Enzymology . How enzymes work - mechanisms.
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Transcript of Enzymology . How enzymes work - mechanisms.
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 a20 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 catalysis1. Metalloenzymes
Tighly bound metal ions(Fe2+, Fe3+, Cu2+, Zn2+,Mn2+, Co3+)
Metal-activated enzymes(Na+, K+, Mg+, Ca2+)
Role of metal ions in catalysis1) Binding of substrate and their properlyorientation to reaction
2) Mediating of oxidation-reductionreactions through reversible changes inthe metal ion oxidation state3) Charge stabilization, shielding negativecharges
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 ofbonds and E-NADH- aldehyd complex formations
Metal Ion catalysisMediating of oxidation-reduction reactions
3. Release of product and reduced NADH coenzyme, rearrangement
Metal Ion catalysisMetal 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. Oxidoreductases2. Transferases3. Hydrolases4. Lyases5. Isomerases6. 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, D-alanine
• 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