3/9/2011

Lecture 5

Enzymes and the nature of enzyme catalysis

BIOSCI 101: Cellular and Molecular Biology

Enzymes and catalysis

Cells are chemical factories - anabolic reactions - large molecules synthesised from small ones - catabolic reactions - break down of large molecules yielding energy and raw materials

BIOSCI 101: Cellular and Molecular Biology

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Energy-yielding processes are coupled with energy-consuming ones

Campbell 8.11

BIOSCI 101: Cellular and Molecular Biology

ATP is an energy-rich compound

Campbell 8.8b

BIOSCI 101: Cellular and Molecular Biology

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ATP drives cellular processes

Transport protein Solute

ATP P Pi

ADP

Pi

Solute transported
Vesicle Cytoskeletal track

ATP

ATP

ADP

Pi

Motor protein

Protein and vesicle moved

Campbell 8.10

BIOSCI 101: Cellular and Molecular Biology

Enzymes and catalysis (contd)

Activation energy in chemical reactions - most chemical reactions require energy to get started (need energy to break chemical bonds) -activation energy is the energy which must be put into the system in order for a reaction to commence ie. the energy molecules need to react with each other

BIOSCI 101: Cellular and Molecular Biology

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An energy profile of a chemical reaction

A C B D

Transition state

Free energy

A C

B D

EA

Reactants

A C

B
G O

Products Progress of the reaction

Campbell 8.12

BIOSCI 101: Cellular and Molecular Biology

Enzymes and catalysis (contd)

Enzymes as catalysts - a catalyst is a substance that increases the rate of a reaction by lowering the activation energy required by the interacting molecules - catalysts are recovered after the reaction unchanged and may be used again - cells ll use enzymes as catalysts l to speed d up o chemical reactions because at 37 C, most reactions would go very slowly

BIOSCI 101: Cellular and Molecular Biology

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Enzymes lower the activation energy

Course of reaction without enzyme EA without enzyme

EA with enzyme is lower

Free energy F

Reactants Course of reaction with enzyme G is unaffected by enzyme

Products

Progress of the reaction Campbell 8.13

BIOSCI 101: Cellular and Molecular Biology

Enzymes and catalysis (contd)

Almost all enzymes are proteins - have 1, 2, 3, 4 structure - the 3D shape of the enzyme controls its activity

BIOSCI 101: Cellular and Molecular Biology

3/9/2011

Enzymes and catalysis (contd)

Enzymes may contain cofactors - non-protein low molecular weight substances. a) prosthetic groups - organic molecules tightly bound to the protein (eg. haeme) b) coenzymes - small organic molecules, noncovalently bound to protein (eg. many vitamins) c) metal ions -inorganic ions that are essential for enzyme activity eg. Cu2+, Mg2+
BIOSCI 101: Cellular and Molecular Biology

Enzymes and catalysis (contd)

Types of enzymes - names of many enzymes end in -ase and often describe the reaction catalysed eg isomerases catalyse intramolecular rearrangements - some important enzymes in molecular biology: - ribonuclease and deoxyribonuclease hydrolyse RNA and DNA - RNA and d DNA polymerase l synthesise th i RNA and DNA - DNA ligase and DNA gyrase and topoisomerase are involved in DNA replication
BIOSCI 101: Cellular and Molecular Biology

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Enzymes and catalysis (contd)

Types of enzymes (contd) - restriction enzymes hydrolyse phosphodiester bonds in DNA at defined sequences eg. Bam H1 - from Bacillus amyloliquefaciens GGATCC (sticky ends) Eco RV - from Eschericia coli strain R (5th isolate) GATATC (blunt ends) - recognition sequences are palindromic (sequence on top strand is same as reverse on bottom strand)
BIOSCI 101: Cellular and Molecular Biology

DNA sequences contain restriction enzyme sites

BIOSCI 101: Cellular and Molecular Biology

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Enzymes and catalysis (contd)

Enzyme-substrate interactions E + S --------> ES ---------> E + P
Sucrase

Sucrose (C12H22O11)

Glucose (C6H12O6)

BIOSCI 101: Cellular and Molecular Biology

The catalytic cycle of an enzyme

1 Substrates enter active site. 2 Substrates are held in active site by weak interactions.

Substrates Enzyme-substrate complex

6 Active site is available for two new substrate molecules. 3 Active site can lower EA and speed up a reaction.

Enzyme

5 Products are released.

4 Substrates are converted to products.

Products

Campbell 8.15

BIOSCI 101: Cellular and Molecular Biology

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Enzymes and catalysis (contd)

Enzyme-substrate interactions (contd) a) the enzyme and substrate combine at the active site - the bonds holding the enzyme and substrate together are weak (non-covalent) - the interaction is very specific due to the configuration of the active site of the enzyme (involves only a few amino acids) - various models proposed for how this might work (lock and key vs induced fit) - the enzyme exerts catalytic activity by bringing reacting molecules close together - once reaction has occurred, E + P dissociate, E recycled for next time
BIOSCI 101: Cellular and Molecular Biology

Induced fit between an enzyme and its substrate

Campbell 8.14

BIOSCI 101: Cellular and Molecular Biology

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3 amino acids are essential in the active site of chymotrypsin

BIOSCI 101: Cellular and Molecular Biology

Enzymes and catalysis (contd)

Enzyme Enzyme-substrate substrate interactions (contd) b) the union between enzyme and substrate is very brief - enzymes are very efficient. eg catalase catalyses degradation of peroxide at a rate of 5 6 x 106 molecules/min 5.6 l l / i

BIOSCI 101: Cellular and Molecular Biology

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Enzymes and catalysis (contd)

Enzyme Enzyme-substrate substrate interactions (contd) c) factors affecting reaction rates (i) temperature, pH and ionic strength (affect 2 3 and 4 structure) - enzymes have an optimum temperature - each enzyme also has a favoured pH for operation called pH optimum

BIOSCI 101: Cellular and Molecular Biology

Optimal temperature for Optimal temperature for typical human enzyme (37C) enzyme of thermophilic (heat-tolerant) bacteria (77C) Rate of reaction

20

40

60 80 Temperature (C)

100

120

(a) Optimal temperature for two enzymes

Optimal pH for pepsin (stomach enzyme) Rate of reac ction

Optimal pH for trypsin (intestinal enzyme)

Environmental f factors affecting enzymes

5 pH

10

(b) Optimal pH for two enzymes

Campbell 8.16

BIOSCI 101: Cellular and Molecular Biology

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Enzymes and catalysis (contd)

c) factors affecting reaction rates (contd) (ii) enzyme and substrate concentrations - enzyme concentration is directly proportional to reaction rate (the more enzyme the faster the reaction) - substrate concentration can also be critical in cells because the available enzyme can become saturated - different enzymes have differing affinities for their substrates
BIOSCI 101: Cellular and Molecular Biology

Enzyme and substrate concentration affect reaction rates

BIOSCI 101: Cellular and Molecular Biology

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Enzymes and catalysis (contd)

Regulation of enzyme activity a) by activation (conversion from an inactive to an active form) eg. chymotrypsinogen ---> chymotrypsin via action of trypsin in duodenum b) by sequestration (enzymes may be locked into certain organelles inside the cell) eg. lysosomes

BIOSCI 101: Cellular and Molecular Biology

Enzymes and catalysis (contd)

Regulation of enzyme activity (contd) c) allosteric enzymes and feedback inhibition - the function of an allosteric enzyme is affected by the binding of a regulatory molecule - these enzymes can exist in two distinct spatial conformations, one active, the other inactive - many metabolic pathways are regulated by feed-back inhibition of allosteric enzymes d) multienzyme complexes - related enzymes close together, substrates passed directly on from one reaction to the next eg DNA replication enzymes, mitochondria and chloroplasts BIOSCI 101: Cellular and Molecular Biology

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Allosteric regulation

Campbell 8.19a

BIOSCI 101: Cellular and Molecular Biology

Feedback inhibition

Campbell 8.21

BIOSCI 101: Cellular and Molecular Biology

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Test question
Which of the following statements about enzymes is INCORRECT? 1 1. enzymes may be inactivated by disruption of their three-dimensional structure 2. the substrate binds to the enzyme at the active site 3. enzymes increase the rate of chemical reaction by lowering activation energy barriers 4. enzyme function is influenced by temperature but not by pH 5. some enzymes require a cofactor for catalytic activity
BIOSCI 101: Cellular and Molecular Biology

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