Lecture 19
– Quiz next Friday (Oct. 28) on glycolysis.
– Metabolism and thermodynamics
– (This material forward not on exam)
Summary: various methods to increase rate
• Increase frequency of the correct group in the correct place
e.g. proximity effect
• Lower EA by specific catalysis -acid-base catalysis,
nucleophile or electrophile
• Raise energy of reactants (closer to EA) - ring distortion,
transition state analog
• Provide alternate low EA pathway - covalent catalysis.
• Michaelis Menten vs. Allosterism
• Lineweavear Burk
• Eadie Hofstee
• Competitive inhibition
• Noncompetitive inhibition
• Uncompetitive inhibition
Terms to review for enzymes
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Cofactor
Coenzyme
Prosthetic group
Holoenzyme
Apoenzyme
Lock and Key
Transition analog model
Induced fit
Active site, binding site, recognition site, catalytic site
To be included on the quiz
Introduction to metabolism: Main Functions
•
To obtain energy for growth
•
From the degradation of energy rich compounds
(chemotrophy)
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Organotrophs-organism obtains H or e- from organic
compounds
Lithotrophs-uses an inorganic substrate to obtain reducing
equivalents.
From light (photosynthesis) Phototrophs
To use energy to assemble precursors into building
blocks of the cell and to convert those building blocks
into proteins, carbohydrates, lipids, etc.
• Autotrophs (starting carbon source is CO2)
• Heterotroph (use preformed complex organics)
To degrade and recycle unnecessary metabolites or
compounds no longer in use by the cell.
Metabolism
• The sum of the chemical changes that
convert nutrients into energy and the
chemically complex products of cells
• Hundreds of enzyme reactions organized
into discrete pathways
• Substrates are transformed to products
via many specific intermediates
• Metabolic maps portray the reactions
• Intermediary metabolism
A Common Set of Pathways
• Organisms show a marked similarity
in their major metabolic pathways
• Evidence that all life descended from
a common ancestral form
• There is also significant diversity
The Sun is Energy for Life
• Phototrophs use light to drive
synthesis of organic molecules
• Heterotrophs use these as building
blocks
• CO2, O2, and H2O are recycled
Metabolism
• Metabolism consists of catabolism and
anabolism
• Catabolism: degradative pathways
– Usually energy-yielding!
• Anabolism: biosynthetic pathways
– energy-requiring!
Metabolism is divided into 2 types
A.
Catabolism - degradative metabolism to yield CO2,
simple metabolites, and energy. Accompanied by the
release of G stored in complex molecules - stored as ATP,
NADPH (generate free energy)
B.
Anabolism - Biosynthesis. Energy requiring step-uses
building blocks, ATP and NADH/NADPH to form complex
molecules.
I.
Primary Metabolism - metabolism used for the
construction of essential building blocks and energy
metabolism.
Secondary Metabolisms - Other nonessential
metabolism.
“Chemical reactions that create diverse byproducts often
unique to a taxon and generally not essential for survival
and have no known metabolic role.”
II.
Catabolism and Anabolism
• Catabolic pathways converge to a
few end products
• Anabolic pathways diverge to
synthesize many biomolecules
• Some pathways serve both in
catabolism and anabolism
• Such pathways are amphibolic
Organization in Pathways
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•
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Pathways consist of sequential steps
The enzymes may be separate
Or may form a multienzyme complex
Or may be a membrane-bound
system
• New research indicates that
multienzyme complexes are more
common than once thought
Mutienzyme complex
Separate
enzymes
Membrane
Bound System
Organization of Pathways
Closed Loop
(intermediates recycled)
Linear
(product of rxns are
substrates for
subsequent rxns)
Spiral
(same set of
enzymes used
repeatedly)
5 principal characteristics of
metabolic pathways
1. Metabolic pathways are irreversible.
(large negative free energy change)
2. Catabolic and anabolic pathways must
differ.
3. Every metabolic pathway has a first
committed step.
4. All metabolic pathways are regulated.
5. Metabolic pathways in eukaryotic cells
occur in specific cellular locations.
Metabolism Proceeds in
Discrete Steps
•Enzyme specificity defines
biosynthetic route
•Controls energy input and
output
•Allow for the establishment
of control points.
•Allows for interaction
between pathways
Regulation of Metabolic Pathways
• Pathways are regulated to allow the organism to
respond to changing conditions.
• Most regulatory response occur in millisecond
time frames.
• Most metabolic pathways are irreversible under
physiological conditions.
• Regulation ensures unidirectional nature of
pathways.
• Flow of material thru a pathway is referred to as
flux.
• Flux is regulated by supply of substrates,
removal of products, and activity of enzymes
Enzyme Regulation of Flux
Common mechanisms
• Feedback inhibition – product of pathway down regulates
activity of early step in pathway
• Feedforward activation – metabolite produced early in
pathway activates down stream enzyme
Metabolic Control Theory
• Pathway flux is regulated by multiple enzymes in
a pathway.
• Control coefficient determined for each enzyme. =
 activity /  enzyme concentration.
• Enzymes with large control coefficients impt to
overall regulation.
• Recent finding suggest that the control of most
pathways is shared by multiple pathway enzymes
Regulating Related Catabolic and
Anabolic Pathways
• Anabolic & catabolic pathways involving the
same compounds are not the same
• Some steps may be common to both
• Others must be different - to ensure that each
pathway is spontaneous
• This also allows regulation mechanisms to turn
one pathway and the other off
Gibb’s Free Energy
•
Useful to know how likely a reaction will occur. The
measurement of driving force for all reactions is the
decrease in G.
G = H -TS
Spontaneous reactions all have -G, but imply nothing
about the rate! (G = 0 at equilibrium)
This is related to the G of the entire system.
For our purposes, dependent upon Gf of formation for
the reactants and products.
Gibb’s Free Energy
Oxaloacetate (OAA) + H+
CO2 (g) + pyruvate
G for the reaction = Gf(products) - Gf(reactants)
Gf (kcal/mol)
OAA
Pyr
CO2(g)
H+
-190.62
-113.44
-94.45
0(-9.87)
1M
pH 7
G = (-113.44 + -94.45) - (-190.62 + 0) = -17.27 kcal/mol
In biochemistry-usually deal with physiological conditions (10-7M H+)
G ‘ = (-113.44 + -94.45) - (-190.62 + -9.87) = -7.4 kcal/mol
Gibb’s Free Energy
Since the spontaneity of a reaction is independent of
the pathway, we can force G of an overall reaction
to be negative even though it has a positive step by
coupling it to a step with greater -Gf.
Reaction A
Method A
(steps 6,7)
C
Pi +
NAD+
same as
NADH
Gº = +1.5
GAP
MgADP
1,3-BPG
Glyceraldehyde-3phosphate dehydrogenase
A
B
MgATP
Gº = -4.5
3PG
3-phosphoglycerate
kinase
Gº = -3.0
Net reaction
GAP
3PG
C
Gibb’s Free Energy
Method B
(step 1)
C
D
A
B
Glucose + Pi
MgATP
Glucose + Pi + MgATP
Net reaction
G6P
Gº = +3.3
MgADP + Pi
Gº = -7.3
G6P + MgADP + Pi Gº = -4.0
• ATP is the energy
currency of cells
• In phototrophs, light
energy is transformed
into the light energy of
ATP
• In heterotrophs,
catabolism produces
ATP, which drives
activities of cells
• ATP cycle carries
energy from
photosynthesis or
catabolism to the
energy-requiring
processes of cells
ATP
ATP is the general “high energy” coupler molecule
used in biochemistry
N
N
O
N
N
O
CH2
O
O
-O-P-O-P-O-P-OO- O- O-
MgATP
MgADP
MgATP
AMP
HO
OH
Mg++
Mg Adenosine triphosphate (MgATP)
ADP + Pi
AMP + Pi
AMP + Pi
Adenosine
+ Pi
Gº
-7.3
-7.4
-7.4
-3.0
ATP
O
Why is the presence of the
energetically unfavorable?
O
P-O-P anhydride so
(1) Ability of products to delocalize electrons (all phosphates)
O
O
P=O
-O
P
O-
O
-O
O-
Free ATP
(2) Close proximity of charges (bond strain)
O
O
+
O
+
pH 6.0
pH 9.0
Gº
7.89
9.56
0.0 M Mg
0.001 M
Mg
0.01 M Mg
-8.4
-7.7
-7.5
Adenosine-O-P-O-P-O-P-OO-
O-
OpK2
ATP-4
Phosphoric Acid Anhydrides
• ADP and ATP are
examples of phosphoric
acid anhydrides
• Large negative free
energy change on
hydrolysis is due to:
– electrostatic repulsion
– stabilization of
products by ionization
and resonance
– entropy factors
Phosphoryl-group Transfer
• Energy produced from a rxn can be coupled to
another rxn that requires energy to proceed.
• Transfer of a phosphate group from high energy
phosphorylated compounds can activate a substrate
or intermediate of an energy requiring rxn.
A-P + ADP -> A + ATP, ATP +C-> ADP + C-P
• The ability of a phosphorylated compound to transfer
a phosphoryl group is termed its phosphoryl-grouptransfer-potential.
Phosphoryl-group Transfer
Redox chemistry
In addition to energetics -must balance redox
chemistry
6 CO2 + 6 H2O
Glucose (C6H12O6) + 6 O2
Broken down into “half pathways”
Glycolysis
Glucose
Active hydrogen 2H+ + 2e-
2 pyruvate + 2 (2H)
Mitochondria
(2H) + 1/2 O2
H2O
Common carrier of (H)
O
N
N
O
N
N
O
HO
C-N-H2
O
CH2-O-P-O-P-CH2
O
O- OOH
HO
N(+)
OH
Pi
NAD(P) Nicotinamide adenine dinculeotide (phosphate)
(oxidized form)
NAD+ + 2e-
NADH + H+
Common carrier of (H)
H
H
N
N
O
N
N
O
HO
C-N-H2
O
CH2-O-P-O-P-CH2
O
O- OOH
O
HO
N
OH
Pi
NAD(P) Nicotinamide adenine dinculeotide (phosphate)
(reduced form)
NADH + H+
NAD+ + 2e-
Eº ‘ = 0.31 volt
Thermodynamically
2e- + 2H+ + 1/2 O2
NADH + H+
H2O
NAD+ + 2H+ + 2e-
NADH + H+ + 1/2 O2
Eº’ = +0.82 volt
Eº’ = +0.31 volt
NAD+ + H2O Eº’ = +1.13 volt
Convert using the Nernst Equation
Ease at which molecule
donates electron(s)
Gº ‘ = -nF Eº‘ F = faraday= 23,086 cal
aka electromotive force
n=mol e-
Gº ‘ = -2(
23,086 cal
mol  e-  volt
mol  e-  volt
)131 volt)
Gº ‘ = -56 kcal/mol
ATP and NAD(P)H
So in metabolism, ATP formed in reaction sequences where
Gº‘ > Gº‘ hydrolysis of ATP (catabolism)
Used to drive reaction with Gº‘ < Gº‘ hydrolysis (<0)
NAD(P)H production and ATP production are usually coupled
ATP and NAD(P)H are coenzymes and therefore need to be
recycled.
Thermodynamics and
Metabolism
• Standard free energy A + B <-> C + D
•
Go’ =-RT ln([C][D]/[A][B])
•
Go’ = -RT ln Keq
•
Go’ < 0 (Keq>1.0) Spontaneous forward rxn
•
Go’ = 0 (Keq=1.0) Equilibrium
•
Go’ > 0 (Keq <1.0) Rxn requires input of energy