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).