Transcript Microbial Metabolism- Energy and Enzymes

Chapter 8: Energy, enzymes, and regulation
Energy and life
1st law of thermodynamics:
Law of Conservation of Energy.
Energy cannot be created or destroyed
Then why do we talk about the “energy crisis?”
What does it mean to be phototrophic vs chemotrophic?
(Light as energy source vs chemical energy source)
What does ATP synthetase or photosynthetic reaction center do?
Energy transduction
Enzymes can convert one form of energy into another form.
* Examples?
Myosin in muscle:
chemical to mechanical energy
ATP synthase:
transmembrane gradient into chemical energy
Flagellum:
transmembrane gradient into motion
Photosynthetic
reaction center:
light into transmembrane gradient
Electron transfer chain
in mitochondria:
chemical energy into transmembrane proton gradient
2nd law of thermodynamics: entropy (disorder)
of an isolated system always increases
Is a living organism in a relatively low or high state?
How to grow from a seed or an embryo to an adult organism?
Decrease in entropy?
Entropy:
A measure of the
randomness or disorder
of a system
The greater the disorder
the greater the entropy
Energy = The capacity to do work or to cause particular changes.
Chemical work
The synthesis of complex biological molecules from simpler
precursors
Transport work
The ability to transport molecules against a concentration
gradient (uptake of nutrients, elimination of waste, maintenance
of ion balance)
Mechanical work
Changing the location of organisms (e.g., flagellum), cells
and structures within cells
Efficiency of energy conversion?
Less than 100%. Question: Where does the rest go?
Heat: thermal motion of molecules without (strong) thermal
gradient. It is often difficult to capture this form of energy for
doing work
Idea: Eventual thermal death of the universe. Is being debated.
Bottom line for biology: living systems need input of energy
to keep functioning.
Question: what is the overall energy source driving the
biosphere on earth?
Sun light: photosynthesis
Free energy G and chemical reactions
G = H - TS
G = change in free energy (amount of energy available to do work)
Describes direction of spontaneous processes. Reactions with a negative
G value will occur spontaneously
H = change in enthalpy (heat content)
T = temperature in Kelvin (C + 273)
S = change in entropy
Standard free energy (G )
and the equilibrium constant
When G is determined under standard conditions of concentration,
pressure, and temperature the G is called the standard free energy
change (G)
If the pH is set to 7, the standard free energy change is indicated by
the symbol G´
A+B⇄ C+D
G´ = -RT ln Keq
Keq = [C] [D] / [A] [B]
Reactions proceed in the direction
of negative G´
Reaction will proceed
to the right (downhill process)
Reaction will proceed
to the left (uphill process)
Key issue: how can cells achieve essential
reactions with a positive G´?
Examples:
Nutrient uptake
DNA replication
Amino acid biosynthesis
CO2 fixation
Flagellar motion
ATP synthesis
By coupling an uphill process to a
downhill process
This makes the overall reaction downhill, so it will proceed
Free energy input is needed to sustain life and growth
Main downhill processes? ATP hydrolysis and proton motive force
A major role of ATP is to
drive otherwise endergonic
reactions
Energy cycle
Note: this is simplification, because it ignores coupling of
proton motive force to all three forms of work
Adenosine 5´-triphosphate (ATP)
ATP serves as the major energy
currency of cells
“Contains 2 high energy bonds”.
Note: there is nothing particularly
special about these two bonds
except that cells happen to use
them.
ATP  ADP + Pi + Energy
Pi = orthophosphate
Note: ATP is complexed to Mg2+
Oxidation-reduction reactions
Oxidation-reduction reactions are key in almost all energy
metabolism of life (respiration, photosynthesis, and also
fermentation, glycolysis):
Coupled to the generation of ATP, proton motive force.
Loss of electrons is oxidation (LEO)
Gain of electrons is reduction (GER)
Aerobic respiration is when O2 acts as the final electron
acceptor (O2  H2O)
Acceptor + ne- ⇄ donor, n = number of electrons transferred
Quantifying redox reactions
1. Split redox reactions into two half reactions involving two redox pairs.
Example: Fe3+ + Cu+  Fe2+ + Cu2+
Fe3+ + e-  Fe2+ (electron acceptor)
Cu+  Cu2+ + e- (electron donor)
2. Redox potential E (similar to G) = EA - ED
The equilibrium constant of a redox reaction is called the standard
reduction potential (E). G = -nFE F=constant of Faraday
2. Define hydrogen half reaction as the absolute reduction reduction
potential: 2H+ + 2e- ⇄ H2
The reference standard for reduction potentials is the hydrogen system with
an E´ of - 0.42 volts (at pH 7).
Note: a positive E corresponds to a negative G: electrons will flow to
the compound with the most positive E
In our mitochondria:
NADH + H+ ⇄ NAD+ + 2H+ + 2eO2 + 2H+ + 2e- ⇄ H2O
-0.32 V
0.82 V
E = EA - ED
so: 0.82 - - 0.32 = 1.14V
G = -nFE
G = -2*23*1.14 = -54.4
kcal/mol
ATP hydrolysis: -7.3 kcal/mol
Respiration in our
mitochondria yields 1.14V of
driving force to convert into
other forms of energy (pmf)
Electrons flow to more positive redox potential
Electrons flow from donors
with more negative redox
potential to acceptors with
more positive redox potential.
Key electron carriers
Electron carriers serve to transport electrons between different
chemicals
Example - Nicotinamide adenine dinucleotide (NAD)
NADH + H+ + 1/2 O2  H2O + NAD+
NAD+/ NADH is more negative than 1/2 O2/ H2O, so electrons
will flow from NADH (donor) to O2 (acceptor)
Structure of NAD
Water soluble electron carrier
Photosynthesis
Flavin adenine dinucleotide (FAD)
Proteins bearing FAD (or
FMN) are referred to as
flavoproteins
FAD is usually
bound to proteins
Coenzyme Q (CoQ) or ubiquinone
Transports electrons and
protons in respiratory
electron transport chains.
Residues in membrane
(hydrophobic molecule)
Note:
* One-versus two-electron
processes
* In some cases electron
transfer is coupled to
protonation/deprotonation
Cytochromes
Cytochromes are redox
proteins that bind a heme.
They use the iron atoms in
the heme to reversibly
transport a single electron
Iron atoms in cytochromes
are part of a heme group
Nonheme iron proteins carry
electrons but lack a heme
group (e.g. Ferrodoxin)
Enzymes
Enzymes are protein catalysts
* Enzymes catalyze an astonishing array of different reactions
* Enzymes speed up reactions without altering their
equilibrium position. Note: they can couple down-hill and
uphill reactions
* Enzymes are permanently chemically altered during
catalysis
* Enzymes tremendously speed up reactions: typically 109
*Enzymes are highly specific
Reacting molecules = substrates
Substances formed = products
Enzymes can have cofactors
Some enzymes are composed purely of protein)
Some enzymes contain both a protein and a nonprotein
component: a cofactor (like FAD)
The protein component = apoenzyme
The nonprotein component = cofactor
Apoenzyme + cofactor = holoenzyme
Cofactor tightly attached to apoenzyme = prosthetic group
Loosely bound cofactor = coenzyme
Classification of enzymes
Enzymes can be placed in six classes and are usually named in
terms of substrates and reactions catalyzed.
Mechanisms of enzyme activity
Central effect: enzymes speed up the rate at which a reaction
proceed to equilibrium by lowering the activation energy
Activation energy required to from the transition state (AB‡)
Lock-and-key model
Some enzymes are rigid and
shaped to precisely fit the
substrate(s)
Binding to substrate
positions it properly for
reaction
Referred to as the lock-andkey model
Induced fit model
Some enzymes change shape when they bind their substrate so
that the active site surrounds and precisely fits the substrate
This is referred to as the induced fit model
Glucose binding to hexokinase
Describing enzyme activity: Km and Vmax
* Add various concentrations of substrate [S] to a constant amount of
enzyme and measure the initial rate V0 (or v) of the reaction.
Question: why the initial rate?
* Repeat this for various substrate concentrations and plot V0 versus [S].
Question: what will the curve look like?
And: Where have we seen this curve before?
Michaelis-Menten kinetics: Km and Vmax
Hyperbolic dependence of V0 on [S]
Saturation behavior: why?
Effect of temperature on enzyme activity
Enzymes are most active at optimum temperatures; deviation
from the optimum can slow activity and damage the enzyme
Question: Where have we seen this before?
Effect of pH on enzyme activity
Enzymes often have pH optimium.
Question: how to explain this?
Active site of serine protease
Change in protonation state
of active site residues. Here:
Asp and His. pKa values
Enzyme inhibition
Many poisons and antimicrobial agents are enzyme inhibitors
Can be accomplished by competitive or noncompetitive inhibitors
Competitive inhibitors - compete with substrate for the active site
Noncompetitive inhibitors - bind at another location
Competitive inhibitors
Usually resemble the
substrate but cannot be
converted to products
Malonate is competitive inhibitor of succinate dehydrogenase
Noncompetitive inhibitors
Bind to the enzyme at some location other than the active site
Do not compete with substrate for the active site
Binding alters enzyme shape and slows or inactivates the enzyme
Heavy metals often act as noncompetitive inhibitors
(e.g. Mercury)
Metabolic regulation
Important to conserve energy and resources
Cell must be able to respond to changes in the environment
Changes in available nutrients will result in changes in
metabolic pathways
Control of enzyme activity
* Allosteric control
* Covalent modification
Allosteric enzymes
Activity of enzymes can be
altered by small molecules
known as effectors or
modulators
Effectors bind reversibly and
noncovalently to the
regulatory site
Binding alters the
conformation of the enzyme
Positive and negative
effectors
Example: regulation of aspartate carbamyltransferase
Regulation of aspartate carbamyltransferase is a well studied
example of allosteric regulation
CTP inhibits activity and ATP stimulates activity
ACTase regulation
Binding of effectors cause conformational changes that result in
more or less active forms of the enzyme
Top view
Side view
T state
Less active
R state
More active
ACTase regulation
CTP inhibits activity and ATP stimulates activity
Binding of substrate also
increases enzyme activity
(more than one active site)
Velocity vs. substrate curve
is sigmoid
Covalent modification of enzymes
Attachment of group to
enzyme can result in
stimulation or inhibition of
activity
Attachment is covalent and
reversible
Example: phosphorylase b
from Neurospora crassa
Question: where have we
seen this?
Feedback inhibition
Metabolic pathways can
contain a pacemaker enzyme
(rate-limiting step)
Usually catalyzes the first
reaction in the pathway
Activity of the enzyme
determines the activity of the
entire pathway
Feedback inhibition occurs
when the end product
interacts with the pacemaker
enzyme to inhibit its activity