Download 1. Metabolic pathways 2. Basic enzyme kinetics 3. Metabolic

Regulation of Metabolic Pathways
1. Metabolic pathways
2. Basic enzyme kinetics
3. Metabolic pathway regulation
Basic Functions of Metabolic Pathways
Classification of Reactions
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Fueling reactions (catabolic pathways)
» Produce precursor metabolites needed for biosynthesis
» Generate energy (ATP) for cellular functions
» Produce reducing power (NAPDH) for biosynthesis
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Biosynthetic reactions (biosynthetic pathways)
» Produce building blocks for macromolecular synthesis
» Produce coenzymes & signaling molecules
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Polymerization reactions
» Form macromolecules from building blocks
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Assembly reactions
» Chemical modifications of macromolecules to form
cellular structures (cell wall, membranes)
http://www.expasy.org/cgi-bin/show_thumbnails.pl
Membrane Transport Processes
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Free diffusion
» Species transported down concentration gradient
» Transport driven by chemical potential difference
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Facilitated diffusion
» Species transported down concentration gradient
» Specific carrier or transmembrane protein
involved
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Active transport
» Species can be transported up concentration
gradient
» Specific proteins or permeases involved
Catabolic Pathways
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Basic functions
» Generate energy & reducing power
» Produce precursors for biosynthesis
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Participating pathways
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Glycolytic pathway
Pentose phosphate pathway (PPP)
Fermentative pathways
Tricarboxylic acid (TCA) cycle
Anaplerotic pathways
Pathways involved in catabolism of fats,
organic acids & amino acids
Glycolytic Pathway
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Also called the Embden-Meyerhof-Parnas (EMP)
pathway
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Converts glucose to pyruvate
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Generates ATP & NADH
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Glucose + 2 NAD+ + 2 ADP + 2 Pi " 2 pyruvate
+ 2 NADH + 2 H+ + 2 ATP + 2 H20
Also produces three precursors for biosynthesis
» Glyceraldehyde 3-phosphate (G3P)
» 3-phoshoglycerate (3-PG)
» Phosphoenolpyruvate (PEP)
Simplified Picture of Glycolytic Pathway
Pentose Phosphate Pathway (PPP)
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Converts G6P to NAPDH & two precursors
for biosynthesis
» Ribose-5-phosphate (R5P)
» Erythrose-4-phosphate (E4P)
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Also produces glycolytic intermediates F6P &
G3P
Relative flux through EMP & PPP varies
» Energy & reducing power requirements
» Need for precursor metabolites
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PPP stoichiometry depends on extent carbon is
recycled back to EMP
Oxidative PPP
Non-Oxidative PPP
Overall PPP
Fermentative Pathways
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Fermentation
» Occurs under oxygen limited conditions
» Pyruvate converted into metabolic products (lactic
acid, acetic acid, ethanol)
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Bacteria
» Several different metabolic products formed
» Mixed acid fermentation
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Yeasts
» Ethanol is the main metabolic product
» Alcohol fermentation
» Limited acetate & succinate also formed
Yeast Fermentation
TCA Cycle
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Also called the citric acid cycle or Krebs
cycle
Completely oxidizes pyruvate to produce
ATP via oxidative phosphorylation
Location
» Bacteria – occurs in cytosol
» Yeast – occurs in mitochondria
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Stoichiometry
» Pyruvate + CoA + NAD " Acetyl-CoA + CO2 +
NADH + H+
» Acetyl-CoA + 3 NAD + FAD + GDP + Pi + 2H20
" 2 CO2 + 3 NADH + FADH2 + GTP + 2H+ +
CoA
TCA Cycle in Yeast
Simplified Picture of TCA Cycle
Oxidative Phosphorylation in Yeast
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Basic functions
» Regenerate NAD+ for glycolysis
» Generate ATP for biosynthesis
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Basic mechanism
» Electrons are transported from NADH & FADH through the electron
transport chain to oxygen
» Electron transport causes protons to be released into the
intermembrane space
» These electrons can be transported back into mitochondrial matrix by a
proton conducting ATP-synthase
» The detailed mechanistic steps are not completely understood
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Theoretical yields
» P/O ratio: 3 ATP/NADH & 2 ATP/FADH
» Overall: 15 ATP/pyruvate
» Actual yields are lower due to incomplete coupling of the oxidative &
phosphorylation processes
Oxidative Phosphorylation
Biosynthetic Pathways
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Generate 12 precursor metabolites needed for
cellular synthesis
Amino acid biosynthesis
» Forms 20 common amino acids
» Well characterized in bacteria & yeast
» Consumes considerable energy & reducing power
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Biosynthesis of other building blocks
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Nucleotides " RNA & DNA
Fatty acids " lipids
UDP-glucose " storage carbohydrates
Also consume energy & reducing power
Metabolic Pathway Regulation
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Basic concepts
» Pathways must be regulated to compensate for
changes in nutrient availability & cellular demands
» Regulated variables include concentrations of
substrate, enzyme, product & special regulatory
molecules
» Regulation implemented over a very wide range of
time scales (15 orders of magnitude)
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Present focus
» Regulation of enzyme activity at relatively short
time scales
» Best understood form of metabolic regulation
Hierarchy of Regulatory Mechanisms
Time Scale of Regulatory Mechanisms
Enzyme Regulation
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Regulation of enzyme activity
» Achieved at the metabolic level
» Feedback inhibition & activation of enzyme activity
by pathway substrate/products or global metabolites
(ATP, NADH)
» Fast responses (second time scale)
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Regulation of enzyme concentration
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Achieved at the gene level
Repression & induction of enzyme synthesis
Slow responses (hour time scale)
Focus of lecture on signal transduction networks
Michaelis-Menten Kinetics
Reversible Inhibition
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Substrate inhibition
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» Rate inhibited by high
substrate concentrations
v"
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» Inhibitor reversibly binds to
enzyme on non-active
vmax sites
S
regulatory
v"
vmax S
Km ! S ! S 2 / Ki
1 ! I / Ki K m ! S
Competitive inhibition
» Inhibitor competes with
substrate for enzymatic
active sites
vmax S
v"
K m (1 ! I / K i ) ! S
Non-competitive
inhibition
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Uncompetitive inhibition
» Inhibitor
vmax reversibly
S binds to
v"
enzyme-substrate
K m complex
1! I / K
i
1 ! I / Ki
!S
Allosteric Enzymes
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Cooperativity
» Binding to one vacant site induces altered affinities for
remaining vacant sites
» Homotrophic – only substrate involved
» Heterotrophic – involve substrate & regulator
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Allosteric enzymes
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Exhibit cooperativity
Composed of multiple catalyic & regulatory subunits
Characterized by sigmoidal velocity curves
Allow large change in reaction rate for small changes
in substrate concentration
» Facilitate regulation of metabolic pathways where the
substrate concentrations exhibit small variations
Cooperative Binding
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Types of cooperative effects
» Positive – binding of first molecule activates binding of
second molecule
» Negative – binding of first molecule inhibits binding of
second molecule
vmax S n
v"
Hill equation
K ! Sn
n " number of sites
Glycolytic Pathway
Regulation of EMP & PPP in Yeast
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Glycolytic (EMP) pathway
» Hexokinase: inhibited by G6P (product inhibition)
» Phosphofructokinase
– Inhibited by ATP & citrate (signals overabundance of TCA
cycle intermediates)
– Activated by AMP & ADP (signal lack of available energy)
– Activated by ammonia, phosphate & fructose-2,6bisphosphate (regulatory molecule)
» Pyruvate kinase: inhibited by ATP & acetyl-CoA
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Pentose phosphate pathway
» Glucose-6-phosphate dehydrogenase: activity
regulated by NAPDH/NADP+ ratio
Glycolytic Regulation in Yeast
Phosphofructokinase (PFK) Regulation
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PFK is a complex allosteric enzyme
» Inhibited by ATP
» Activated by fructose-2,6-bisphosphate (F-2,6-P)
» F-2,6-P formed by phosphorylation of F6P by ATP
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Cooperativity
» ATP binding decreases affinity for F6P (substrate)
» F-2,6-P binding causes large increase in F6P affinity
» Glycolytic flux stimulated by F-2,6-P & inhibited
by ATP
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Regulatory effects
» Allows PFK to increase activity in response to
increasing F6P concentration
» High energy levels suppress PFK activity
TCA Cycle in Yeast
Regulation of the TCA Cycle in Yeast
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Citrate synthase
» Weakly inhibited by NADH/NAD+ ratio
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Isocitrate dehydrogenase
» Strongly inhibited by NADH/NAD+ ratio
» Activated by AMP
» Inhibited by ATP
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Alpha-ketoglutarate dehydrogenase
» Weakly inhibited by NADH/NAD+ ratio
Regulation at the Gene Level
Summary
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Metabolic pathways
» Classified according to their function
» Focused on yeast central carbon metabolism
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Metabolic pathway regulation
» Essential to control metabolic function
» Implemented over a wide range of time scales
» Focused on short-term regulation of enzymatic
activities
» Can involve complex mechanisms
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Implications
» Complexity necessitates integrative approaches
» Metabolic pathway modeling & analysis
References
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G. H. Braus, ”Aromatic Amino Biosynthesis in the
Yeast Saccharomyces cerevisiae: a Model System
for the Regulation of a Eukaroytic Biosynthetic
Pathway,” Microbiological Reviews, 55, 349-370
(1991).
J. Nielsen & J. Villadsen, Bioreaction Engineering
Principles, Plenum Press, New York, NY (1994).
M. L. Shuler & F. Kargi, Bioprocess Engineering:
Basic Concepts, 2nd edition, Prentice Hall,
Englewood Cliffs, NJ (2002).
G. N. Stephanopoulos, A. A. Aristidou & J.
Nielsen, Metabolic Engineering: Principles and
Methodologies, Academic Press, New York, NY
(1998).