Mechanism of enzyme catalysis How enzymes work

Enzymes existence of catalysis in biological materials was discovered by Swedish chemist Berzelius proteins (the first isolated enzyme was urease) catalyze the chemical reactions necessary for life typical increase of chemical reaction rates 1010 – 1015 times many enzymes for their activity require cofactor called coenzyme

Decreasing of free energy of the transition state by catalysis G activation free energy

The steps in enzyme-catalyzed reaction

The important effects of enzyme catalysis proximity effect general-base and general-acid catalysis electrostatic effects nucleophilic or electrophilic catalysis by functional groups of enzyme structural flexibility

The proximity effect reaction between two reactants is accelerated by holding these ones closer together in a correct orientation

The general-base and general-acid 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

The general-base and general-acid catalysis

Ribonuclease A - example of acid-base catalysis small (124AA) protein that hydrolyzes RNA by cleaving ester bond between P and O of ribose carbon 5

Electrostatic effects stabilization of distribution of electric charge in transition states during enzymatic reactions the changing charges on atoms of substrate in a transition state intearacts with charges on atoms of the surrounding enzyme and also nearby 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 similarly electrophilic group of enzymes is usually its complex of metal cofactor with substrate nucleophic catalysis involves the formation of an intermediate state in which substrate is covalently bound to a nucleophilic group

Nuclephilic catalysis - acetoacetic decarboxylase an cytosolic enzyme involved in the ketone body production pathway in humans

Serine protease - an example of nucleophilic catalysis serine proteases are a large family of proteolytic enzymes using this mechanism the best known serine endoproteases are trypsin, chymotrypsin and elastase of pancreatic juice conserved sequence , about 1/2 trypsin and chymotrypsin identical and 1/4 tr X elastase cleavage - basic , aromatic and (less specifity) small hydrophobic(Ala)

Characteristics of the substrate-binding sites in chymotrypsin, trypsin and elastase Baynes, J.W.,Dominiczak, M.H.: Medical Biochemistry

The mechanism of action of chymotrypsin a) pocket for substrate, trypsin - Asp and attraction of Lys,Arg, chymotrypsin Ser - less polar for aromatics

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

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 measured in serum for diagnostic purposes Typical examples of diagnostically important serum isoenzymes are CK (myocardial infarction), CGT (hepatitis) or LDH (myocardial infarction, hepatitis) aspartate amino transferase(SGOT), creatin kinase, gama glutamyl transferase CK enzyme consists of two subunits, which can be either B (brain type) or M (muscle type) , CK-MM, CK-BB and CK-MB heart muscle expresses CK-MM at 70% and CK-MB at 25-30%

LDH isoenzymes LDH catalyzes the interconversion of pyruvate and lactate with concomitant conversion of NADH and NAD+ tetrameric enzyme from two different subunits (H and M) H – heart , M muscle and liver Baynes, J.W.,Dominiczak, M.H.: Medical Biochemistry

Classifying enzymes (1972 International Union of Biochemistry) Oxidoreductases Transferases Hydrolases Lyases Isomerases Ligases (synthetases)

Oxidoreductases (EC 1.) catalyzes transfer of electrons from one molecule (reductant, electron donor) to another (oxidant, electron acceptor) dehydrogenases catalyze oxidation reaction which involves removing hydrogen from 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 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 e.g. ester bonds (esterases, nucleases, phosphodiesterases, lipases, phosphatases), glycosidic bonds (glycosidases), peptide bonds (proteases and peptidases) Hydrolysis is a chemical reaction during which one or more water molecules are split into hydrogen and hydroxide ions which may go on to participate in further reactions. chemical reaction

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 joining two molecules reaction is usually accompanied by hydrolysis of ATP or another similar triphosphate biotin is cofactor for enzymes catalyzing carboxylation (joining carbon dioxide to molecule) called carboxylases (e.g. pyruvate carboxylase) Nitric oxide synthases (EC 1.14.13.39 ) !!

An enzyme catalyzed reaction proceeds rapidly under mild conditions because involved enzyme decreases the activation energy for a reaction. Enzymes are usually highly specific for the reactions they catalyze.