Enzymes

Part I

Enzymes

• Nearly all are proteins• Catalyse reactions

– Stabilise the transitional state• Some RNA molecules can also catalyse

reactions

Enzymes

• Accelerate reaction rates by up to 106 times– Carbonic anhydrase fastest know enzymes

• Consumes 106 molecules of CO2 per second

Enzymes

• Catalyse specific reaction or a similar type of reactions

• Proteinases– Catalyse hydrolysis of peptide bonds or ester

bonds

Hydrolysis of ester bonds

Proteases

• Papain- unspecific protease enzyme

• Trypsin & Thrombin very specific enzymes– Trypsin → Carboxy

peptide bond next to Lys & Arg

– Thrombin → Peptide bond on carboxy side of Arg in a particular peptide sequence

Enzyme co-factors

• Small molecules that help an enzyme to catalyse a reaction

• Derived from vitamins• Metals or small organic molecules (coenzymes)• Prosthetic group- tightly bound co-factors

– Eg. Haem group of haemoglobin• Apoenzyme + cofactor = holoenzyme• Apoenzyme= Enzyme – co-factor

Enzyme cofactors

Enzyme

• Name– ase as the suffix– word to describe its activity

• Eg. Urease– Enzyme that catalyses the hydrolysis of urea.

• Some enzymes named after action– Eg. Digestive proteolytic enzyme “pepsin”

• From the Greek “pepsis”- digestion

6 classes or enzymes based on type of reaction

1. Oxidoreductases• Catalyse oxidation-reduction reactions• Eg. Lactate dehydrogenase (EC 1.1.1.27)

2. Transferases

• Catalyse group transfer reactions• May require co-enzymes• Eg. Alanine transaminase (EC 2.6.1.2)

3. Hydrolases

• Catalyse hydrolysis• Eg. Pyrophosphatase (EC 3.6.1.1)

4. Lyases• Lysis of a substrate, generates a double bond• Not→ hydrolytic, oxidative• Reverse direction→ + 1 substrate to double bond of 2nd

substrate• Synthase- lyase that catalyses an addition reaction• Eg. Pyruvate decarboxylase (EC 4.1.1.1)

5. Isomerase

• Catalyse structural change within a molecule• Eg. Alanine racemase (EC 5.1.1.1)

6. Ligase• Catalyse ligation• Requires energy input (ATP)• Also called synthethetases• Eg. Glutamine synthetase (EC 6.3.1.2)

EC number

• Introduced by enzyme commission in 1964• 4 digit number that describes the class of an

enzyme and the type of reaction catalysed• Eg. ATP: glucose phosphotransferase

(Hexokinase)• ATP + D-glucose → ADP + D-glucose 6-

phosphate• EC 2.7.1.1.

ATP: glucose phosphotransferase (Hexokinase)EC 2.7.1.1.

• 1st number (2)– Class name- 2- transferase

• 2nd number (7)– Subclass- phosphotransferase

• 3rd number (1)– Phosphotransferase with a hydroxyl group as

the acceptor• 4th number (1)

– D-glucose as the phosphoryl group acceptor.

Enzymes Efficient at transforming energy from one from to another

• Eg. Photosynthesis– Light energy → Chemical bond energy

• Mitochondria– Free energy in chemical bonds → ion gradient

energy → Energy in ATP• Membrane pumps (eg Na+/K+ ATPase)

– Energy → ion gradients– Ion gradients→ transport molecules or

conduct nerve impulses.

Enzymes alter reaction rate but not the equilibrium

• Reaction will reach equilibrium state faster in the presence of an enzyme

• At equilibrium the conc of products & reactants are the same whether or not an enzyme is present

S → X++ → PTransition state

least stable species of a reactionHas highest Free energy

•Free energy of activation

sXGGG

• Accelerate reaction by reducing the free energy of activation

Activation energy & free energy• Reaction rate proportional to conc of transitional

state

• Reduce energy required to go into transitional state more transitional state can be formed

• Higher the conc of transitional state→ the reaction proceeds faster

• Free energy change dependent on free energy of products and reactants. Free energy of transitional state not counted as free energy released when transition state is converted into product.

][X v

What is the common strategy by which catalysis occurs?

1. increasing the probability of product formation2. shifting the reaction equilibrium 3. stabilization of transition state 4. All of the above. 5. None of the above.

Enzyme-substrate complex

• 1st step in catalyzing a reaction is the formation of the substrate-enzyme complex

• Substrate bound to enzyme at active site• Evidence for this

– Saturation of reaction rate with substrate conc– All active sites taken up– X-ray crystallography of enzyme substrate complexes– Change in spectroscopic characters of enzymes &

substrates in the presence of enzymes

Evidence for enzyme-substrate complexX-ray crystallography of enzyme substrate

complexes

Camphor bound in the active site of enzyme cytochrome P450

Change in spectroscopic characters of enzymes & substrates in the presence of

enzymes• Bacterial tryptophan

synthetase– Serine + indole →

tryptophan• Fluorescence

increases with serine bound

• Then decreases with addition of indole which is bound

Common features of active sites• Lowers ∆G of the transition state

• 3D cleft that involves amino acids from different parts of the sequence– Eg. Lysozyme

Important amino acids in the active site

• Active site is a small part of the total protein– Most of amino

acids are not involved in catalysis

– Most serve as scaffolding to create the active site

– Some amino acids involved in regulation of enzyme activity

• Active sites are unique micro-environments– Water usually excluded– Non polar site enhances binding & catalysis– Polar acids in active may take on catalytic

roles

• Substrates bind to enzyme through multiple weak interactions– Change in free energy with

binding typically• -13 to -50 kJ mol-1

– H bonds, van der Waals interactions, hydrophobic interactions

– Interactions are specific hence enzyme & substrate have complementary shapes

• Binding specificity of the enzymes depends on the 3D arrangement of the amino acids– Lock & key model of

enzyme– Induced fit, enzyme

can change shape to accommodate substrate during binding

– Complementary shapes only after binding

Binding energy between enzyme & substrate

• Free energy released when substrate binds to enzyme

• Only correct substrate can interact with the enzyme active site

• Enzyme active site is complementary to the transition state

• Transition state is unstable and collapses to product or substrate depending on the free energy change