Chapter 6: Enzymes, cont'

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Chapter 6 Enzymes, cont.

• Dr. Clower
• Chem 4202

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Part 2 Enzyme Kinetics, Inhibition, and
Regulation

• Enzyme kinetics (section 6.3)
• At what rate do enzymes react?
• How does rate change with varied conditions?
• Enzyme inhibition (section 6.3)
• What prevents interactions between enzymes and
  substrates?
• Enzyme regulation (section 6.5)
• How is enzymatic activity controlled?
• Gives information about catalytic mechanism,
  enzyme structure, biological function
• Provides insight into how the enzyme can be
  regulated for therapeutic purposes

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I. Enzyme Kinetics

• Expression for enzyme catalyzed reaction

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Michaelis-Menton Equation

• Relates rate and S
• Rate V0 k2ES
• V0 initial velocity/rate
• Neglects complications from inhibition,
  deactivation, etc.
• What is the ES?
• To determine, look at rate of formation and
  disappearance of ES

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ES

• Rate of formation k1ES
• Rate of breakdown k-1ES k2ES
• Assume steady state
• As ES is produced, it reacts
• ES remains constant
• Rate formation rate breakdown
• So, k1ES k-1ES k2ES

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• k1ES k-1ES k2ES
• k1ES (k-1 k2)ES
• Rearrange
• ES / ES (k-1 k2) / k1
• Where (k-1 k2) / k1 KM (Michaelis constant)
• Define total enzyme concentration ET E
  ES
• Substitute for E
• (ET ES)S / ES KM
• Solve for ES
• ES ETS / KM S

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• ES ETS / KM S
• Since V0 k2ES
• V0 k2ETS / KM S
• Define maximum velocity Vmax
• Occurs at high S
• Enzyme is all in ES form
• ES ET
• Vmax k2ET
• Therefore
• V0 VmaxS / KM S

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Michaelis-Menton Equation
V0 VmaxS / KM S

• Rate increase with S
• Rate levels off as approach Vmax
• More S than active sites in E
• Adding S has no effect
• At V0 ½ Vmax
• S KM

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KM

• Unique for each E-S pair
• Magnitude varies
• Depends on E and S
• Function of Temperature
• Rate increases with T
• Optimum T 37C for most enzymes
• Function of pH
• Change catalytic activity
• Most enzymes active in narrow pH range close to
  pH of environment of enzyme

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Catalytic Constant kcat

• kcat Vmax / ET
• Turnover number
• Number of reaction processes each active site
  catalyzes per unit time
• Measure of how quickly an enzyme can catalyze a
  specific reaction
• For M-M systems kcat k2
• Rate constant of RDS

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kcat / kM

• Rate constant of rxn E S ? E P
• Specificity constant
• Gauge of catalytic efficiency
• Catalytic perfection 108 - 109 M-1 s-1

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Lineweaver-Burk Plot

• Another method to determine Vmax and KM
• Reciprocal of M-M equation
• Linear equation

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II. Enzyme Inhibition

• What happens if a substrate does not leave the
  active site?
• Enzyme is blocked (inhibited) from further
  interactions with substrate
• Why inhibit?
• To control S or P
• Increase S unreacted
• Decrease P formed

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Examples

• Increase S
• Decreased levels of GABA cause seizures
• GABA aminotransferase degrades GABA
• Inhibition of enzyme raise GABA levels
• Decrease P
• Xanthine converts to uric acid with xanthine
  oxidase
• Excess uric acid leads to gout
• Inhibition of enzyme lowers production of uric
  acid

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Types of Inhibition

• Reversible
• Competitive
• Uncompetitive
• Mixed
• Noncompetitive
• Irreversible
• Suicide inactivators

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Competitive Inhibition

• Most common
• Inhibitor competes with natural substrate for
  binding to active site
• Inhibitor similar in structure to natural
  substrate
• Binds more strongly
• May or may not react
• If reacts, does so very slowly
• Gives info about active site through comparison
  of structures
• Example antifreeze poisoning

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Competitive Inhibition

• I and S compete for E
• Increasing I
• Increases EI
• Reduces E available for substrate binding
• Need to keep I high to ensure inhibition
• Dosage

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Michealis-Menton Equation for Competitive
Inhibition

• Where a 1 I/KI
• Function of inhibitor concentration and affinity
  for enzyme
• a 1 when I 0
• As S increases, V0 will approach Vmax for any
  value of a
• S overcome inhibitor effects saturate
• As I increases, KM increases by a
• More S required to reach ½ Vmax
• Rate does not increase rapidly with S due to
  inhibition

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Lineweaver-Burk Equation for Competitive
Inhibition

• Slope is larger (multiplied by a)
• Intercept does not change (Vmax is the same)
• KM is larger (multiplied by a)

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Uncompetitive Inhibition

• Inhibitor binds to enzyme-substrate complex, not
  to free enzyme
• Inhibitor does not need to resemble substrate
• Active site multisubstrate, or I in second site
• Distorts active site prevents reaction from
  occurring
• Increasing S does not change binding of
  inhibitor to ES (uncompetitive)
• Vmax is affected by I

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M-M and L-B Equations for Uncompetitive Inhibition

• Increasing I
• Lowers Vmax (y-intercept increases)
• Lowers KM (x-intercept decreases)
• Ratio of KM/Vmax is the same (slope)

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Mixed Inhibition

• Inhibitor binds to either E or ES

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M-M and L-B Equations for Mixed Inhibition

• Increasing I
• Lowers Vmax (y-intercept increases)
• Raises KM (x-intercept increases)
• Ratio of KM/Vmax is not the same (slope changes)

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Noncompetitive Inhibition

• Special case of mixed inhibition where
• a a
• KI KI
• Vmax lowers as increase I
• Lines intersect on x-axis
• KM does not change

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Summary of Reversible Inhibition
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Irreversible Inhibition

• Inhibitor
• Binds covalently, or
• Destroys functional group necessary for enzymatic
  activity, or
• Very stable noncovalent binding
• Suicide Inactivators
• Starts steps of chemical reaction
• Does not make product
• Combines irreversibly with enzyme

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III. Regulatory Enzymes

• Catalytic activity modified in response to a
  signal
• Increase or decrease
• Allow cell to meet changing needs
• Energy
• Substrate
• Product
• Usually located early in multi-enzyme reaction
  pathway

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Regulation Mechanisms

• Three main mechanisms
• Often used in combination
• Control enzyme synthesis or degradation
• Noncovalent conformational change
• Allosteric enzymes
• Covalent modification

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Allosteric Enzymes

• Usually large multiple subunits
• Compare to Hb
• Site for allosteric modulator (R regulatory)
  generally different from active site (C
  catalytic)
• Can be positive or negative

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Aspartate Transcarbamoylase

• Synthesis of pyrimidine nucleotides

• Feedback inhibition
• Noncovalent
• reversible

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Kinetics of Allosteric Enzymes

• Differ from Michaelis-Menton
• Sigmoidal curve
• K0.5 instead of KM

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Homotropic Enzyme

• Substrate is positive modulator

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Heterotropic Enzyme

• Modulator is metabolite
• Activator approach hyperbola
• Deactivator more sigmoidal

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Covalent Modification

• Reversible
• Phosphorylation
• Most common
• Catalyzed by kinases
• Change conformation
• Introduce bulky, charged group
• Increase polarity and H-bonding
• Repel negative side chains (Asp and Glu)

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Chapter 6 Problems

• 1, 3-5, 7-10, 12, 15-16, 18