ENZYME- BIOLOGICAL CATALYST

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ENZYME-BIOLOGICAL CATALYST
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Enzyme As Catalyst

• All enzymes are proteins - with the exception of
  some RNAs that catalyze their own splicing all
  enzymes are proteins
• In general, names end with suffix ase
• - tyrosinase (tyrosine), celullase (cellulose),
  protease (protein), lipase (lipid)
• Enzyme a biological catalyst
• enzymes can increase the rate of a reaction by a
  factor of up to 1020 over an uncatalyzed reaction

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• Catalysis
• - the process of increasing the rate of chemical
  reactions
• Catalyst
• - the substance that facilitate in catalysis

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Enzyme As Catalyst

• Enzymes are catalysts
• - increase the rate of a reaction
• - not consumed by the reaction
• - act repeatedly to increase the rate of
  reaction
• - enzymes often specific promote only 1
  particular reaction, others catalyze a family of
  similar reactions
• cellulase cellulose as substrate
• hexokinase any 6 ring monosaccharide -
  fructose, glucose

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General Properties of Enzyme

• Higher reaction rates
• - the rates of enzymatically catalyzed reactions
  are 106 to 1020 gt than uncatalyzed reaction
• Milder reaction rates
• - enzyme catalyzed reactions occur under
  relatively milder conditions lt 100oC,
  atmospheric pressure and nearly neutral pH-
  contrast with chemical catalysis requires high
  temperature, pressures and extremes pH.
• Greater reaction specificity
• - enzyme have greater degree of specificity to
  their substrates and their products rarely have
  side products

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Enzyme Catalysis
Active site - part of enzyme to which the
substrate binds and the reaction takes place
Substrate a reactant in an enzyme-catalyzed
reaction
Product
Enzyme-substrate (ES) complex the intermediate
formed when the substrate is bind at the active
site of an enzyme
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Enzyme Catalysis

• GENERAL FORMULA

E enzyme S substrate P product
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Enzyme catalysis reaction

• Physically interact with their substrates to
  effect catalysis
• E S ES ES EP E P
• Where
• - E enzyme
• - ES enzyme/substrate complex
• - ES enzyme/transition state complex
• - EP enzyme/product complex
• - P product

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Enzyme catalysis reaction

• Substrate bind to the enzymes active site
• pocket in the enzyme
• Catalytic site active site where reaction
  takes place

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Enzyme catalysis reaction

• E S ES ES EP E P
• 1st step enzyme binds to substrate molecule to
  form an enzyme substrate complex
• E S ES

Enzyme
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Enzyme catalysis reaction

• E S ES ES EP E P
• 2nd step Formation of the transition state
  complex where the bound substance is neither
  product nor reactant
• ES ES, ES?ES

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Enzyme catalysis reaction

• E S ES ES EP E P
• 3rd step Formation of the enzyme product
  complex ES EP

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Enzyme catalysis reaction

• E S ES ES EP E P
• 4th step Release of product EP E P

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Enzyme catalysis reaction

• Enzyme can only work on one substrate molecule at
  
• a time
• Not change during the reaction
• One product is release, enzyme is available to
  accept another substrate molecule

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Enzyme Catalysis

• Rate of reaction reaction velocity (V)
• - the rate of enzyme reaction is measured by the
  rate of the appearance of products or the rate of
  disappearance of substrates.
• - dP/dT or dS/dT
• ?mol product/min or ?mol substrate/min
• Enzyme activity?
• 1 unit (U) is the amount of enzyme that catalyses
  the reaction of 1 ?mol of substrate per minute
  under specified conditions.

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Enzyme Catalysis

• The rate of a reaction depends on its activation
  energy, DG
• an enzyme provides an alternative pathway with a
  lower activation energy
• Activation energy the energy required to start
  a reaction
• Transition state the intermediate stage in a
  reaction in which the old bonds break and new
  bonds are formed

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How enzyme work?

• Transition state theory
• The enzyme (E) must approach the substrate (S),
  the substrate attach to the active site through
  noncovalent bond
• Formed the high energy (unstable) ES complex
• In ES complex, the covalent bond in substrate is
  in the process of breaking while the EP complex
  is forming.

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Enzyme Catalysis - Example

• Consider the reaction

catalase

1. No catalyst,
2. with added Fe3 salt,
3. with added catalase

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• (a) a/e for the reaction in the absence of a
  catalyst
• (b) a/e lowered in the presence of an iron
  catalyst
• (c) energy diagram for the catalase-catalyzed
  breakdown of H2O2
• (d) energy diagram for the noncatalysed
  breakdown of H2O2 at elevated temperature

a/e activation energy
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Active site

• Has specificity can discriminate among possible
  substrate molecules
• - others recognize a functional group (group
  specificity)
• - only recognize one type of molecule (eg. D vs
  L isomer)
• (absolute specificity)
• Relatively small 3D region within the enzyme
• - small cleft or crevice on a large protein
• Substrates bind in active site by weak
  non-covalent interactions (Hydrogen bond,
  hydrophobic and ionic interaction)

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Active site

• The interactions hold the substrate in the proper
  orientation for most effective catalysis
• The energy derived from these interactions
  binding energy
• Binding energy is used, in large part to lower
  the activation energy and stabilize the
  transition state complex (ES)
• Each non-covalent interaction provides energy to
  stabilize the transition state

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Binding Models

• Two models have been developed to describe
  formation of the enzyme-substrate complex
• Lock-and-key model substrate binds to that
  portion of the enzyme with a complementary shape
• Induced fit model binding of the substrate
  induces a change in the conformation of the
  enzyme that results in a complementary fit

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Enzyme/substrate interaction

• Lock and key model
• - substrate (key) fits into a perfectly shaped
  space in the enzyme (lock)
• - lots of similarities between the shape of the
  enzyme and the shape of the substrate
• - highly stereospecific
• - implies a very RIGID inflexible active site
• - site is preformed and RIGID

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Enzyme/substrate interaction

• Induced fit model (hand in glove analogy)
• - count the flexibility of proteins
• - substrate fits into a general shape in the
  enzyme, causing the enzyme to change shape
  (conformation) close but not perfect fit of E
  S
• - change in protein configuration leads to a
  near perfect fit of substrate with enzyme

Figure 6.3, pg 148 Campbell Farrell,
Biochemistry, 6th Ed., 2009, Thomson Brooks/Cole
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Key words for today

• You need to understand
• Factors effecting enzyme reaction rate
• 6 classes of enzymes
• Cofactor, coenzymes, holoenzymes, apoenzymes
• Allosteric enzyme, effectors (positive and
  negative), heterotropic and homotropic
  allosterism
• Isoenzyme and multienzyme

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Characteristics of enzyme reactions

• What influence the enzyme reaction rate?
• Substrate concentration
• Temperature
• pH
• Enzyme concentration
• Inhibitor

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Characteristics of enzyme reactions

• Substrate Saturation Increasing the substrate
  increases the rate of reaction (enzyme activity).
  
• enzyme saturation limits reaction rates. An
  enzyme is saturated when the active sites of all
  the molecules are occupied most of the time.
• At the saturation point,
• the reaction will not speed
• up, no matter how much
• additional substrate
• is added. The graph of
• the reaction rate will plateau.

substrate substrate concentration
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Characteristics of enzyme reactions

• Temperature
• - very sensitive to temperature changes
• - low temp, rate of an enzyme-catalysed reaction
  increases proportionally with increasing
  temperature

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Characteristics of enzyme reactions

• Effects of Temperature
• All enzymes work within a range of temperature
  specific to the organism.
• Increases in temperature lead to increases in
  reaction rates - is a limit to the increase
  because higher temperatures lead to a sharp
  decrease in reaction rates - due to the
  denaturating (alteration) of protein structure
  resulting from the breakdown of the weak ionic
  and hydrogen bonding that stabilize the three
  dimensional structure of the enzyme.

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Characteristics of enzyme reactions

• pH
• - enzymes have an optimal pH at which they
  function properly
• - varies to each other but most in the range of
  pH 6-8

pepsin in the stomach works best at a pH of 2 and
trypsin at a pH of 8.
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Characteristics of enzyme reactions

• Effects of pH Most enzymes are sensitive to pH
  and have specific ranges of activity.
• All have an optimum pH. The pH can stop enzyme
  activity by denaturating (altering) the three
  dimensional shape of the enzyme by breaking
  ionic, and hydrogen bonds.

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Characteristics of enzyme reactions

• Enzyme concentration
• - the higher the concentration, the greater
  should be the initial reaction rate will be
  lasting as long as substrate present

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Characteristics of enzyme reactions

• Inhibitor
• - inhibit enzyme by occupy the active site or
  bind to other part of enzyme leading to the
  change of enzyme shape and eventually the active
  site
• - this will decrease the enzymatic reaction rate

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Classification of Enzymes

• Have 6 categories
• Each enzyme has an official international name
  ending with ase and a classification number
• Number consists in 4 digits (referred to a class
  and subclass of reaction

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Classification of Enzymes
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Classification of Enzymes
Table 5.1, pg 136 Boyer, R., Concepts in
Biochemistry, 3rd Ed., 2006, John Wiley Sons
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Enzyme Classes Examples
Class Example Reaction
1 (oxidoreductase) alcohol dehydrogenase
2 (transferase) hexokinase glucose ATP ? glucose-6-phosphate ADP
3 (hydrolase) chymotrypsin polypeptide H2O ? peptides
More examplesRefer Table 5.2, pg 136 , Boyer,
R., Concepts in Biochemistry, 3rd Ed., 2006, John
Wiley Sons
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Enzyme Classes Examples
Class Example Reaction
4 (lyase) pyruvate decarboxylase
5 (isomerases) alanine racemase D-alanine L-alanine
6 (ligases) pyruvate carboxylase
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Enzymes cofactor

• Enzymes require chemical entity in order to
  function properly (assists an enzyme in catalytic
  action)
• Cofactor nonprotein molecule that assist in an
  enzyme catalytic reaction
• Coenzyme smaller organic or organometallic
  molecule derived from vitamin, weakly bound to
  enzyme, temporarily associated with enzymes
• Prosthetic group coenzymes that are covalently
  or noncovalently tightly bound to enzyme and
  always present.

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Enzymes cofactor

• Holoenzyme an enzyme in its complete form
  including polypeptide(s) and cofactor
• Apoenzyme enzyme in its polypeptide form
  without any necessary prosthetic groups or
  cofactors

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Allosteric Enzymes

• Allosteric enzyme an oligomer whose biological
  activity is affected by other substances binding
  to it
• these substances change the enzymes activity by
  altering the conformation(s) of its 4structure
• Allosteric effector a substance that modifies
  the behavior of an allosteric enzyme may be an
• allosteric inhibitor negative effectors
• allosteric activator positive effectors

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Allosteric enzyme in feedback Inhibition

• Formation of product inhibits its continued
  production feedback inhibition

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Allosteric Enzymes (Contd)

• The key to allosteric behavior is the existence
  of multiple forms for the 4structure of the
  enzyme
• allosteric effector a substance that modifies
  the 4 structure of an allosteric enzyme
• homotropic effects allosteric interactions that
  occur when several identical molecules are bound
  to the protein e.g., the binding of aspartate to
  ATCase
• heterotropic effects allosteric interactions
  that occur when different substances are bound to
  the protein e.g., inhibition of ATCase by CTP
  and activation by ATP

ATCase aspartate transcarbomylase CTP
cytidine triphosphate
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Allosteric enzymes

• A change in conformational structure at one
  location of a multisubunit protein that causes a
  conformational change at another location on the
  protein
• Effectors i) serves as stimulants to enzyme
  (ve effectors) increase catalytic
  activity ii) inhibitors (-ve effectors) to
  enzyme
  reduce/inhibit catalytic activity
• - Act by reversible, noncovalent binding to a
  site on the enzyme
• Larger and more complex than nonallosteric enzyme
• Have 2 or more subunits (oligomeric)
• Allosteric enzymes have regulatory sites for
  binding of substrates and reaction (catalytic
  sites)

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HOMOTROPIC ALLOSTERISM

• Eg. Tetrameric allosteric enzyme composed of 4
  identical subunits
• Each subunit has a catalytic site where
  substrate/effector will bound and transformed to
  product
• Once bound to active site, a message will
  transmitted via conformational changes to an
  active site on another subunit which makes it
  easier for a substrate molecule to bind and react
  at that site
• This type (substrate and effector) are the same
  is called cooperative or homotropic

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HETEROTROPIC ALLOSTERISM

• A dimer with nonidentical subunits
• Subunit a contain the active site catalytic
  subunit
• Subunit ß contains the site for effector binding
  regulatory subunit
• Binding of a specific effector molecule to the
  regulatory site on the ß subunit sends a signal
  via conformational changes to the catalytic site
  on subunit a
• Substrate and effector different kinds of
  molecules - heterotropic

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Isoenzymes

• Enzymes that catalyze the same reaction
  (catalytically and structurally similar) but are
  encoded by different genes
• Glycogen phosphorylase-synthesize in liver, brain
  and muscle-involves in degradation of glycogen
• Isoenzymes isoforms

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Multienzymes

• A group of noncovalently associated enzymes that
  catalyze 2 or more sequential steps in
  metabolic/biochemical pathway