Enzymology. How enzymes work mechanisms.
Bruno Sopko
A thermodynamic model of catalysis
A thermodynamic model of catalysis
• The rate of a chemical reaction is related to the activation
energy of the reaction by the following equation:

Therefore, the rate acceleration provided by the catalysis can
simply be calculated::
If, for example, a catalyst can provide 10 kJ/mol1 of transition
stabilisation energy for a reaction at 25º C a 55-fold rate acceleration
will result, whereas a
20 kJ/mol stabilisation will give a 3000-fold acceleration and a 40
kJ/mol stabilisation a 107-fold acceleration!
A thermodynamic model of catalysis
The steps in enzyme-catalyzed reaction
The important effects of enzyme catalysis
•
•
•
•
•
proximity effect
transition state stabilisation
acid/base catalysis
electrostatic effects
nucleophilic or electrophilic catalysis by
functional groups of enzyme
• structural flexibility
Proximity effect
Transition state stabilisation
Acid/base catalysis
• this catalysis avoids the need of extremely low
or high pH
• principle is to make a potentially reactive
group more reactive by increasing their
nucleophilic or electrophilic character by
adding or removing a proton
Acid/base catalysis
Mechanism for ketosteroid isomerase. Example of acid/base
catalysis
Acid/base catalysis
Metal Ion catalysis
1. Metalloenzymes
Tighly bound metal ions
(Fe2+, Fe3+, Cu2+, Zn2+,Mn2+, Co3+)
Metal-activated enzymes
(Na+, K+, Mg+, Ca2+)
Role of metal ions in catalysis
1) Binding of substrate and their properly
orientation to reaction
2) Mediating of oxidation-reduction
reactions through reversible changes in
the metal ion oxidation state
3) Charge stabilization, shielding negative
charges
Metal Ion catalysis
Mediating of oxidation-reduction reactions
Lactatedehydrogenase
1) Fast bond of NAD+ in enyzmatic domain, isomerization and deprotonisation (H
bonds Ser 48 and His 51)
2) Alcoholic substrate changes OH- coupled with Zn2+ reorganisation of
bonds and E-NADH- aldehyd complex formations
Metal Ion catalysis
Mediating of oxidation-reduction reactions
3. Release of product and reduced NADH coenzyme, rearrangement
Metal Ion catalysis
Metal Ion
Enzyme
Iron
Cytochromes
Aconitase
Catalase
Zinc
Carbonate dehydrogenase
Superoxide dismutase
Manganese
Pyruvate Carboxylase
Superoxide dismutase
Molybdenum
Xanthine oxidase
Copper
Superoxide dismutase
Cytochrome oxidase
Electrostatic effects
• stabilization of electric charge distribution in
transition states during enzymatic reactions
• the changing atom charges of substrate in a
transition state interacts with atom charges of
the surrounding enzyme and also neighbour
water molecules
Nucleophilic or electrophilic catalysis
• enzymatic functional groups provide nucleophilic and
electrophilic catalysts
• typical nucleophilic groups are amino, hydroxyl and
thiol groups of AA residues but imidazol group of His or
carboxyl group of Asp, Glu can serve as well
• electrophilic group of enzymes is usually complex of
metal cofactor with substrate
• nucleophilic catalysis involves the formation of an
intermediate state in which substrate is covalently
bound to a nucleophilic group
Nucleophilic catalysis
Electrophilic catalysis
Nucleophilic catalysis - acetoacetic decarboxylase
Serine proteases - examples of nucleophilic catalysis
• serine proteases belong to large family of proteolytic enzymes
using this mechanism
• the best known serine endoproteases are trypsin,
chymotrypsin and elastase of pancreatic juice
Characteristics of the substrate-binding sites in
chymotrypsin, trypsin and elastase
The mechanism of chymotrypsin action
Hexokinase - example of structural flexibility increasing
the specifity of enzymes
Hexokinase catalyzes the transfer of phosphate group from ATP to glucose:
ATP + Glc → ADP + Glc-6-phosphate
Enzyme flexibility – the use of strain energy
Isoenzymes
• Isoenzymes are enzymes that catalyse the same
reaction, but differ in their primary structure
and/or subunit composition
• Amounts of some tissue-specific enzymes are
determined in serum for diagnostic purposes
• Typical examples of diagnostically important
serum isoenzymes are CK (myocardial infarction),
GGT (hepatitis) or LDH (myocardial infarction,
hepatitis)
LDH isoenzymes
• LDH catalyzes the interconversion of pyruvate and lactate with
accompanying conversion of NADH and NAD+
• tetrameric enzyme made of two different subunits (H and M)
Classifying enzymes
(1972 International Union of Biochemistry)
1.
2.
3.
4.
5.
6.
Oxidoreductases
Transferases
Hydrolases
Lyases
Isomerases
Ligases (synthetases)
Oxidoreductases (EC 1.)
• catalyze transfer of electrons from one molecule (reductant,
electron donor) to another (oxidant, electron acceptor)
• dehydrogenases catalyze oxidation reaction which involves
removing hydrogen from the reductant
• typical coenzymes are nicotine nucleotides (NADH, NADPH),
flavin nucleotides (FMN, FAD), hemins, coenzyme Q
(ubichinone) and lipoic acid
• typical representants are alcohol dehydrogenase,
glucosooxidase etc.
Transferases (EC 2.)
• catalyze the transfer of a functional group
(e.g. methyl, acyl, phospho, glycosyl etc.) from
one molecule (donor) to another (acceptor)
• donor molecule is often a coenzyme
• typical coenzymes of transferases are ATP,
pyridixalphosphate (amino group), tetrafolic
acid (formyl group), adenysylmethionine
(methyl), coenzyme A (acetyl)
Hydrolases (EC 3.)
• catalyzes the hydrolysis of a chemical bond:
A–B + H2O → A–OH + B–H
• cleave, for instance, ester bonds (esterases,
nucleases, phosphodiesterases, lipases,
phosphatases), glycosidic bonds
(glycosidases), peptide bonds (proteases and
peptidases)
Lyases (EC 4.)
• cleave C-C, C-O, C-N and other bonds by other
means than by hydrolysis or oxidation
• require only one substrate for the reaction in one
direction, but two substrates for the reverse
reaction (e.g. adenyl cyclase catalyzes ATP →
cAMP + PPi )
• decarboxylases (EC 4.1.1) are lyases cleaving C-C
bond and liberates carbon dioxide from carboxyl
group
Isomerases (EC 5.)
• catalyze reactions involving a structural
rearrangement of a molecule
• e.g. alanine racemase catalyzes the conversion of
L-alanine into its isomeric (mirror-image) form, Dalanine
• isomerase called mutarotase catalyzes the
conversion of a-D-glucose into b-D-glucose.
• UDP-Glc-epimerase : UDP-Glc ⇌ UDP-Gal
Ligases (synthetases) (EC 6.)
• catalyze synthesis of a new bond between two
molecules
• reaction is usually accompanied by hydrolysis of
ATP or another similar triphosphate
• biotin is a cofactor for enzymes catalyzing
carboxylation binding carbon dioxide to
molecule) called carboxylases (e.g. pyruvate
carboxylase)
Literature
• Baynes, J.W.,Dominiczak, M.H.: Medical
Biochemistry, Elsevier 2004
• Bugg, T.:Introduction to Enzyme and
Coenzyme Chemistry, Blackwell Publishing,
2004