HOW ENZYMES WORK

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HOW ENZYMES WORK
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ENZYMES SPEED UP CHEMICAL REACTIONS
Enzymes are biological catalysts substances
that speed a reaction without being altered in
the reaction.
Most enzymes are proteins.
Enzymes are essential for life.
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• Enzymes
• ?? Cofactors
• ?? Coenzymes
• ?? Holoenzyme
• ?? Apoenzyme

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How Enzymes Work?

• Body conditions(temperature, pressure etc.) not
  good for reaction
• Only enzymes can catalyse the reactions
• in this conditions
• A special environment inside enzymes for
• reaction ACTIVE SITE
• Molecule binds active site SUBSTRATE

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Enzymes Lower a Reactions Activation Energy
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Each reaction has a transition state where
thesubstrate is in an unstable,
short-livedchemical/structural state.
Free Energy of Activation is symbolized by ?G.
Enzymes act by lowering the free energy of the
transition state
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Enzymes speed up metabolicreactions by lowering
energy barriers

• Enzyme speed reactions by lowering EA.
• The transition state
• can be reached at
• moderate temperatures.
• Enzymes do not change
• delta G.
• It speed-up reactions
• that would occur eventually.
• Because enzymes are
• so selective, they determine which
• chemical processes will occur
• at any time

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• Enzymes lower the free energy of activation by
  binding the transition state of the reaction
  better than the substrate
• The enzyme must bind the substrate in the
  correct orientation otherwise there would be no
  reaction
• Not a lock key but induced fit the enzyme
  and/or the substrate distort towards the
  transition state

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Induced Fit

• A change in the shape of an enzymes active site
• Induced by the substrate

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Lock and Key Model

• An enzyme binds a substrate in a region called
  the active site
• Only certain substrates can fit the active site
• Amino acid R groups in the active site help
  substrate bind
• Enzyme-substrate complex forms
• Substrate reacts to form product
• Product is released

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Enzyme Kinetics

• - Kinetics The study of the rate of
  change.
• - Enzyme Kinetics Rate of chemical
• reactions mediated by enzymes. Enzymes can
  increase reaction rate by favoring or
• enabling a different reaction pathway with
• a lower activation energy, making it easier
• for the reaction to occur.

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Michaelis-Menten kinetics
Vmax approached asymptotically
V0 varies with S
V0 is moles of product formed per sec. when
P is low (close to zero time)
E S?ES?E P
Michaelis-Menten Model
V0 Vmax xS/(S Km)
Michaelis-Menten Equation
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Determining initial velocity (when P is low)
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Steady-state pre-steady-state conditions
At pre-steady-state, P is low (close to zero
time), hence, V0 for initial reaction velocity
At equilibrium, no net change of S P or of
ES E
At pre-steady state, we can ignore the back
reactions
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Michaelis-Menten kinetics (summary)

• Enzyme kinetics (Michaelis-Menten Graph)
• At fixed concentration of enzyme, V0 is
  almost linearly proportional to S when S is
  small, but is nearly independent of S when S
  is large

Proposed Model E S ? ES ? E P
ES complex is a necessary intermediate
Objective find an expression that relates rate
of catalysis to the concentrations of S E, and
the rates of individual steps
Start with V0 k2ES, and derive, V0 Vmax
xS/(S Km) This equation accounts for graph
data. At low S (S lt Km), V0 (Vmax/Km)S At
high S (S gt Km), V0 Vmax When S Km, V0
Vmax/2. Thus, Km substrate
concentration at which the reaction rate (V0) is
half max.
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Range of Km values
Km provides approximation of S in vivo for many
enzymes
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Lineweaver-Burk plot (double-reciprocal)
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Eadie-Hofstee plot
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Hanes-Woolf Plot
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Allosteric Enzymes

• Allosteric enzymes have one or more allosteric
  sites
• Allosteric sites are binding sites distinct
  from an enzymes active site or substrate-binding
  site
• Molecules that bind to allosteric sites are
  called effectors or modulators
• Binding to allosteric sites alters the activity
  of the enzyme. This is called cooperative
  binding. Allosteric enzymes display sigmoidal
  plot of Vo vs S
• Effectors may be positive or negative
• Effectors may be homotropic or heterotropic
• Regulatory enzymes of metabolic pathways are
  allosteric enzymes (eg feedback inhibition)

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Allosteric enzymes

• Allosteric enzymes tend to be
• multi-sub unit proteins
• The reversible binding of an
• allosteric modulator (here a
• positive modulator M) affects
• the substrate binding site

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Mechanism and Example of Allosteric Effect
Allosteric site
R Relax (active)
Homotropic () Concerted
Allosteric site
A
Heterotropic () Sequential
X
Heterotropic (-) Concerted
T Tense (inactive)
I
X
X
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Enzyme Inhibitors

• Specific enzyme inhibitors regulate enzyme
  activity and help us understand mechanism of
  enzyme action. (Denaturing agents are not
  inhibitors)
• Irreversible inhibitors form covalent or very
  tight permanent bonds with aa at the active site
  of the enzyme and render it inactive. 3 classes
  groupspecific reagents, substrate analogs,
  suicide inhibitors
• Reversible inhibitors form an EI complex that
  can be dissociated back to enzyme and free
  inhibitor. 3 groups based on their mechanism of
  action competitive, non-competitive and
  uncompetitive.

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Enzyme Inhibition
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Competitive inhibitors

• Compete with substrate for binding to enzyme
• E S ES or E I EI . Both S and I cannot
  bind enzyme at the same time
• In presence of I, the equilibrium of E S ES
  is shifted to the left causing dissociation of
  ES.
• This can be reversed / corrected by increasing
  S
• Vmax is not changed, KM is increased by (1
  I/Ki)
• Eg AZT, antibacterial sulfonamides, the
  anticancer agent methotrexate etc

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Competitive Inhibition
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Kinetics of competitive inhibitor
Increase S to overcome inhibition Vmax
attainable, Km is increased
Ki dissociation constant for inhibitor
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V max unaltered, Km increased
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Non-competitive Inhibitors

• Inhibitor binding site is distinct from
  substrate binding site. Can bind to free enzyme E
  and to ES
• E I EI, ES I ESI or EI S ESI
• Both EI and ESI are enzymatically inactive
• The effective functional E (and S) is
  reduced
• Reaction of unaffected ES proceeds normally
• Inhibition cannot be reversed by increasing S
• KM is not changed, Vmax is decreased by (1
  I/Ki)

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Mixed (Noncompetitive) Inhibition
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Kinetics of non-competitive inhibitor
Increasing S cannot overcome inhibition Less E
available, V max is lower, Km remains the
same for available E
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Km unaltered, V max decreased
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Uncompetitive Inhibitors

• The inhibitor cannot bind to the enzyme
  directly, but can only bind to the
  enzyme-substrate complex.
• ES I ESI
• Both Vmax and KM are decreased by (1I/Ki).

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Uncompetitive Inhibition
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Substrate Inhibition

• Caused by high substrate concentrations

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Substrate Inhibition

• At low substrate concentrations S2/Ks1ltlt1 and
  inhibition is not observed
• Plot of 1/v vs. 1/S gives a line
• Slope Km/Vm
• Intercept 1/Vm

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Substrate Inhibition

• At high substrate concentrations, Km/Sltlt1, and
  inhibition is dominant
• Plot of 1/v vs. S gives a straight line
• Slope 1/KS1 Vm
• Intercept 1/Vm

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Competitive
Uncompetitive
Substrate Inhibition
Non-Competitive
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Enzyme Inhibition (Mechanism)
Non-competitive
Uncompetitive
Competitive
E
Substrate
E
X
Cartoon Guide
Compete for active site
Inhibitor
Different site
Equation and Description
I binds to free E only, and competes with
S increasing S overcomes Inhibition by I.
I binds to ES complex only, increasing S
favors the inhibition by I.
I binds to free E or ES complex
Increasing S can not overcome I inhibition.
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Enzyme Inhibition (Plots)
Vmax
vo
I
I
Km
Km
S, mM
Km
Vmax unchanged Km increased
Vmax decreased Km unchanged
Both Vmax Km decreased
I
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• Factors Affecting Enzyme Kinetics

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Effects of pH

• - on enzymes
• - enzymes have ionic groups on their active
  sites.
• - Variation of pH changes the ionic form of the
  active sites.
• - pH changes the three-Dimensional structure of
  enzymes.
• - on substrate
• - some substrates contain ionic groups
• - pH affects the ionic form of substrate
• affects the affinity of the substrate to the
  enzyme.

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Effects of Temperature

• Reaction rate increases with temperature up to a
  limit
• Above a certain temperature, activity decreases
  with temperature due to denaturation
• Denaturation is much faster than activation
• Rate varies according to the Arrhenius equation

Where Ea is the activation energy (kcal/mol) E
is active enzyme concentration
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Factors Affecting Enzyme Kinetics

• Temperature
• - on the rate of enzyme catalyzed reaction
• k2Aexp(-Ea/RT)
• T k2
• - enzyme denaturation
• T

Denaturation rate
kdAdexp(-Ea/RT)
kd enzyme denaturation rate constant Ea
deactivation energy
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REFERENCES

• Michael L. Shuler and Fikret Kargi, Bioprocess
  Engineering Basic Concepts (2 nd
  Edition),Prentice Hall, New York, 2002.
• 1. James E. Bailey and David F. Ollis,
  Biochemical Engineering Fundementals (2 nd
  Edition), McGraw-Hill, New York, 1986.
• www.biochem.umass.edu/courses/420/lectures/Ch08B.p
  pt -

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• class.fst.ohio-state.edu/fst605/605p/Enzymes.pdf
  
• www.horton.ednet.ns.ca/staff/selig/powerpoints/bio
  12/biochem/enzymes.pdf
• www.siu.edu/departments/biochem/som_pbl/SSB/powerp
  oint/enzymes.ppt
• www.associazioneasia.it/adon/files/2005_luisi_05_w
  hy_are_enzymes.pdf
• www.fatih.edu.tr/abasiyanik/Chapter6_enzymes.pdf
  -

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• http//www.authorstream.com/presentation/kkozar-14
  001-enzymes-enzyme-ppt-education-powerpoint/
• http//highered.mcgraw-hill.com/sites/0072495855/s
  tudent_view0/chapter2/animation__how_enzymes_work.
  html
• http//www.wiley.com/college/pratt/0471393878/stud
  ent/animations/enzyme_kinetics/index.html