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Chapter 3: Enzymes: Structure and Function Enzymes actt as the E th body’s b d ’ catalysts t l t by b complexing l i the th reaction's ti ' participants ti i t in i the th correctt arrangement to react, lowering the activation energy, Ea, to react, but G stays the same. H HO B + O TS1 + C H enzyme active site O O C H3C enzyme active site B complex forms = O C C H3C O lactic acid = Product C H3C + + O TS3 disassociate O C C H3C + O TS2 O C enzyme active site H HO NH2 O NAD+ N O Pyruvic acid = Substrate Lactate dehydrogenase LDH (enzyme) H R O O S + E Enzymes lower the Ea of a reaction Substrate G = PE potential energy TS1 Substrate exergonic TS3 Ea2 G = Product Product POR = progress of reaction with the enzyme POR = progress of reaction without the enzyme -Ea2 10 2.3RT -Ea1 10 2.3RT -Ea2 2 3RT = 10 2.3RT -(4 - 20) 14 1.4 = 10 H HO TS2 11 43 = 1011.43 = 3 x 1011 Enzyme catalyzed reactions are always l f faster. A Assumed, d Ea1 = 20 kcal/mole and Ea2 = 4 kcal/mole. N attack site (2 faces) O NH2 H H P EP OH HO O O ES N H H H H Ea is much lower so reaction is much faster. Ea1 kenz = kno enz P N O O Ea is too high so reaction is too slow. TS exergonic O NAD+ Nicotinamide adenosine dinucleotide (reducing agent ) cofactor site P H O NADH (cofactor) H PE potential energy N R N N H OH 1. Provides a reaction surface (the active site) 2. Provides a suitable environment (hydrophobic) 3. Brings reactants together 4. Positions reactants correctly for reaction 5. Weakens bonds in the reactants 6. Provides acid / base catalysis 7. Provides nucleophilic groups 8 Stabilises 8. St bili the th transition t iti state t t with ith intermolecular bonds © Oxford University Press, 2013 1 The active site is often a hydrophobic hollow or cleft with key polar (or nonpolar) amino acids in key locations on the enzyme surface that can accept substrates and cofactors. The enzyme contains amino acids that interact with the substrate and cofactor in the usual way (ionic interactions, H bonds, dipole-dipole, dispersion forces and covalent bonds) which all help repeatedly catalyze the reaction (catch and release). It is usually proposed that the transition state complex is stabilized, lowering the activation energy which accelerates the reaction rate. Rather than the old 'lock and key' model, it is proposed that the enzyme and substrate influence one another to form a stronger interaction. This is called the 'induced fit' model. Identification of active sites is crucial in the process of drug discovery. The 3-D structure of the enzyme is analysed to identify active sites and design drugs which can fit into them. The most common ways to do this are x-ray crystollography, NMR analysis and computer modeling. Inhibitors bind to an enzyme's active site and block interaction with natural substrates. Knowing the strength of binding between the active site and an enzyme inhibitor is an important strategy in drug design. © Oxford University Press, 2013 2 active site Interactions between the substrate, substrate cofactors and the enzyme can be very complicated. © Oxford University Press, 2013 3 Substrate binding uses the usual forces of interaction. 1. Ionic 2. H-bonding 3. Dispersion forces 4 Dipole-dipole 4. Dipole dipole vdw 5. Covalent bonds interaction 6. Pi stacking S H-bond Active site H Ser ionic bond O Phe CO2 Asp Enzyme 4 © Oxford University Press, 2013 Induced fit - active site of the enzyme and the substrate alter shapes to maximise intermolecular bonding. bonding S Ser O S Ser Phe H CO2 Asp Intermolecular bonds not optimum length for maximum bonding Induced fit O H Phe CO2 Asp Intermolecular bond lengths optimised Susceptible bonds in optimised. substrate strained. Susceptible bonds in substrate more easily broken 5 © Oxford University Press, 2013 Binding of pyruvic acid in LDH (lactic dehydrogenase enzyme) 1. 2. 3. Ionic bonding H-bonding Dispersion i i forces f O H-Bond H O O C H3C dispersion vdw-interactions vdw interactions C O + H3N Ionic bond 6 © Oxford University Press, 2013 Catalysis mechanisms – necessary functions Acid/base catalysis Histidine H O N C CH Protein Protein O N C pKa 6.0 p H B H N B H H Non-ionised Acts as a basic catalyst (proton 'sink') N N Cysteine H O H b C CH Protein CH2 a H O C CH Protein H Where is the best nucleophile? a. serine oxygen b. cysteine sulfur c. tyrosine i oxygen d. all are similar CH2 b H a. mainly on a b. mainly on b c. on both a and b Tyrosine H O N C O N Protein N Where is the + charge? H Protein N H Ionised Acts as an acid catalyst (proton source) Nucleophilic residues Serine N CH2 a N Protein CH Protein N CH2 H Protein CH CH2 N Protein N H H S H c O 7 © Oxford University Press, 2013 Serine acting as a nucleophile S i Serine O H C N CH Protein B H H S N C H B acyl CoA H H N C CH R B S H thiol H O B H B O C Protein N CH2 O H O H H O O H Protein O C R R Protein N CH2 R C Serine CH Protein H S Threonine is also possible. O R N CH2 O Protein H R B H O O As carboxylate at body pH. C O 8 © Oxford University Press, 2013 R Mechanism for chymotrypsin uses 3 amino acids at the active site Catalytic triad of serine, histidine and aspartate H N N H O O O Serine Histidine Aspartic acid Chymotrypsin 9 © Oxford University Press, 2013 Mechanism for chymotrypsin Chymotrypsin C N O H O H Protein Chymotrypsin N R H C N Protein CH Protein CH Protein N H R N H N H O H O Serine Aspartic acid Histidine Chymotrypsin Chymotrypsin Chymotrypsin O C O H Protein O N C CH R N N H H O H CH R H O Protein H2 N C N Protein R H O H CH O H N O O Serine Aspartic acid Histidine Chymotrypsin N O O Serine Protein H N O O H N N H O O O Histidine Chymotrypsin Aspartic acid Serine Histidine Aspartic acid Chymotrypsin © Oxford University Press, 2013 10 Mechanism for chymotrypsin :O: C NH protein Ser C C H Ser His Asp Ser C :O : O .. His O O N H Ser Asp His Asp O :O : Ser N H His protein C O O Asp OH O H O :N :O : C :N H O C .. : O : .. OH protein .. H H O N H N :O : O .. H :O: protein protein H O Asp : :O O NH protein protein N H :N O : .. :O : NH protein Ser His .. :O : protein N H :N H : .. :O : protein N N H His O Asp O .. : OH Ser :N N H His O O Asp © Oxford University Press, 2013 11 Overall Process of Enzyme Catalysis S P S EE E catch E+S P E react ES E release EP E+P 1 Binding interactions must be strong enough to hold the substrate 1. sufficiently long for the reaction to occur 2. Interactions must be weak enough to allow the product to depart 3. Interactions stabilize the transition state,, loweringg Ea 4. Designing molecules with stronger binding interactions results in enzyme inhibitors which block the active site 12 © Oxford University Press, 2013 Regulation of Enzymes 1.. Many a y eenzymes y es aaree regulated egu a ed by age agentss w within thee cell ce 2. Regulation may enhance or inhibit the enzyme 3. The products of some enzyme-catalysed reactions may act as inhibitors 4. Often they bind to a binding site called an allosteric binding site NH2 Example N N O O P O O O H H OH H OH H O AMP = negative feedback reduces enzyme activity HO HO H H H = stimulates enzyme activity H O H O HO N H OH H OH Glycogen N OH OH Phosphorylase a H H H OH O Glucose-1-phosphate O P HO OH © Oxford University Press, 2013 n 13 Regulation of Enzymes Active A ti site it unrecognisable Active site ACTIVE SITE (open) Enzyme ENZYME Allosteric bindingg site Induced fit (open) Enzyme ENZYME Allosteric inhibitor 1. Inhibitor binds reversibly to an allosteric binding site (molecule near end of pathway 2 Intermolecular bonds are formed (the usual kinds) 2. 3. Induced fit of allosteric inhibitor alters the shape of the enzyme 4. Active site is distorted and is not recognised by the substrate (catalysis slows or stops) 5. Increasing substrate concentration does not reverse inhibition 6. Inhibitor differs in structure to the substrate (different enzyme location) © Oxford University Press, 2013 14 Regulation of Enzymes Biosynthetic pathway S P P’ P’’ P’’’ (open) Enzyme ENZYME Inhibition Feedback control Enzymes with E i h allosteric ll i sites i are often f at the h start off a biosynthetic bi h i pathway. The enzyme is controlled by the final product of the pathway. The final product binds to the allosteric site and switches off the enzyme as it builds up in concentration concentration. 15 © Oxford University Press, 2013 Regulation of Enzymes 1 External 1. E t l signals i l can regulate l t the th activity ti it off enzymes (e.g. neurotransmitters or hormones) 2. Chemical messenger initiates a signal cascade which activates enzymes called protein kinases 3. Protein kinases phosphorylate target enzymes to affect activity Example Phosphorylase b (inactive) Protein kinase Signal cascade p y a Phosphorylase (active) Gl Glycogen Gl Glucose-1-phosphate 1 h h t inside cell Adrenaline, outside cell, reacts with different types of cells in different ways. 16 © Oxford University Press, 2013 Enzyme kinetics can be used to study factors important to enzyme behavior. Michaelis-Menton derivation k1 E + k2 S ES E k-1 dP dt = o = k2 [ES] + P rate of product formation assume: k-1 >> k2 assume: [S] >> [E] so [ES] constant = steady state assumption d[ES] = 0 = k [E][S] - k [ES] - k [ES] 1 -1 2 dt k1[E][S] = k-1[ES] + k2 [ES] = (k-1 + k2)[ES] Etotal = [ET] = [E] + [ES] (rearranged) [E] = [ET] - [ES] [ES] = [ET] - [E] k1([ET] - [ES]) ([S]) = (k-1 + k2) [ES] substitution: [E] = [ET] - [ES] k1 [ET] [S] - k1 [ES] [S] = (k-1 + k2) [ES] k1 [ET] [S] = k1 [ES] [S] + (k-1 + k2) [ES] 1 k1 x k1 [ET] [S] = k1 [ES] [S] + (k-1 + k2) [ES] ( k1 [ET] [S] = k1 [ES] [S] + (k-1 + k2) [ES]) (k1) (k1) (continued on next slide) 17 © Oxford University Press, 2013 [ET] [S] = [ES] = (k1[S] + k-1 + k2) k1 k1 (k1[S] + k-1 + k2) 1 [ES] = dP dt [S] + KM = o = k2 [ES] defined KM = (k-1 + k2) k1 [ES] [ET] [S] (rearranged) (1/k1) k1 (1/k1) (k1[S] + k-1 + k2) algebra (substitution) [ET] [S] = k2 [ET] [S] [S] + KM = Vmax[S] [S] + KM 1 = ([S]) + = (k-1 + k2) k1 1 [S] + KM Michaelis-Menton Eq. Vmax = k2 [ET] 18 © Oxford University Press, 2013 dP dt k1 [E ][S] T = k2 [S] + KM = Vmax[S] Michaelis-Menton Eq. [S] + KM Vmax = k2[ET] Vmax The smaller the KM, the tighter the binding. when KM = [S] (1/2) Vmax KM Vmax[S] dP = dt Vmax[S] = [S] + KM [S] + [S] = defined (k-1 + k2) = k1 KM 1 2 Vmax unbound bound KM 1 Another way to plot the data (inverse). initial rate y= = Vmax[S] dP dt [S] + KM Vmax[S] = KM + Vmax[S] [S] Vmax[S] [S] + KM 1 Lineweaver-Burk Plots 1 = 1 = y = KM Vmax 1 [S] m (x) + 1 Vmax + b y intercept x intercept _ 1 Vmax 1 KM m = slope = 1 x= [S] KM Vmax x © Oxford University Press, 2013 19