Enzymes

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Enzymes

• Packet 10

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Properties of Enzymes

• Active Sites
• A special pocket that contains amino acid side
  chains that are complementary to the substrate
• Catalytic Efficiency
• Enzymes catalyze reactions 103 to 106 faster than
  uncatalyzed reactions
• Lower the activation energy
• Work in only one direction as they will not
  catalyze a reverse reaction
• Specificity
• Enzymes are very specific
• Interacting with one, or few, specific substrates
  and catalyzing only one type of chemical reaction

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Properties of Enzymes II

• Cofactors
• Some enzymes associate with a nonprotein cofactor
  that is needed for enzymic activity
• Zn2
• Fe2
• and with organic molecules that are often
  derivatives of vitamins
• Regulation
• Enzyme activity can be regulated
• Can be activated or inhibited so that the rate of
  product formation responds to the needs of the
  cell

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Properties of Enzymes III

• Location within the cell
• Many enzymes are localized in specific organelles
  within the cell
• Allows isolation of substrate or product from
  other competing reactions
• Provides a favorable environment for the reaction
• Allows organization of the 1000s of enzymes
  present in the cell into purposeful pathways.

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Gibbs Free Energy

• Free Energy
• The portion of a systems energy that can perform
  work when temperature and pressure are uniform
  throughout the system.

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Activation Energy

• Activation Energy
• The energy difference between reactants and the
  transition state
• Determines how rapidly the reaction occurs at a
  given temperature
• The lower the activation energy, the faster the
  reaction will occur
• The higher the activation energy, the slower the
  reaction will occur
• Transition State
• Represents the highest-energy structure involved
  in the process of a chemical reaction
• A chemical reaction must have enough energy to
  overcome the transition state.

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Changing Shape of Enzyme

• Temperature
• Increases the kinetic motion
• Breaks the hydrogen bonds
• pH
• Changes the ionic charges
• Alters the shape
• If the pH becomes basic, the acidic amino acid
  side chains will lose H ions
• If the pH becomes acidic, the basic amino acid
  side chains will gain H ions
• Causes the ionic bonds, that help stabilize the
  tertiary structures of proteins, to break.
  Resulting in the denaturation of the enzyme.

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Changing the Shape of Enzymes II

• Inhibitors
• Chemicals that binds to enzyme and changes its
  activity
• Competitive
• Non-competitive
• More to come later
• Poisons
• Organo-phosphorous compounds
• Insecticides
• Bind to enzymes of the nervous system and kills
  the organism

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Factors Affecting the Rate of Production of
Enzymatic Product

• Concentration of Substrate to Enzyme
• Discussed already in class

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Enzyme Kinetics

• Way of describing properties of enzymes
• Mathematical
• Graphical expression
• Expression of reaction rates of enzymes
• A ? B C
• Please read Chapter 8
• Section 4

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Graphical Curves of Enzyme Activity

• Rate vs. Enzyme
• Ml substrate/min ?Rate
• Rate vs. pH
• Reveals the optimum pH
• Rate vs. Temperature
• Reveals the optimum temperature
• Rate vs. Substrate
• Shows a saturation curve
• Most definitive curve of enzyme activity

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Michaelis-Meten Enzyme Curve

• Michaelus and Menten proposed a simple model that
  accounts for most of the features of
  enzyme-catalyzed reactions.
• In this model, the enzyme reversibly combines
  with its substrate to form an Enzyme-Substrate
  Complex that subsequently breaks down to product.
• Results in the regeneration of a free enzyme.
• E S ? ES ? E P
• S substrate
• E Enzyme
• ES Enzyme-substrate complex
• K1, k-1, k2 rate constants

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Michaelis-Menten Equation

• Describes how reaction velocity varies with
  substrate concentration
• Rate (Reaction Velocity) vs. Substrate
  Concentration
• V0 Vmax S/Km S
• V0 initial reaction velocity
• Vmax maximal velocity
• Km Michaelis constant (k-1 k2)/k1
• Is the substrate concentration at which rate is
  one-half the maximal velocity
• A measure of affinity of enzyme for a substrate
• S Substrate Concentration

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Michaelis-Menten Equation

• Assumptions (3)
• The concentration of substrate is greater than
  the concentration of enzymes
• Remember, only one substrate is able to bind at
  the active site of an enzyme at any time.
• The rate of formation of the enzyme-substrate
  complex is equal to the breakdown of the
  enzyme-substrate complex
• To either
• E S
• E P
• Recall equation from earlier slide.
• Initial velocity
• Only used in the analysis of enzyme reactions
• Meaning, the rate of reaction is measured as soon
  as enzyme and substrate are mixed

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Conclusions about Michaelis-Menten Kinetics

• Characteristics of Km
• Km ½ Vmax
• Does not vary with the concentration of enzyme
• Small Km
• Reflects high affinity(an attraction to or liking
  for something) of the enzyme for substrate
• Why?
• Because a low concentration of substrate is
  needed to reach a velocity of ½ Vmax
• Large Km
• Reflects low affinity of the enzyme for substrate

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Conclusions about Michaelis-Menten Kinetics

• Relationship of Velocity to Enzyme Concentration
• Rate of the reaction is directly proportional to
  the enzyme concentration at all substrate
  concentrations
• Example
• If the enzyme concentration is halved, the
  initial rate of the reaction (v0) is reduced to
  one half that of the original

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Conclusions about Michaelis-Menten Kinetics

• Order of Reaction
• Recall from Chemistry
• Will leave the details of this conclusion out.

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Lineweaver-Burke Plot

• When the reaction velocity is plotted against the
  substrate concentration, it is not always
  possible to determine when Vmax has been
  achieved.
• Due to the gradual upward slope of the hyperbolic
  curve at high substrate concentration.
• However, if 1/V0 is plotted vs 1/S , a straight
  line is obtained.
• This plot is known as the Lineweaver-Burke Plot
• Can be used to calculate
• Km
• Vmax
• Determines the mechanism of action of enzyme
  inhibitors

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Lineweaver-Burke Equation

• 1/V0 Km/Vmaxs 1/Vmax
• The intercept on the x axis
• -1/Km
• The intercept on the y axis
• 1/Vmax

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Inhibition of Enzyme ActivityReduction in Enzyme
Activity

• Enzyme Inhibitors
• Competitive Inhibitors
• Resemble the substrate molecule for that specific
  enzyme
• Competes for the active site
• Reduces the productivity of enzymes by blocking
• Non Competitive Inhibitors
• Does not directly bond to the active site of the
  enzyme
• Binds at another location and alters the shape of
  the enzyme so that the active site is no longer
  fully functional

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Competitive Inhibition

• Effect on Vmax
• Vmax is the same in the presence of a competitive
  inhibitor
• Effect on Km
• Michaelis constant, Km, is increased in the
  presence of a competitive inhibitor
• Effect of Lineweaver-Burke Plot
• Vmax is unchanged

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Non-Competitive Inhibition

• Effect on Vmax
• Vmax is decreased
• Cannot overcome by increasing the amount of
  substrate
• Effect on Km
• Michaelis constant, Km, is the same
• Non-competitive inhibitors do not interfere with
  the binding of substrate to enzyme
• Effect of Lineweaver-Burke Plot
• Vmax decreases
• Km is unchanged

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Feedback Inhibition

• An end product inhibits an initial pathway enzyme
  by altering efficiency of enzyme action

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Real-Life Applications of Inhibitors

• Competitive Inhibitor
• Important Information
• Enzyme
• Succinate dehydrogenase
• Catalyzes the oxidation of succinate to fumarate
• Cell Respiration
• Malonate
• Structurally similar to the substrate succinate
• Binds at the active site of the enzyme
• Results in an increase of the substrate succinate
  in the cell
• However, the probability of the active site being
  occupied by the substrate, instead of the
  inhibitor, increases

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Real-Life Applications of Inhibitors

• Non-Competitive Inhibitors
• Lead poisoning
• Lead forms covalent bonds with the sulfhydryl
  side chains of cysteine in proteins
• The binding of the heavy metal shows
  non-competitive inhibition
• Drugs
• Can behave as enzyme inhibitors
• Lactam antibiotics
• Penicillin
• Amoxicillin
• Inhibit one or more enzymes of bacteria walls

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Regulation of Enzyme Activity

• The regulation of the reaction velocity of
  enzymes is essential if the organism is to
  coordinate its numerous metabolic pathways
• The control of metabolism

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Allosteric Regulation

• Results in changes an enzymes shape and function
  by binding to an allosteric site
• Specific receptor site on some part of the enzyme
  molecule remote from the active site
• Allosteric inhibitor, binds at the allosteric
  site, and stabilizes the inactive form of the
  enzyme
• Makes the enzyme non-functional
• Activator, also binds at the allosteric site, and
  stabilizes the active form on the enzyme
• Makes the enzyme functional
• ATP and ADP are examples

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Naming of Enzymes

• Most historically
• Substrate ase
• Sucrase
• Catalase
• Mallerase
• International Union Biochemistry and Molecular
  Biology
• 4 digit Nomenclature Committee Numbering System

• 1st
• Major Class of Activity
• Only six classes recognized
• 2nd
• Subclass
• Type of bond acted on
• 3rd
• Subclass
• Group acted upon
• Cofactor required
• 4th
• Serial Number
• Sequence order

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Classes of Enzymes

• Oxidoreductases
• Catalyze oxidation-reduction reactions
• Transferases
• Catalyze transfer of C, N or P containing groups
• Hydrolases
• Catalyze cleavage of bonds by addition of water
• Lyases
• Catalyze cleavage of C-C, C-S and certain C-N
  bonds

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Classes of Enzymes

• Isomerases
• Catalyze racemization of optical or geometric
  isomers
• Catalyze isomerization
• Change from one isomer to another
• Ligases
• Catalyze formation of bonds between carbon and O,
  S, N coupled with hydrolysis of high energy
  phosphates (ATP)
• Condensation of 2 substrates with splitting of ATP

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Exergonic Reaction