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ENZYMES

Prof.Dr. A. Süha Yalçın

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Enzymes are highly specialized proteins, they have evolved to catalyze reactions in biological systems and organisms.

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History of enzymes
1800s........... Digestion of meat by secretions of the stomach. 1850s........... Fermentation of sugar into alcohol by yeast. 1850s........... Yeast extracts ferment sugar to alcohol. 1926............ Isolation and crystallization of urease. 1930s........... Crystallization of pepsin and trypsin Today...........Nearly two thousand different enzymes identified, hundreds have been crystallized.

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Properties
Extraordinary catalytic power High degree of specificity for substrates No by-product formation Function under mild conditions of temperature and pH

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All enzymes are proteins (except for some catalytic RNA molecules) The primary, secondary, tertiary and quaternary structures of enzymes are all essential to their catalytic activity MW of enzymes range from 12,000 to > 1,000,000

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Some enzymes require an additional chemical component other than their amino acid residues for activity. Such additional groups are called cofactors Cofactor(s) may be
one or more inorganic ions a complex organic or metallo-organic molecule

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12.2008 Cytochrome oxidase Peroxidase DNA polymerase Hexokinase Arginase Pyruvate kinase Urease ASY/Enzymes 7 .Fe2+ Cu2+ Zn2+ Mg2+ Mn2+ K+ Ni2+ 06.

2008 Transfer of aldehydes Transfer of hydrogen atoms Transfer of hydride ion Transfer of acyl groups Transfer of amino groups Transfer of H / alkyl groups Transfer of 1 C atoms ASY/Enzymes 8 .12.TPP FAD NAD CoA PP Co-B12 THF 06.

• Prosthetic group – covalently bound organic molecule or metal ion • Coenzyme (Cosubstrate) – tightly but not covalently bound organic molecule 06.12.2008 ASY/Enzymes 9 .

The protein part of an enzyme is called the apoenzyme or apoprotein.12. catalytically active enzyme together with its coenzyme and/or metal ions is called a holoenzyme.2008 ASY/Enzymes 10 . Apo Holoenzyme 06.A complete.

trypsin 06.12.Enzyme Names End in –ase Identifies substrate sucrase – reacts with sucrose lipase .2008 ASY/Enzymes 11 .reacts with lipid Describes function of enzyme oxidase – catalyzes oxidation hydrolase – catalyzes hydrolysis Common names are still used Pepsin.

Classification 1. Oxidoreductases Transfer of electrons Transferases Group-transfer reactions Hydrolases Hydrolysis reactions Lyases Addition to double bonds Isomerases Group-transfer in molecules Ligases Bond forming reactions (ATP) 06. 5.2008 ASY/Enzymes 12 . 4. 2. 6.12. 3.

7.1.1 2: 7: 1: 1: Class name (transferase) Subclass (phosphotransferase) Sub-subclass (hydroxyl group as acceptor) Glucose as phosphate group acceptor 06.12.2008 ASY/Enzymes 13 .ATP + Glucose ADP + Glucose-P Trivial name: Hexokinase Systematic name: ATP : glucose phosphotransferase Classification no.: EC 2.

2008 ASY/Enzymes 14 . 06.12.Enzymes and catalysis Under biologically relevant conditions. uncatalyzed reactions tend to be slow. Enzymes provide a specific environment within which a given reaction is energetically more favorable.

forming an active site.2008 ASY/Enzymes 15 . Folding of the protein into its tertiary structure brings sidechains of various amino acids that may be far apart in the primary sequence into close juxtaposition.Active site Enzymes are proteins that catalyze chemical reactions. 06.12.

Water S P Active site Enzyme 06.12.2008 ASY/Enzymes 16 .

i. decarboxylation.Reactive groups at the active site catalyze reactions by: donating or withdrawing electrons stabilizing or generating free radical intermediates forming temporary covalent bonds (a transition state intermediate) There is high degree of specificity for the reaction catalyzed.12. 06. transamination or sidechain elimination but an enzyme will normally catalyze one of these reactions.e.2008 ASY/Enzymes 17 . an amino acid bound to pyridoxal phosphate may undergo: isomerization.

Amino acids at the active site will make non-covalent interactions between their side-chains and substrate molecule(s): acidic groups (Asp. Met.2008 . Trp) hydrophobic interactions (Ala.1. Glu) basic groups (Lys. Thr.12. carbohydrates and lipids bound to the enzyme may also interact with substrates. Arg) hydrophilic interactions with –OH groups (Ser. Leu. ASY/Enzymes 18 06. Tyr. Binding may result in considerable conformational change. Val. Ile. 3. His. Pro)Amin 2. Tyr) hydrophilic interactions with –SH groups (Cys) hydrophilic interactions with amide groups (Asn. Gln) aromatic interactions (Phe. Metal ions.

2008 ASY/Enzymes 19 .12. enzymes can readily distinguish between isomers H H C C O OH H COO C CH 3 D -alanine NH 3+ CH 2OH D -glyceraldehyde H HO C C O H +H 3N COO C CH 3 L -alanine H CH 2OH L -glyceraldehyde 06.There is specificity in binding to the active site Because of multiple interactions in binding to the active site.

2008 ASY/Enzymes 20 .12.Enzymes can also distinguish between isomers cis trans 06.

06.2008 ASY/Enzymes 21 . but they do not alter the position of the equilibrium.Enzymes act by lowering the activation energy of the reaction energy level non-enzymic + enzyme excited final initial They increase the speed at which equilibrium is achieved.12.

Activation energy barrier Activation energy of the uncatalyzed reaction Free energy of system Activation energy of the catalyzed reaction Initial state Overall freeenergy change Final state at equilibrium Progress of reaction 06.2008 ASY/Enzymes 22 .12.

they loose their activity at low or high pH R groups of amino acids at the active site determine pH profile 06.2008 ASY/Enzymes 23 .Effect of pH Enzymes have maximum activity at their optimum pH Tertiary structure of enzyme must be maintained Most enzymes have a narrow range of activity.12.

2008 ASY/Enzymes 24 .Pepsin Glucose 6-phosphatase Relative rate 2 4 6 pH 8 10 12 06.12.

2008 ASY/Enzymes 25 .Effect of temperature Enzymes will have very little activity at low temperatures Reaction rate increases with temperature Enzymes are most active at their optimum temperatures (usually 37°C in humans) At high temperatures activity will be lost due to denaturation of protein 06.12.

2008 ASY/Enzymes 26 .Relative rate 20 30 40 50 60 70 Temperature o C 06.12.

Effect of substrate: [S] Increasing substrate concentration increases the rate of reaction (at constant enzyme concentration) Maximum activity is reached when all the enzyme combines with substrate 06.2008 ASY/Enzymes 27 .12.

M 06.12.2008 ASY/Enzymes 28 .Initial rate Substrate concentration.

Michaelis-Menten Equation E+S k1 k-1 ES k2 k-2 E+P E: enzyme S: substrate P: product ES: enzyme-substrate complex 06.12.2008 ASY/Enzymes 29 .

[ES]) [S] 2.12. Rate of formation of ES Rate of formation = k1([Et] . Rate of breakdown of ES Rate of breakdown = k-1[ES] + k2[ES] 06.2008 ASY/Enzymes 30 .E+S k1 k-1 ES k2 k-2 E+P 1.

E+S k1 k-1 ES k2 k-2 E+P 3.[ES]) [S] = k-1[ES] + k2[ES] 06. The steady state Rate of formation = Rate of breakdown k1([Et] .2008 ASY/Enzymes 31 .12.

Separation of the rate constants k1[Et] [S] = (k1 [S] + k-1 + k2) [ES] [Et] [S] [ES] = [S] + (k2 + k-1) / k1 06.2008 ASY/Enzymes 32 .12.E+S k1 k-1 ES k2 k-2 E+P 4.

2008 ASY/Enzymes 33 . Definition of initial velocity vo = k2 [ES] k2 [Et] [S] vo = [S] + (k2 + k-1) / k1 06.12.E+S k1 k-1 ES k2 k-2 E+P 5.

Definition of Vmax and Km Vmax = k2 [Et] Km = (k2 + k-1) / k1 06.2008 ASY/Enzymes 34 .E+S k1 k-1 ES k2 k-2 E+P 6.12.

Vmax= maximum rate.2008 ASY/Enzymes 35 . KM = Michaelis-Menten constant 06.12.Michaelis-Menten Equation Vmax [S] vo = KM + [S] vo= initial rate.

2008 Substrate concentration. M ASY/Enzymes 36 .Vmax o o o Initial rate o o o o o o 1/2 Vmax o o KM 06.12.

Determination of Km and Vmax 1 0.4 0.8 0.6 0.2008 ASY/Enzymes 37 .2 0 0 200 400 [substrate] 600 800 Vmax maximum rate of reaction when the enzyme is saturated Units: mol product / time relative activity Km [Substrate] required to achieve ½ Vmax Units: mol substrate / L 06.12.

12.2008 ASY/Enzymes 38 .Lineweaver-Burk plot 1 / rate Slope= Km / V 1 / Vmax 1 / [substrate] max -1 / Km 06.

12.2008 Km low compared with physiological range of [substrate] small increase in rate of reaction for a large increase in [substrate] Km high compared with physiological range of [substrate] large increase in rate of reaction for a small increase in [substrate] 100 200 ASY/Enzymes 300 400 39 [substrate]. mmol /L . µmol /min 120 100 80 60 40 20 0 0 06.A high or low value of Km is relative to the physiological range of concentration of substrate in cells rate of reaction.

12.enzyme A Two enzymes “competing” for substrate S P X S enzyme B rate of reaction. mmol /L 06. µmol /min 120 100 80 60 40 20 0 0 enzyme A low Km enzyme B high Km 100 200 300 400 [substrate].2008 ASY/Enzymes 40 .

Bisubstrate reactions Single-displacement reactions Double-displacement or ping-pong reactions S1 S2 E P 06.2008 ASY/Enzymes 41 .12.

12.2008 ASY/Enzymes 42 .Single displacement: (A + B A B A E E BA E C + D) E +C+D 06.

Double displacement: (AX + B AX E B X E BX E AX E A + BX) A X E BX E 06.2008 ASY/Enzymes 43 .12.

Enzyme Inhibitors cause loss of catalytic activity change the protein structure of enzyme may be competitive or noncompetitive some are irreversible 06.12.2008 ASY/Enzymes 44 .

2008 ASY/Enzymes 45 .12.Enzyme inhibitors Irreversible Reversible • Competitive • Non-competitive 06.

12.mechanism-dependent (suicide) inhibitors .rational drug design 06.2008 ASY/Enzymes 46 .Enzyme inhibitors irreversible bind to enzyme covalently may undergo part of reaction transition state intermediate does not breakdown reversible non-covalent (equilibrium) binding to enzyme many are substrate analogues may be relatively unspecific some inhibitors are used as drugs .highly specific for target enzyme .

2008 ASY/Enzymes 47 .12.Irreversible inhibition I E I E Inactive enzyme 06.

2008 ASY/Enzymes .12.Competitive inhibition E-S complex S S E E Inactive enzyme 48 I I E 06.

Competitive Inhibitors have a structure similar to substrate occupy the active site compete with substrate for the active site their effect is reversed by increasing substrate concentration 06.2008 ASY/Enzymes 49 .12.

2008 ASY/Enzymes 50 .12.Competitive inhibition rate of reaction 1 / rate of reaction Vmax unchanged Km increased [substrate] 1 / [substrate] 06.

Noncompetitive Inhibitors do not have a structure like substrate bind to the enzyme but not to the active site change the shape of enzyme and active site so that substrate can not fit altered active site their effect is not reversed by adding substrate 06.12.2008 ASY/Enzymes 51 .

2008 ASY/Enzymes I E Inactive enzyme I 52 .Noncompetitive inhibition S E S E-S complex S S E I 06.12.

12.Noncompetitive inhibition 1 / rate of reaction [substrate] 1 / [substrate] Km unchanged 06.2008 ASY/Enzymes Vmax decreased 53 rate of reaction .

2008 ASY/Enzymes S E I 54 .Uncompetitive inhibition S E S E E-S complex I Inactive enzyme 06.12.

2008 ASY/Enzymes 55 .How can we determine whether an inhibitor is reversible or irreversible ? 06.12.

dialysis semi-permeable membrane small molecules equilibrate across the membrane proteins are too large to cross the membrane 06.2008 ASY/Enzymes 56 .12.

2008 ASY/Enzymes 57 .dialysis semi-permeable membrane small molecules equilibrate across the membrane proteins are too large to cross the membrane 06.12.

dialysis semi-permeable membrane small molecules equilibrate across the membrane proteins are too large to cross the membrane inhibitor removed activity restored ASY/Enzymes 58 06.2008 .12.

dialysis semi-permeable membrane small molecules equilibrate across the membrane proteins are too large to cross the membrane inhibitor bound covalently to protein 06.12.2008 ASY/Enzymes 59 .

dialysis semi-permeable membrane inhibitor not removed activity not restored 06.12.2008 ASY/Enzymes 60 .

2008 ASY/Enzymes 61 . not by their mass.12.Enzymes are measured by their catalytic activity. 06.

Factors affecting enzyme activity pH of incubation or environment temperature time of incubation concentration of enzyme concentration of substrate covalent modification of enzyme inhibitors and activators 06.12.2008 ASY/Enzymes 62 .

12. Optimum pH 6. Substrate concentration dependency 5.2008 ASY/Enzymes 63 . Equation of the reaction 2. Cofactor requirement 4. Temperature dependency 06. Analytical procedure 3.Information required for quantitative enzyme measurement 1.

limiting factor in product formation is [enzyme].2008 ASY/Enzymes 64 .12.Excess substrate is used so that enzyme is saturated. 120 rate o f reaction 100 80 60 40 20 0 0 100 200 300 400 500 600 700 800 [substrate] 06.

2008 ASY/Enzymes 65 .12.Determining initial rates at increasing [enzyme] Initial rates 3xE Progress of reaction 2xE E Time 06.

Enzyme activity vs. units 14 06.12.2008 ASY/Enzymes 66 . initial rate 4 3 Initial 2 rate 1 o o o o o 2 4 o o 6 8 10 12 Enzyme activity.

06. The specific activity is the number of enzyme units per milligram of protein.2008 ASY/Enzymes 67 .12.Enzyme Activity (IU) One unit of enzyme activity is defined as that amount causing transformation of 1 μmol of substrate per minute under optimal conditions of measurement.

2008 ASY/Enzymes 68 .12.An example of enzyme catalysis: serine proteases H H2N N H C R1 O C N H R2 C H C O H2O COOH H H2N N H C R1 O C OH H N H R2 C H C O COOH In vitro: 10 – 12 hours in 12 mol /L HCl at 105ºC random hydrolysis of peptide bonds In vivo: 1 – 2 hours at 37ºC. specific bonds hydrolysed 06.

2008 ASY/Enzymes 69 .Bonds hydrolysed: Trypsin: esters of basic amino acids Chymotrypsin: esters of aromatic amino acids Elastase: esters of small neutral amino acids bond to be cleaved lies over catalytic site substrate sits in a groove on the enzyme surface 06.12.

HN - OOC CH 2 CH C O Aspartate-102 HN HO CH 2 CH C O Serine-195 HN CH 2 N N CH C O 06.2008 ASY/Enzymes 70 Histidine-57 .12.

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2008 ASY/Enzymes 72 .12.Bonds hydrolysed: peptide in groove on enzyme surface trypsin esters of basic amino acids chymotrypsin esters of aromatic amino acids Gly elastase esters of small neutral amino acids Asp Gly + -- trypsin 06.

Bonds hydrolysed: peptide in groove on enzyme surface trypsin esters of basic amino acids chymotrypsin esters of aromatic amino acids elastase esters of small neutral amino acids Gly Gly Ser chymotrypsin 06.2008 ASY/Enzymes 73 .12.

Bonds hydrolysed: peptide in groove on enzyme surface trypsin esters of basic amino acids chymotrypsin esters of aromatic amino acids elastase esters of small neutral amino acids Val Thr Gly elastase 06.12.2008 ASY/Enzymes 74 .

Lock-and-key Model Enzyme binds the substrate in the active site Only certain substrates can fit the active site R-groups of amino acids forming the active site aid substrate binding Enzyme-substrate complex forms subtrate changes to product product is released from the enzyme 06.12.2008 ASY/Enzymes 75 .

12.2008 E ES ASY/Enzymes E P2 E + P 76 .P1 + S S + E E + S 06.

Catalytic Efficiency of Enzymes Proximity and orientation of the substrate in relation to the catalytic group Strain and distortion of the susceptible bond (induced fit of the enzyme) General acid-base catalysis Covalent catalysis 06.12.2008 ASY/Enzymes 77 .

2008 ASY/Enzymes E Favorable orientation Favorable proximity 78 .12.Unfavorable orientation Favorable proximity E E Unfavorable orientation Unfavorable proximity 06.

S + A S Relaxed enzyme molecule Induced fit of enzyme to the bound substrate 06.12.2008 ASY/Enzymes 79 .

Induced-fit model Enzyme structure is flexible.2008 ASY/Enzymes 80 .12. not rigid Enzyme and active site adjust their shape to bind substrate Both the enzyme and substrate undergo conformational change Shape change increases range of substrate specificity improves catalysis during reaction 06.

P1 E + S E S E + P2 E + S 06.2008 ES ASY/Enzymes E + P 81 .12.

In the induced fit model; when substrate binds the shape of the enzyme adapts to the substrate.

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Proton donors/acceptors
carboxyl group (-COOH) amino group (-NH2) sulfhydryl group (-SH) imidazole group

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Enzymes act in organized sequences

A
E1

B
E2

C
E3

D

Some enzymes participating in cellular metabolism are regulatory enzymes.

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2008 ASY/Enzymes 85 .Regulation of Enzyme Activity Inhibition Allosteric regulation Covalent modification Isoenzymes Synthesis/degradation 06.12.

2008 ASY/Enzymes .Allosteric modulation Increased enzyme activity E M M+ E M- E M Decreased enzyme activity 86 06.12.

2008 ASY/Enzymes 87 .12.+ v Normal rate - [S] 06.

2008 ASY/Enzymes 88 .12.rate of reaction [substrate] Allosteric regulation is instantaneous activation by precursors inhibition by end-products 06.

12.06.2008 ASY/Enzymes 89 .

2008 ASY/Enzymes 90 .activation due to decreased cooperativity rate of reaction inhibition due to increased cooperativity substrate concentration 06.12.

12.2008 ASY/Enzymes 91 .A SA C C A A B B B 06.

Aspartate Transcarbamoylase 06.12.2008 ASY/Enzymes 92 .

2008 ASY/Enzymes 93 . glycosylation and other processes. These alterations effect enzyme activity. and are involved in the regulation of enzymes. 06.12.Covalent modification Some enzymes are modified by phosphorylation.

H2O CH2OH HN CH serine CO ATP P O CH2 HN CH CO H2O OH H3PO4 CH2OH HN CH CO phosphoserine serine Covalent modification of an enzyme is fast: time course of seconds – minutes commonly in response to: fast acting hormones (peptides. etc) 06.2008 ASY/Enzymes 94 .O HO ADP.12.

12.OH CH2 OH CH2 OP CH2 OP CH2 Phosphorylase a (inactive) 06.2008 ASY/Enzymes Phosphorylase b (active) 95 .

or even in the same cell.2008 ASY/Enzymes 96 . creatine kinase (CK) 06.12. Such multiple forms of enzymes are called isoenzymes or isozymes. Typical examples are: lactate dehydrogenase (LDH).Multiple forms of enzymes Many enzymes occur in more than one molecular form in the same species. in the same tissue.

but differing in structure: may have different charges at a given pH may have different affinity for substrate may preferentially catalyse reaction in one direction may differ in temperature sensitivity may differ in inhibitor sensitivity may differ in coenzyme specificity They may be found in different tissues and in different organelles 06.12.Isoenzymes Enzymes catalysing the same reaction.2008 ASY/Enzymes 97 .

2008 type 2 type 3 ASY/Enzymes type 4 type 5 98 .12.Lactate dehydogenase pyruvate reduction in skeletal muscle NAD+ NADH CH3 C O NAD NADH lactate oxidation in heart muscle + CH3 CHOH COOH lactate COOH pyruvate type 1 06.

12.2008 Type 1 ASY/Enzymes 99 + .Separation of LDH isoenzymes Type 2 Type 3 Type 4 Type 5 The different isoenzymes have different charges. 06. and can be separated by electrophoresis.

Physiological control of enzyme activity Change in the rate of synthesis of the enzyme (change in gene expression) slow: time course of hours or days commonly in response to: slow acting hormones (steroids) long-term adaptation 06.2008 ASY/Enzymes 100 .12.

2008 ASY/Enzymes 101 .Use of enzymes in medicine measurement of metabolites in plasma and urine measurement of enzymes in plasma • assessment of tissue damage physiological control of enzyme activity use of enzyme inhibitors as drugs 06.12.

swf 06.SWF http://www.uk/animations/model.us/biology/biology1111/ animations/enzyme.kscience.mn.stolaf.swf http://www.swf http://cble.12.2008 ASY/Enzymes 102 .Animations http://www.northland.chem.nl/biolip/SERPROTE.uu.edu/people/giannini/flashanimat/ enzymes/allosteric.co.cc.