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ENZYMES
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Overview: The Energy of Life
• The living cell is a miniature chemicalfactory where thousands of reactions occur • The cell extracts energy and applies energy
to perform work• Some organisms even convert energy to
light, as in bioluminescence
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An organism’s metabolismtransforms matter and energy,
subject to the laws ofthermodynamics
• Metabolism is the totality of an organism’schemical reactions
• Metabolism is an emergent property of lifethat arises from interactions betweenmolecules within the cell
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Organization of the Chemistry ofLife into Metabolic Pathways
• A metabolic pathway begins with a specificmolecule and ends with a product
• Each step is catalyzed by a specific enzyme
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Enzyme 1 Enzyme 2 Enzyme 3
DCBA Reaction 1 Reaction 3Reaction 2Startingmolecule
Product
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• Anabolic pathways consume energy tobuild complex molecules from simpler ones• The synthesis of protein from amino acids is
an example of anabolism• Bioenergetics is the study of how
organisms manage their energy resources
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Forms of Energy
• Energy is the capacity to cause change• Energy exists in various forms, some of
which can perform work
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• Kinetic energy is energy associated with motion• Heat (thermal energy) is kinetic energy
associated with random movement of atoms ormolecules
• Potential energy is energy that matter possessesbecause of its location or structure
• Chemical energy is potential energy available for
release in a chemical reaction• Energy can be converted from one form to another
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Climbing up converts the kineticenergy of muscle movement
to potential energy.
A diver has less potentialenergy in the water than on the platform.
Diving convertspotential energy tokinetic energy.
A diver has more potentialenergy on the platformthan in the water.
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The Laws of EnergyTransformation
• Thermodynamics is the study of energytransformations• A closed system, such as that approximated
by liquid in a thermos, is isolated from itssurroundings• In an open system, energy and matter can
be transferred between the system and itssurroundings
• Organisms are open systems
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The First Law ofThermodynamics
• According to the first law ofthermodynamics , the energy of theuniverse is constant: – Energy can be transferred and transformed,
but it cannot be created or destroyed
• The first law is also called the principle ofconservation of energy
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(a) First law of thermodynamics (b) Second law of thermodynamics
Chemicalenergy
Heat CO 2
H2O
+
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Biological Order and Disorder
• Cells create ordered structures from lessordered materials• Organisms also replace ordered forms of
matter and energy with less ordered forms• Energy flows into an ecosystem in the form
of light and exits in the form of heat
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• The evolution of more complex organismsdoes not violate the second law ofthermodynamics
• Entropy (disorder) may decrease in anorganism, but the universe’s total entropyincreases
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The free-energy change of areaction tells us whether or not
the reaction occursspontaneously
• Biologists want to know which reactionsoccur spontaneously and which requireinput of energy
• To do so, they need to determine energychanges that occur in chemical reactions
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Free Energy and Metabolism
• The concept of free energy can be appliedto the chemistry of life’s processes
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Exergonic and EndergonicReactions in Metabolism
• An exergonic reaction proceeds with a netrelease of free energy and is spontaneous• An endergonic reaction absorbs free
energy from its surroundings and isnonspontaneous
Fig 8 6
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Fig. 8-6
Reactants
Energy
F r e e e n e r g y
Products
Amount of energy
released(∆G < 0)
Progress of the reaction
(a) Exergonic reaction: energy released
Products
ReactantsEnergy
F r e e e n
e r g y
Amount of energyrequired(∆G > 0)
(b) Ender gonic reaction: energy req uired
Progress of the reaction
Fig 8 6a
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Fig. 8-6a
Energy
(a) Exergonic reaction: energy released
Progress of the reaction
F r e e e n e r g y
Products
Amount of energyreleased(∆G < 0)
Reactants
Fig 8 6b
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Fig. 8-6b
Energy
(b) Endergonic reaction: energy required
Progress of the reaction
F r e e e n e r g y
Products
Amount of energyrequired
(∆G > 0)Reactants
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Equilibrium and Metabolism
• Reactions in a closed system eventually reachequilibrium and then do no work
• Cells are not in equilibrium; they are open systemsexperiencing a constant flow of materials
• A defining feature of life is that metabolism isnever at equilibrium
• A catabolic pathway in a cell releases free energy
in a series of reactions
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ATP powers cellular work by couplingexergonic reactions to endergonic reactions
• A cell does three main kinds of work: – Chemical – Transport – Mechanical
• To do work, cells manage energy resources byenergy coupling , the use of an exergonic process
to drive an endergonic one• Most energy coupling in cells is mediated by ATP
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The Structure and Hydrolysis of ATP
• ATP (adenosine triphosphate) is the cell’senergy shuttle• ATP is composed of ribose (a sugar),
adenine (a nitrogenous base), and threephosphate groups
Fig 8-8
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Fig. 8-8
Phosphate groupsRibose
Adenine
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• The bonds between the phosphate groupsof ATP’s tail can be broken by hydrolysis• Energy is released from ATP when the
terminal phosphate bond is broken• This release of energy comes from thechemical change to a state of lower freeenergy, not from the phosphate bondsthemselves
Fig 8-9
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Fig. 8 9
Inorganic phosphate
Energy
Adenosine triphosphate (ATP)
Adenosine diphosphate (ADP)
P P
PP P
P ++
H2O
i
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• ATP drives endergonic reactions byphosphorylation, transferring a phosphategroup to some other molecule, such as areactant
• The recipient molecule is nowphosphorylated
Fig. 8-11 M b i
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g. 8
(b) Mechanical work: ATP binds noncovalently
to mot or proteins, then is hydrolyz ed
Membrane protein
Pi
ADP
+
P
Solute Solute transported
P i
Vesicle Cytoskeletal track
Motor protein Protein moved
(a) Transport work: ATP phosphorylatestransport proteins
ATP
ATP
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g
P iADP +
Energy fromcatabolism (exergonic,energy-releasing
processes)
Energy for cellular work (endergonic,energy-consuming
processes)
ATP + H2O
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nzym s
1. Enzymes speed up metabolic reactions by lowering energy barriers2. Enzymes are substrate specific3. The active site in an enzyme’s catalytic center 4. A cell’s physical and chemical environment affects enzyme activity
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• A catalyst is a chemical agent that changesthe rate of a reaction without being consumedby the reaction.
– An enzyme is a catalytic protein.
• Enzyme: An organic catalyst (usually protein) that accelerate a specificchemical reaction by lowering the activation energy required for that
reaction.
1. Enzymes speed up metabolic reactionsby lowering energy barriers
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• Anabolism: Aspect of metabolism
simpler substances are combined to form complex substancesfor storage of energyfor the production of new cellular materials and growth.
• Catabolism: Aspect of metabolismcomplex substances are broken down to simpler substancereleasing chemical energy.• Metabolism = Catabolism + Anabolism
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• Exergonic reaction:release of energy.
• Endergonic reaction Absorb energy.
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• Chemical reactions between moleculesinvolve both bond breaking and bond
forming. – To hydrolyze sucrose, the bond between
glucose and fructose must be broken and thennew bonds formed with a hydrogen ion andhydroxyl group from water.
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• Activation energy is the amount of energynecessary to push the reactants over an
energy barrier. – At the summit themolecules are atan unstable point,the transition state.
– The difference betweenfree energy of theproducts and the freeenergy of the reactantsis the delta G.
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• For some processes, the barrier is not highand the thermal energy provided by room
temperature is sufficient to reach thetransition state.• In most cases, E A is higher and a significant
input of energy is required. – A spark plug provides the energy to energize
gasoline. – Without activation energy, the hydrocarbons of
gasoline are too stable to react with oxygen.
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• The laws of thermodynamics would seemto favor the breakdown of proteins, DNA,
and other complex molecules. – However, in the temperatures typical of the cellthere is not enough energy for a vast majorityof molecules to make it over the hump ofactivation energy.
– Yet, a cell must be metabolically active. – Heat would speed reactions, but it would also
denature proteins and kill cells. – Denature: To alter the physical properties and three dimensionalstructure of protein, usually by treating it with excess heat, strongacid or strong base.
– Reactant: substrate that participate in a chemical reaction.
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• Enzyme speed reactions by lowering E A. – The transition state can then be reached even
at moderate temperatures.• Enzymes do not change delta G.
– Because enzymesare so selective,they determinewhich chemicalprocesses will
occur at any time.
Fig. 8
-14
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Progress of the reaction
Products
Reactants
∆G < O
Transition state
EA
DC
BA
D
D
C
C
B
B
A
A
Th A i i E B i
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The Activation Energy Barrier
• Every chemical reaction between moleculesinvolves bond breaking and bond forming• The initial energy needed to start a chemical
reaction is called the free energy ofactivation , or activation energy (E A)
• Activation energy is often supplied in theform of heat from the surroundings
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H E L h E
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How Enzymes Lower the E ABarrier
• Enzymes catalyze reactions by lowering theE A barrier • Enzymes do not affect the change in free
energy (∆ G); instead, they hasten reactionsthat would occur eventually
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Progress of the reaction
Products
Reactants
∆G is unaffectedby enzyme
Course of reaction
withoutenzymeE
Awithoutenzyme EA with
enzymeis lower
Course of reactionwith enzyme
2 E b ifi
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• A substrate is a reactant which binds to an
enzyme.• When a substrate or substrates binds to an
enzyme, the enzyme catalyzes the
conversion of the substrate to the product. – Sucrase is an enzyme that binds to sucrose and
breaks the disaccharide into fructose andglucose.
2. Enzymes are substrate specific
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• The shape of active site matches the shape of a specific part ofthe substrate molecule.
• Induced Fit – Substrate molecule upon binding to the active site of the enzyme
induces a slight change in the shape of both molecule, substrateand enzyme.
– Once substrate molecule has made contact with the active site and
has initiated the change in shape, the enzyme initiates the actualreaction of the substrate molecule.
– Once the reaction complete, attraction between substrate andenzyme
cease, freeing the enzymes from the product enabling to catalyzedthesecond reaction of the same type.
Th i i f i
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• The active site of an enzymes is a grooveon the surface of the protein into which thesubstrate fits.
• The specificity of an enzyme is due to thefit between the active site and thesubstrate.
• As the substrate binds, the enzymechanges shape leading to a tighterinduced fit , bringing chemical groups inposition to catalyze the reaction.
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• The reason a particular enzyme will combine onlywith one kind of substrate is that the fit between thetwo is very precise .
• In fact, the fit is so precise, the combining of theenzyme and substrate results in a slight change inthe shape of both. This precise fit that modifies bothoriginal molecules is called an induced fit .
• This "strained" fit acts to break old chemical bondsand form new ones, resulting in the formation of theproduct from the substrate.
• Once this change has occurred, the product isreleased from the enzyme, and the enzyme cancombine with another molecule of substrate.
3 Th i i i
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• In most cases substrates are held in theactive site by weak interactions, such ashydrogen bonds and ionic bonds.
– R groups of amino acids on the active sitecatalyze the conversion of substrate to product.
3. The active site is anenzyme’s catalytic center
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C t l sis in the En me’s Acti e
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Catalysis in the Enzyme’s ActiveSite
• In an enzymatic reaction, the substratebinds to the active site of the enzyme• The active site can lower an E A barrier by
– Orienting substrates correctly – Straining substrate bonds – Providing a favorable microenvironment – Covalently bonding to the substrate
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Enzymes speed up metabolic reactions by
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Enzymes speed up metabolic reactions bylowering energy barriers
• A catalyst is a chemical agent that speedsup a reaction without being consumed bythe reaction
• An enzyme is a catalytic protein• Hydrolysis of sucrose by the enzyme
sucrase is an example of an enzyme-catalyzed reaction
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Fig. 8-16
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Substrate
Active site
Enzyme Enzyme-substratecomplex
(b)(a)
A i l l l t l
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• A single enzyme molecule can catalyzethousands or more reactions a second.
• Enzymes are unaffected by the reactionand are reusable.• Most metabolic enzymes can catalyze a
reaction in both the forward and reversedirection. – The actual direction depends on the relative
concentrations of products and reactants. – Enzymes catalyze reactions in the direction of
equilibrium.
E i t f h i t
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• Enzymes use a variety of mechanisms tolower activation energy and speed a
reaction. – As the active site binds the substrate, itmay put stress on bonds that must bebroken, making it easier to reach the
transition state. – R groups at the active site may create a conducive
microenvironment for a specific reaction.
– The active site orients substrates in the
correct orientation for the reaction.
• The rate that a specific number of enzymes
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• The rate that a specific number of enzymesconverts substrates to products depends inpart on substrate concentrations.
• At low substrate concentrations, an increasein substrate speeds binding to availableactive sites.
• However, there is a limit to how fast areaction can occur.
• At some substrate concentrations, the active
sites on all enzymes are engaged, calledenzyme saturation.• The only way to increase productivity at this
point is to add more enzyme molecules.
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• Michaelis-Menten
• E + S --- k1k2 ES--- k3 E + P
S reacts with E, and turns into ES, with rate constantk1, which turns into P and regenerates E, with rateconstant k 3. Now, ES can turn back into S and E,
with rate constant k 2. The Michaelis-Menten
mechanism for enzymic catalysis follows this path.
• E = free enzymes, S = substrate, ES = enzymes-substrate complex, P = product, e = total enzymeconcentration.
• K1 = rate constant for formation ES
• K2 = rate constant for dissociation of ES to E and S.
• K3 = rate constant for dissociation of ES to E and P
• Effect of enzyme concentration on reaction velocity
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• Effect of enzyme concentration on reaction velocity – If the substrate concentration constant, velocity of reaction is proportional to
the enzyme concentration. – Enzyme concentration is propotional to velocity of reaction
– Vmax
Reaction velocity(V)
½ V max
Substrate concentration at ½ Vmax
[S]Km
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P iADP +
Energy fromcatabolism (exergonic,energy-releasing
processes)
Energy for cellular work (endergonic,energy-consuming
processes)
ATP + H2O
Fig. 8-13
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Sucrose (C 12 H22 O 11 )
Glucose (C 6H12 O 6) Fructo se (C 6H12 O 6)
Sucrase
Substrate Specificity of Enzymes
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Substrate Specificity of Enzymes
• The reactant that an enzyme acts on iscalled the enzyme’s substrate
• The enzyme binds to its substrate, formingan enzyme-substrate complex
• The active site is the region on the enzymewhere the substrate binds
• Induced fit of a substrate brings chemicalgroups of the active site into positions thatenhance their ability to catalyze the reaction
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Fig. 8-17
Substrates enter active site; enzymechanges shape such that its active site
21
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Substrates
Enzyme
Products arereleased.
Products
Substrates areconverted toproducts.
Active site can lower E Aand speed up a reaction.
Substrates held inactive site by weakinteractions, such ashydrogen bonds andionic bonds.
changes shape such that its active siteenfolds the substrates (induced fit).
Activesite is
availablefor two new
substratemolecules.
Enzyme-substratecomplex
5
3
2
6
4
Effects of Local Conditions on
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Effects of Local Conditions onEnzyme Activity
• An enzyme’s activity can be affected by – General environmental factors, such as
temperature and pH – Chemicals that specifically influence the
enzyme
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Effects of Temperature and pH
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Effects of Temperature and pH
• Each enzyme has an optimal temperature inwhich it can function
• Each enzyme has an optimal pH in which itcan function
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Optimal temperature for f h hili
Optimal temperature for i l h
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R a
t e o f r e a c
t i o n
enzyme of thermophilic(heat-tolerant)bacteria
typical human enzyme
(a) Optimal temperature for two enzymes
(b) Optimal pH fo r two enzymes
R a
t e o
f r e a c
t i o n
Optimal pH for pepsin(stomach enzyme)
Optimal pHfor trypsin(intestinalenzyme)
Temperature (ºC)
pH543210 6 7 8 9 10
0 20 40 8060 100
Cofactors
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Cofactors
• Cofactors are nonprotein enzyme helpers• Cofactors may be inorganic (such as a
metal in ionic form) or organic
• An organic cofactor is called a coenzyme• Coenzymes include vitamins
Enzyme Inhibitors
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Enzyme Inhibitors
• Competitive inhibitors bind to the activesite of an enzyme, competing with thesubstrate
• Noncompetitive inhibitors bind to anotherpart of an enzyme, causing the enzyme tochange shape and making the active siteless effective
• Examples of inhibitors include toxins,poisons, pesticides, and antibiotics
Fig. 8-19
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(a) Normal binding (c) Noncompetitive inhibition(b) Competitive inhibition
Noncompetitive inhibitor
Active siteCompetitive
inhibitor
Substrate
Enzyme
• inhibitors , prevent enzymes from catalyzing
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inhibitors , prevent enzymes from catalyzingreactions. – If binding involves covalent bonds, then
inhibition is often irreversible. – If binding is weak, inhibition may be reversible.
• If the inhibitor binds to the same site as the
substrate, then it blocks substrate bindingvia competitive inhibition .
• If the inhibitor binds somewhere other than the active site,
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it blocks substrate binding via noncompetitive inhibition .• Binding by the inhibitor causes the enzyme to change
shape, rendering the active site unreceptive or lesseffective at catalyzing the reaction.
• Reversible inhibition of enzymes is a natural part of theregulation of metabolism. – Reversible inhibitor: A substance that forms a weak bonds with an
enzyme, temporarily interfering with its function. A reversibleinhibitor can be competitive or non competitive.
– Irreversible inhibitor: A substance that permanently inactivates anenzyme.
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• Competitive inhibition reversible – Example: succinate dehydrogenase inhibit
by malonate and oxaloacetate. It isactivated by ATP, P i and succinate.
• Succinate + FAD -- Fumarate + FADH 2
Irreversible inhibitors:These molecules bind permanently with the enzyme molecule andso effectively reduce the enzyme concentration, thus limiting therate of reaction, fo r examp le , cyanide irreversibly inhibits theenzyme cytochrome oxidase found in the electron transport chain
used in respiration. If this enzyme cannot be used, death willoccur.
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Regulation of enzyme activityhelps control metabolism
• Chemical chaos would result if a cell’smetabolic pathways were not tightlyregulated
• A cell does this by switching on or off thegenes that encode specific enzymes or byregulating the activity of enzymes
Allosteric Regulation of Enzymes
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Allosteric Regulation of Enzymes
• Allosteric regulation may either inhibit orstimulate an enzyme’s activity
• Allosteric regulation occurs when aregulatory molecule binds to a protein at onesite and affects the protein’s function atanother site
Allosteric Activation and Inhibition
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Allosteric Activation and Inhibition
• Most allosterically regulated enzymes aremade from polypeptide subunits
• Each enzyme has active and inactive forms• The binding of an activator stabilizes the
active form of the enzyme• The binding of an inhibitor stabilizes the
inactive form of the enzyme
Fig. 8-20 Allosteric enyzmewith four subunits
Active site(one of four)
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Regulatorysite (oneof four)
Active formActivator
Stabilized active form
Oscillation
Non-functionalactivesite
Inhibitor Inactive form Stabilized inactive
form
(a) Allosteric activators and inhibitors
Substrate
Inactive form Stabilized activeform
(b) Coope rativity: another type of allosteric activation
Fig. 8-20a Allosteric enzymewith four subunits
Active site(one of four)
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Free Powerpoint Templates Page 80(a) Allosteric a ctivators and inhibitors
Inhibitor Non-functionalactivesite
Stabilized inactiveform
Inactive form
Oscillation
Activator Active form Stabilized active form
Regulatorysite (oneof four)
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• Cooperativity is a form of allostericregulation that can amplify enzyme activity
• In cooperativity, binding by a substrate toone active site stabilizes favorableconformational changes at all other subunits
Fig. 8-20b
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(b) Cooperativity: another type of allosteric activation
Stabilized activeform
Substrate
Inactive form
• In enzymes with multiple catalytic subunits,
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y p ybinding by a substrate to one active site stabilizesfavorable conformational changes at all other
subunits, a process called cooperativity .• This mechanism amplifies the response of
enzymes to substrates, priming the enzyme toaccept additional substrates
Identification of Allosteric
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de t cat o o oste cRegulators
• Allosteric regulators are attractive drug
candidates for enzyme regulation
• Some allosteric regulators, activators,
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g , ,stabilize the conformation that has afunctional active site.
• Other regulators, inhibitors, stabilize theconformation that lacks an active site.
Metabolic control often depends on
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• In many cases, the molecules that naturallyregulate enzyme activity behave likereversible noncompetitive inhibitors.
• These molecules often bind weakly to aallosteric site , a specific receptor on theenzyme that is not the active site.
• Binding by these molecules can either inhibitor stimulate enzyme activity.
allosteric regulation
• Most allosterically regulated enzymes are
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y g yconstructed of two or more polypeptidechains.
• Each subunit has its own active site andallosteric sites are often located wheresubunits join.
• The whole protein oscillates between twoconformational shapes, one active, oneinactive.
• As the chemical conditions in the cell shift,
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,the pattern of allosteric regulation will shift aswell.
• In many cases both inhibitors and activatorsare similar enough in shape that theycompete for the same allosteric sites. – These molecules may be products and
substrates of a metabolic pathway. – For example, some catabolic pathways have
allosteric sites that are inhibited when ATPbinds and activated when AMP binds. – When ATP levels are low, AMP levels are
high, and the pathway is turned on until ATP
levels rise, A MP levels fall and inhibition by
Fig. 8-21 EXPERIMENT
Caspase 1 Active Substrate
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RESULTS
Caspase ct vesite
SHKnown active form
Subst ate
SHActive form canbind substrate
SH Allostericbinding site
Known inactive formAllostericinhibitor Hypothesis: allosteric
inhibitor locks enzymein inactive form
S – S
Caspase 1
Active for m Allostericallyinhibited form
Inhibitor Inactive form
Fig. 8-
21aEXPERIMENT
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SH
Substrate
Hypothesis: allostericinhibitor locks enzymein ina ctive form
Active form canbind substrate
S – SSH
SH
Activesite
Caspase 1
Known active form
Known inactive form
Allostericbinding site
Allostericinhibitor
Fig. 8-21b
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Caspase 1
RESULTS
Active form
Inhibitor
Allosterically
inhibited form
Inactive form
Specific Localization of Enzymes
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p yWithin the Cell
• Structures within the cell help bring order tometabolic pathways
• Some enzymes act as structuralcomponents of membranes
• In eukaryotic cells, some enzymes reside inspecific organelles; for example, enzymesfor cellular respiration are located inmitochondria
Copyright © 2008 Pearson Education, Inc., publishing as Pearson BenjaminCummings
Fig. 8-23
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Mitochondria
C f
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Progress of the reaction
Products
Reactants
∆G is unaffectedby enzyme
Course of reactionwithout
enzyme
EAwithout
enzyme EA withenzymeis lower
Course of reactionwith enzyme
A cell’s physical and chemical environment
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• The three-dimensional structures of enzymes(almost all proteins) depend onenvironmental conditions.
• Changes in shape influence the reactionrate.
• Some conditions lead to the most active
conformation and lead to optimal rate ofreaction.
affects enzyme activity
• 1. Temperature has a major impact on
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reaction rate. – As temperature increases, collisions between
substrates and active sites occur morefrequently as molecules move faster. – However, at some point thermal agitation begins
to disrupt the weak bonds that stabilize theprotein’s active conformation and the proteindenatures.
– Each enzyme has an optimal temperature.
• 2. Because pH also influences shape and
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therefore reaction rate, each enzyme hasan optimal pH too.
• This falls between pH 6 - 8 for mostenzymes.
• However, digestive enzymes in thestomach are designed to work best at pH 2while those in the intestine are optimal atpH 8, both matching their workingenvironments.
Fig. 8-UN4
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• 3 Amount of enzyme present
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• 3. Amount of enzyme present – The amount of enzyme present is
determined by its rate of synthesis (k s) andthe rate of degradation (k d). Enzymeturnover.
• Many enzymes require nonprotein helpers
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Many enzymes require nonprotein helpers,cofactors , for catalytic activity.
– They bind permanently to the enzyme orreversibly.
– Some inorganic cofactors include zinc, iron,and copper.
• Organic cofactors, coenzymes , includevitamins or molecules derived fromvitamins.
Feedback Inhibition
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• In feedback inhibition , the end product of ametabolic pathway shuts down the pathway
• Feedback inhibition prevents a cell fromwasting chemical resources by synthesizingmore product than is needed
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• One of the commonmethods of metabolic
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methods of metaboliccontrol is feedbackinhibitionmetabolic pathway isturned off by its endproduct.
• The end product actsas an inhibitor of anenzyme in the pathway.
• When the product is
abundant the pathwayis turned off, when rarethe pathway is active.
• Systematic names for each enzyme
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have been made in an internationalclaasification of enzymes. – Oxidoreductase: are involved in oxidation/reduction. – Transferase : transfer functional groups between donor and
receptor. – Hydrolases – transfer water that is they catalysed the hydrolysis of
a substrate. – Lyases : add or remove the element of water, ammonia or CO 2 to
form double bond. – Isomerase : catalyzed changes within one molecule. – Ligase (synthetases): join two molecule together at the expense
of a high-energy phosphate bond of ATP.