Enzyme Kinetics

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Enzyme Kinetics
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Enzyme Kinetics

• Kinetics is the study of the rate and mechanism
  of a reaction.
• Rates are usually measured in terms of how many
  moles of reactant or product are changed per time
  period.
• A mechanism is a detailed step-by-step
  description of how a reaction occurs at the
  molecular level.

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Determining Initial Velocity (Vo)
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Michaelis-Menten Plot
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What are Vmax and KM?
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Vmax and KM

• Vmax is limited by
• Time required for product to be released
• How many E molecules are present
• KM gives an idea about affinity
• ?KM ?substrate affinity
• ?KM ?substrate affinity

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Determining KM and Vmax
Lineweaver-Burk (Double Reciprocal Plot)
o
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Enzyme Inhibition

• Inhibitors
• Many substances like drugs, toxins reduce or stop
  reaction rate of enzymes
• Important control mechanism in cells
• Responsible for down regulation in physiological
  processes
• Irreversible
• Covalent bonding of inhibitor with enzyme
• Reversible
• Competitive
• Noncompetitive

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Irreversible Inhibitors

• Group specific agents
• React with specific amino acid R groups
• Penicillin
• Inhibitor of serine-containing enzymes
• Acetylcholinesterase
• Organophosphates cause paralysis

http//www.tvacres.com/images/bug_raid2.jpg
Organophosphate insecticides inhibit
acetylcholinesterase.
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Reversible Inhibition Competitive Inhibitors

• Inhibitor resembles substrate and binds at active
  site.
• Can overcome by adding additional substrate.

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Competitive Inhibitor

• Increases KM
• (? enzymes apparent affinity for substrate)
• Does not affect Vmax

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Competitive Inhibitor Example

• Ethanol for antifreeze poisoning
• Competitive inhibitor of alcohol dehydrogenase

http//www.prestone.com/products/antifreezeCoolant
.php
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Reversible Inhibition Noncompetitive Inhibitors

• Inhibitor binds at site other than active site.
• Cannot overcome by adding extra substrate.

http//www.steve.gb.com/science/enzymes.html
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Noncompetitive Inhibitor

• Decreases Vmax
• (? apparent E)
• Does not affect KM

http//ccnmtl.columbia.edu/projects/biology/lectur
e7/lec7_01.htm
Inhibitor substrate
1/v
substrate
1/S
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Review of Reversible Inhibitors
http//users.rcn.com/jkimball.ma.ultranet/BiologyP
ages/E/EnzymeKinetics.html
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Problem

• The enzyme O-diphenol oxidase causes apple slice
  to turn brown in presence of air.
• What type of inhibtor is PHBA (para-hydroxy
  benzoic acid)?
• What type of inhibitor is phenylthiourea?

http//users.rcn.com/jkimball.ma.ultranet/BiologyP
ages/E/EnzymeKinetics.html
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Enzyme Regulation

• Methods used by cell to regulate rate of
  reactions
• S, P (substrate level regulation)
• pH, salt (various ions)
• Inhibitors (presence and concentration)
• Allosteric modification
• Covalent modification

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Allosteric Enzymes

• Do not exhibit Michaelis-Menten kinetics.
• Plot of Vo vs. S is sigmoidal not hyperbolic.
  Why?

S
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Allosteric Enzymes
Relaxed (R) state (? Km)

• Generally are multisubunit enzymes.
• Exhibit cooperativity.
• The binding of substrate to one active site
  changes the 3-D conformation, which alters the
  affinity other subunits have for the substrate.

Tense (T) state (? Km)
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One Model of Allosteric Mechanism

• The binding of substrate switches conformation of
  only the subunit to which it is bound.
• Conformational change in one subunit may ? or ?
  the affinity of other subunits have for the
  substrate.
• Allows for or - cooperativity.

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Allosteric Enzyme RegulationFeedback Inhibition

• A ? B ? C ? D ? P
• E1 E2 E3 E4

Inhibition
Threonine deaminase is regulated by isoleucine
in the cell. Enzyme exists as 2
states Isoleucine acts as allosteric
effector and binds to enzyme at allosteric site.
Allosteric inhibitor or activator.
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Allosteric Inhibition Activation
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Allosteric Regulation
effector
no effector
- effector
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Types of Subunits

• Catalytic subunits (C)
• Where active sites are located.
• Substrate binds here.
• Regulatory subunits (R)
• Effectors bind here.

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Aspartate Transcarbamoylase (ATCase) Structure

• Important in pyrimidine biosynthesis
• r6c6
• 3 regulatory subunits (consisting of 2 chains
  each)
• 2 catalytic subunits (consisting of 3 chains each)

c
c
c
r
r
r
r
r
r
c
c
c
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ATCase Inhibition
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ATCase Regulation
ATP
no effector
- CTP
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Oxygen Transport Proteins

• Myoglobin
• Exhibits Michaelis-Menten properties
• Hemoglobin
• Exhibits allosteric properties

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Myoglobin

• Single polypeptide
• 16,700 daltons
• 8 a helices (A-H)
• Located in skeletal cardiac muscle
• high in diving mammals like whale seals

Heme prosthetic group
http//www.agen.ufl.edu/chyn/age2062/lect/lect_02
/3_27.gif
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Myoglobin O2 Binding Curve

• Myoglobin has high affinity for O2.
• P50 2.8 Torr
• Allows myoglobin to act as O2 storage reserve.
• Releases O2 when pO2 becomes low indicating O2
  deprivation.

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Hemoglobin

• Heterotetramer
• HbA (95-98 of adults)
• a2b2
• HbA2 (lt3 of Hb)
• a2?2
• HbF (Fetal Hb, 2 of adults)
• a2?2
• 2 dimers

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Hemoglobin Structure

• Each polypeptide chain resembles myoglobin
  tertiary structure but 1 sequence varies.
• Hb exhibits cooperativity

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Hb Variants

• HbA2
• a2d2
• Present in 2 of adults
• Embryonic Hb
• a2e2
• Has ? affinity for O2
• Fetal Hb
• a2g2
• Has ? affinity for O2

http//oregonstate.edu/instruction/bb450/stryer/ch
10/Slide27.jpg
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Regulation Via Protein Modification

• Covalent modification
• Phosphorylation
• Acetylation
• ADP-ribosylation
• Proteolytic Cleavage

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Regulation by Phosphorylation
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Glycogen Metabolism

• Two opposing pathways operating
• Synthesis of glycogen
• Breakdown of glycogen
• Do we want both pathways operating
  simultaneously?

NO!
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Glycogen Synthesis
ATP
ADP
GS kinase
Glycogen synthase inactive
Glycogen synthase active
Pi
HO
phosphatase
Pi
H2O
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Glycogen Degradation
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Proteolytic Cleavage

• Proenzyme or zymogen is an inactive form of
  enzyme.
• Cleavage of zymogen produces active enzyme.

Becker, W. M., L. J. Kleinsmith, and J. Hardin.
(2002) The World of the Cell, 5th ed. Benjamin
Cummings, San Fransico, CA. p. 151.
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Blood Clotting
Campbell, N.A. and J. B. Reece. (2005) Biology,
7th ed. Benjamin Cummings, San Francisco, CA, p.
882