sign in sign up
<<
Home , Active site

Chapter 3: : Structure and Function

Enzymes act as the body’scatalysts bycomplexing thereaction'sparticipants in the correct arrangement to react, lowering the , Ea, to react, but G stays the same. HO H B H B TS3 C O enzyme active site O + enzyme O active site HO H H3C C active site disassociate TS1 TS2 C O C O C O O H3C C lactic acid = Product H3C C + complex H3C C = forms + NH2 + O O + O NAD+ N O N Pyruvic acid = Lactate H O P O N O LDH R N H N (enzyme) R N O H H H NADH H H attack site () (2 faces) NAD+ HO OH P O Nicotinamide O O cofactor adenosine dinucleotide site (reducing agent ) O NH2 N E is too high so O a E is much lower so reaction is much faster. reaction is too slow. a H H TS S +E ES EP P H H HO OH Enzymes TS2 PE lower the E PE TS TS3 1. Provides a reaction surface Ea1 a 1 potential of a reaction potential (the active site) Substrate energy energy Substrate Ea2 2. Provides a suitable environment (hydrophobic) exergonic G = exergonic G = Product 3. Brings reactants together Product 4. Positions reactants correctly for reaction POR = progress of reaction POR = progress of reaction without the enzyme with the enzyme 5. Weakens bonds in the reactants 6. Provides acid / base catalysis -Ea2 -E -(4 - 20) Enzyme catalyzed reactions 7. Provides nucleophilic groups kenz 2.3RT a2 10 2.3RT 1.4 11.43 11 arealways faster. Assumed, 8. Stabilises the transition statewith = -E = 10 = 10 = 10 = 3 x 10 kno enz a1 2.3RT Ea1 = 20 kcal/mole and intermolecular bonds 10 Ea2 = 4 kcal/mole.

1 © Oxford University Press, 2013 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 . 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 . 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 is an important strategy in drug design. 2 © Oxford University Press, 2013 active site Interactions between the substrate , cofactors and the enzyme can be very complicated.

3 © Oxford University Press, 2013 Substrate binding uses the usual forces of interaction. 1. Ionic 2. H-bonding 3. Dispersion forces 4. Dipole-dipole vdw 5. Covalent bonds interaction 6. Pi stacking S H-bond Active site H ionic Phe O Ser bond

CO2

Asp

Enzyme

4 © Oxford University Press, 2013 Induced fit - active site of the enzyme and the substrate alter shapes to maximise intermolecular bonding .

S S

Ser O H Phe H Phe Ser O CO CO 2 2 Induced

Asp fit Asp

Intermolecular bonds not optimum Intermolecular bond lengths length for maximum bonding optimised. Susceptible bonds in substrate strained. Susceptible bonds in substrate more easily broken

5 © Oxford University Press, 2013 Binding of pyruvic acid in LDH (lactic dehydrogenase enzyme)

1. Ionic bonding 2. H-bonding 3. Diifispersion forces

O H-Bond H O

C O H C C 3 + H N O 3 vdwdispersion-interactions Ionic bond

6 © Oxford University Press, 2013 Catalysis mechanisms – necessary functions

Acid/base catalysis

Histidine H O H O

N C N C Protein Protein CH N Protein CH N

pKa  6.0 CH2 H CH2 H

a b BH N N H B N N H Where is the + charge? H Non-ionised Ionised a. mainlyona Acts as a basic catalyst Acts as an acid catalyst b. mainly on b c. on both a and b ( 'sink') (proton source)

Nucleophilic residues H O

Serine H O H O N C Protein Protein CH N N C Protein N C Protein Protein CH N Protein CH N CH2 H

CH a 2 H b CH2 H O S H Where is the best ? H a. c H b. cysteine sulfur O c. tyrosine oxygen d. all are similar 7 © Oxford University Press, 2013 Serine acting as a nucleophile

Serine H O H O H O R Serine H N C S N C Protein Protein N C Protein Protein CH N Protein CH N Protein CH N

CH2 H CH H 2 CH2 H R O O O B H R C H H B S O H B BH O O C O H Threonine is H also possible. H C R R O R B B O acyl CoA S C R H As O at body pH.

8 © Oxford University Press, 2013 Mechanism for uses 3 amino acids at the active site

Catalytic triad of serine, and aspartate

H H N N O O O

Serine Histidine Chymotrypsin

9 © Oxford University Press, 2013 Chymotrypsin Mechanism for chymotrypsin Chymotrypsin

H O H O

N C Protein N C Protein Protein CH N Protein CH N H R H R

H H H N N O N N O H O O O O

Serine Histidine Aspartic acid Serine Histidine Aspartic acid Chymotrypsin Chymotrypsin

Chymotrypsin Chymotrypsin

H O H O Protein H H2N N C O N C Protein CH Protein CH O H R R H

H H N N H H O O N N O O O H O O N C O Protein CH

R Serine Histidine Aspartic acid Serine Histidine Aspartic acid Chymotrypsin Chymotrypsin 10 © Oxford University Press, 2013 Mechanism for chymotrypsin

:O:

C protein NH protein

.. :N N H :O H OO

Ser His Asp

.. .. :O: :O: H H

C protein : C : O O protein NH protein protein NH protein : : C H H :N N N N H :N N H :O H OO :O : OO :O : OO

Ser His Asp Ser His Asp Ser His Asp

protein OH .. H .. C ::O ::O .. protein O.. H protein O..H O C C H H :N N H N N H .. :N N :O : OO :O : OO :OH OO

Ser His Asp Ser His Asp Ser His Asp

11 © Oxford University Press, 2013 Overall Process of

S P S P

EE E E E catch react release E + S ES EP E + P

1. Binding interactions must be strong enough to hold the substrate 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,,g lowering 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. Ma ny ye enz ym esaees are r eguaedbyageegulated by agent sws within th ecee cell 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 called an allosteric binding site

NH2

Example N N

O N N = stimulates enzyme activity O P O O AMP = negative feedback reduces O H H enzyme activity H H OH OH

H OH H OH H O H O HO Glycogen O H H HO O HO H OH H OH a H O P H OH HO OH Glucose-1-phosphate n

13 © Oxford University Press, 2013 Regulation of Enzymes

AtiActive sit e Active site unrecognisable

Induced fit ACTIVE SITE (open) ENZYMEEnzyme (open) ENZYMEEnzyme Allosteric binding site

Allosteric inhibitor 1. Inhibitor binds reversibly to an allosteric binding site (molecule near end of pathway 2. Intermolecular bonds are formed (the usual kinds) 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) 14 © Oxford University Press, 2013 Regulation of Enzymes

Biosynthetic pathway

S P P’ P’’ P’’’

(open) ENZYMEEnzyme

Inhibition Feedback control

EihlliifhfbihiEnzymes with allosteric sites are often at the start of a biosynthetic 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.

15 © Oxford University Press, 2013 Regulation of Enzymes 1. E xt ernal si gnal s can regul at e th e acti v ity of enzymes (e.g. or hormones) 2. Chemical messenger initiates a signal cascade which activates enzymes called protein 3. Protein kinases phosphorylate target enzymes to affect activity

Example

Phosphorylase b (inactive) Signal Protein cascade Adrenaline, outside cell, Phosppyhorylase a reacts with (active) different types of cells in different ways. inside Glycogen Glucose-1-phhhtosphate cell 16 © Oxford University Press, 2013 can be used to study factors important to enzyme behavior. Michaelis-Menton derivation k 1 k2 E +S ES E +P k-1

dP =  = k [ES] dt o 2 rate of product formation

assume: k-1 >> k2

assume: [S] >> [E] so [ES]  constant = steady state assumption

d[ES] = 0 = k1[E][S] - k-1[ES] - k2 [ES] dt Etotal = [ET] = [E] + [ES]

[E] = [ET] - [ES] k1[E][S] = k-1[ES] + k2 [ES] = (k-1 + k2)[ES] (rearranged) [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 x k1 [ET] [S] = k1 [ES] [S] + (k-1 + k2) [ES] k1

( k [E ] [S] = k [ES] [S] + (k + k ) [ES]) 1 T 1 -1 2 (continued on next slide) 17 (k1) (k1) © Oxford University Press, 2013 (k-1 + k2) (k [S] + k + k ) defined KM = 1 -1 2 k [ET] [S] = [ES] 1 k1 (1/k1) k 1 1 = k (1/k ) 1 1 (k1[S] + k-1 + k2) (k-1 + k2) [ES] = [ET] [S] (rearranged) ([S]) + (k1[S] + k-1 + k2) k1 1 = 1 algebra (substitution) [S] + KM [ES] = [ET] [S] [S] + KM

[ET] [S] Vmax[S] dP =  = k [ES] = k = dt o 2 2 Michaelis-Menton Eq. Vmax = k2 [ET] [S] + KM [S] + KM

18 © Oxford University Press, 2013 k1 [ET][S] Vmax[S] dP = k = dt 2 Michaelis-Menton Eq. Vmax = k2[ET] [S] + KM [S] + KM Vmax

The smaller the KM, defined the tighter the binding. (k + k ) when K = [S] K = -1 2 (1/2) V M M max k1

Vmax[S] V [S] unbound dP = max 1 KM  = = V bound dt [S] + KM [S] + [S] 2 max

KM

1 1 [S] + KM K [S] Another way to plot the data (inverse). = = = M + dP Vmax[S] Vmax[S] Vmax[S] Vmax[S] dt initial [S] + KM rate 1 KM 1 1 = + Lineweaver-Burk Plots  V 1  max [S] Vmax y =  y = m (x) + b y intercept 1 x intercept Vmax _ 1 KM m = slope = KM Vmax

x 1 x = 19 [S] © Oxford University Press, 2013