lactase enzyme, which breaks down the milk sugar - lactose

Symptoms of lactose intolerance

◼ stomach cramps,

◼ bloating,

◼ nausea,

◼ diarrhoea after consumption of milk products

Treatment

• lactose-free diet, • pills with lactase enzyme Sucrose (saccharose) intolerance

Sucrose intolerance – sucrase enzyme needed for proper metabolism of saccharose (sucrose) and is not produced or the enzyme produced is either partially functional or non-functional in the small intestine.

Symptoms: • chronic, watery, acidic diarrhea; • gas; • bloating • abdominal pain. Sucrose digestion

Small intestine

Large intestine THERAPEUTIC ENZYMES

Many enzymes are produced on a large scale in microorganisms like E. coli, or purified from other sources to treat diseases and enzyme deficiencies.

Streptokinase and urokinase - dissolve dangerous blood clots in people suffering from strokes and heart attacks. Lysozyme - is used as an antibacterial agent as it specifically dissolves bacterial cell walls

Chitinase is an antifungal which dissolves chitin in the cell walls of fungi.