History of enzymes

1835 Jacob Berzelius – catalytic function of diastase

pol. 18.stol. Luis Pasteur – vitalistic theory

1878 Frederic W. Kühn – „enzyme“

1894 Emil Fischer –„lock and key theory“

1897 Büchner’s experiment

1926 James Sumner – crystalization of urease

30. léta Northrop and Kunitz – enzyme activity is

proportional to protein concentration

1963 primary structure of ribonuclease A

1965 X ray analysis of lysozyme structure

Enzymes = biocatalysts Nearly each (metabolic) reaction has its own enzyme

Enzymes – biological catalysts

Catalyst is not either reactant or product of the reaction, does not undergo permanent structural change

Increases reaction rate in both directions, decreases activation energy of both reactions

It implies, that catalyst shorten the time required for reaching the equilibrium, does not influence the equilibrium itself!

Catalyst can accelerate the only reaction which would proceed in its absence.

Enzym = protein or apoenzyme (protein ) + cofactor = holoenzyme

Kofactor: non proteinaceous part of the enzyme, directly involved in catalysed reaction (often vitamines)

Prosthetic group – covalently bound to the peptide chain

Coenzym - loosely-bound

prosthetic group (ex. FAD, PLP, heme)

E-Pr + S1 E-Pr* + P1

E-Pr* + S2 E-Pr + P2

_____________________

E-Pr

S1 + S2 P1 + P2

β-D-glucose + O2 → δ-D-gluconolactone + H2O2

coenzym (second substrate of the reaction) (ex. NAD(P),CoA, ATP)

E1

S1 + K P1 + C*

E2

C* + S2 K + P2

________________

CH3-CH2OH + NAD+ → CH3-CHO + NADH + H+

NADH + H+ + Q → NAD+ + QH2

Enzymes – biological catalysts …….have additional features: effective decrease of the activation energy specifita (účinku, substrátová) regulovatelnost účinnosti (aktivity)

Catalyst Reaction rate(mol.l-1.s-1) Ea (kJ.mol-1)

None 10-8 71,1

HBr 10-4 50,2

Fe(OH)2-triethylen

tetraamin

103 29,3

Catalase 107 8,4

Activation energy of hydrogen peroxide

decomposition

H2O2 → 2H2O + O2

Enzymes – biological catalysts …….have additional features: effective decrease of the activation energy specificity can be regulated (next lecture)

Active Site of an Enzyme

• The active site is a region

within an enzyme that fits the

shape of substrate molecules

• Amino acid side-chains align

to bind the substrate through

H-bonding, salt-bridges,

hydrophobic interactions, etc.

• Products are released when

the reaction is complete (they

no longer fit well in the active

site)

Enzymes

active site – binding groups

- catalytic groups

stereospecificity

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

Lock-and-Key Model

• In the lock-and-key model of enzyme action:

- the active site has a rigid shape

- only substrates with the matching shape can fit

- the substrate is a key that fits the lock of the active site

• This is an older model, however, and does not work for all enzymes

Induced Fit Model

• In the induced-fit model of enzyme action:

- the active site is flexible, not rigid

- the shapes of the enzyme, active site, and substrate adjust to maximize the fit, which improves catalysis

- there is a greater range of substrate specificity

• This model is more consistent with a wider range of enzymes

Conformational change of hexosekinase caused by thr

presence of substrate in the active site – induced fit

D-glc + ATP → D-glc-6-fosfát + ADP

Enzyme Effectivity

Proximity and orientation of substrate(s) in active site

lower water concentration / lower polarity of the

environment

conformational changes

electrostatic effects

concentration effect

Molecular mechanism of

chymotrypsine cleavege of

peptide bond:

1. Nucleophilic attack of Ser oxygen

2. Formation and stabilisation of the

first reaction intermediate

3. Formation of the first reaction

product (peptide)

4. Nucleophilic attack of water

oxygen

5. Formation of the second

intermediate

6. Release of the second product

(peptide)

Enzyme classification

http//www.rrz.uni-hamburg.de/biologie/b_online/e18_1/ec.htm

http://www.expasy.org/enzyme/

ExPASy Proteomics Server

(Expert Protein Analysis System) proteomics server of the Swiss Institute of

Bioinformatics

Enzyme Commission (EC) IUBMB EC 1.1.1. 1 alkoholdehydrogenase

INTERNATIONAL UNION OF BIOCHEMISTRY AND MOLECULAR BIOLOGY

http://www.chem.qmul.ac.uk/iubmb/enzyme/

EC X.Y.Z.W

Class 1 - 6

Type of reaction

Subclass

substrate

subsubclass

Special characteristic (Acceptor etc)

Ordinal number

Common names, “half-systemic", systemic, -ase

Systemic: substrate 1: (substrate 2) class (ase)

examples:

H2O2 + H2O2 = O2 + 2 H2O

hydrogen peroxide:hydrogen peroxide oxidoreductase (catalase)

β-D-glucose + O2 → δ-D-gluconolactone + H2O2

β-D-glucose:oxygen 1-oxido-reductase (glucose oxidase)

Enzyme nomenclature

Enzyme classes

1. Oxidoreduktasy

2. Transferasy

3. Hydrolasy

4. Lyasy

5. Isomerasy

6. Ligasy

Enzyme classification

Class Reaction catalyzed Typical reaction Enzyme example(s) with

trivial name

EC 1

Oxidoreductases

Catalyze oxidation/reduction

reactions; transfer of H and O

atoms or electrons from one

substance to another

AH + B → A + BH

A- + B → A + B-

Dehydrogenase, oxidase,

oxygenase

EC 2

Transferases

Transfer of a ffunctional

groupsfrom one substance to

another. The group may be

methyl-, acyl-, amino- or

phosphate group

AB + C → A + BC Transaminase, kinase

EC 3

Hydrolases

Hydrolysis of substrate AB + H2O → AOH + BH Lipase, amylase, protease,

peptidase

EC 4

Lyases

Non-hydrolytic addition or

removal of groups from

substrates. C-C, C-N, C-O or C-

S bonds may be cleaved

RCOCOOH → RCOH + CO2

or [x-A-B-Y] → [A=B + X-Y]

decarboxylase

EC 5

Isomerases

Intramolecule rearrangement,

i.e. isomerisation changes

within a single molecule

AB → BA Isomerase, mutase,

racemase

EC 6

Ligases

Join together two molecules by

synthesis of new C-O, C-S, C-N

or C-C bonds with

simultaneous breakdown of

ATP

X + Y+ ATP → XY + ADP +

Pi

Synthetase

1. Oxidoreduktases

CH3-CH2-OH

NAD+ NADH + H+

CH3-CHO

acceptor

donor

EC 1.1.3.4 -D-Glukosa:O2-1-oxidoreduktasa, glukosaoxidasa

-D-glucose + FAD -D-glucono-1,5-lactone + FADH2

FADH2 + O2 FAD + H2O2

-D-glucose + O2 -D-glukono-1,5-lakton + H2O2

Cofactors of oxidoreduktases

Nikotinamidadenindinukleotid

- PO3

2- = NADP

+

Flavine cofaktors

Flavine adenine dinucleotide - FAD

Flavine mononucleotide - FMN

Riboflavine, vitamin B2

Flavine adedine dinucleotide (FAD) oxidized form

Flavine adedine dinucleotide - reduced form (FADH2)

N

N

N

N

O O O O

Fe

Heme – prosthetic group

2. Transferases

glycerol-3 phosphate acyltransferase

+ acyl-SCoA + HSCoA

hexokinase

ATP + D-hexose → ADP + D-hexose-6-phosphate

CH2

CH

CH2

OH

OH

O P

O

O

O

CH2

CH

CH2

O

OH

O P

O

O

O

C

O

R1

Cofactors of transferases

Adenosine triphosphate (ATP)

Cofactors of transferases

Coenzyme A (CoA, CoASH)

β-mercaptoethylamine

Panthotenic

acid

Pyridoxalphosphate (B6 – pyridoxol, pyridoxamine)

Ex. transfer of amino groups

Lipoamide (transferases, oxidoreductases)

Biotin, tranfers carboxyl group, prosthetic group (transferases, ligases)

Growth factor of yeasts, avidin-biotin komplex

Thiaminediphosphate (vitamine B1), transfer of 2C residues, aldehydes

Cereal grains, beri-beri

Good news – 3. hydrolases does not need cofactors for their action

Lyases :

pyruvate decarboxylase

CH3-CO-COOH CH3-CHO + CO2

carbonate anhydrase

H2CO3 CO2 + H2O

Adenylate cyclase

cAMP

H

P

O

OH

O

O

H

O

H

H

CH2

H

NN

NN

NH2

OP

O

O

OP

O

O

OO

HP O

O

O

H

O

H

H

CH2

H

NN

NN

NH2

O

O

P

O

O

OP

O

O

OO

ATP cAMP + PPi

+

Isomerases

• Triosaphosphate isomerase

• D-glyceraldehyde-3-fphosphate dihydroxyacetonphosphate

• EC 5.3.1.5 glukosaisomerasa

Glucose fructose

CH

CH

CH2

O

OH

O P

O

O

O

CH2

C

CH2

OH

O

O P

O

O

O

6. Ligases

tyrosine-tRNA-ligase

L-Tyr + tRNATyr + ATP L-Tyr-tRNATyr + AMP + PPi

Pyruvate carboxylase

CH3-CO-COO- + HCO3- +ATP -OOC-CH2-CO-COO- + ADP + Pi

DNA-ligase

ATP + (deoxyribonucleotide)n + (deoxyribonucleotide)m

(deoxyribonucleotide)n+m + AMP + PPi

Tetrahydrolistová kyselina, přenáší 1C zbytky vázané na N5

Thiamindifosfate, transfer of 2C residues, aldehydes

Obilné slupky, beri-beri