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Ch3: Energy, Catalysis and

Biosynthesis
BSC 300
Lecture 5
Thursday, September 3

K also indicates the strength of

molecular interacEons
A small change in the number of non-
covalent interacEons can have a huge
impact on binding energy.

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K also indicates the strength of

molecular interacEons
K also predicts spontaneity of non-
covalent interacEons, which are
criEcally important in diverse molecular
interacEons, like enzyme-substrate
binding.
Here, K represents the associaEon (KA)
or disassociaEon (KD) constants
(inversely proporEonal to one another),
and as with Keq also depend on reactant
and product concentraEons.

K also indicates the strength of

molecular interacEons
Two molecules will bind each other if
free energy change is negaEve.
KA reects binding strength between
these two molecules and can be used
to quanEfy the specicity of the
interacEon.
At equilibrium the rates of formaEon of
bound vs unbound complexes are equal
As with Keq a larger KA means a greater
drop in free energy: higher binding
energy

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Changes in free energy are addiEve for

sequenEal reacEons
Enzymes can not only directly couple reacEons, but can also link
sequenEal reacEons through facilitaEng the producEon and
consumpEon of reactants/products
If the overall free energy change for individual reacEons in a
pathway is negaEve then the process moves in the forward
direcEon even if individual steps have a posiEve change in free
energy
-0.2

-2.1

4.1

-1.2

-3.2

Changes in free energy are addiEve for

sequenEal reacEons
How?:

An unfavorable reacEon can be
driven by an favorable reacEon acEng
as a chemical siphon - literally
drawing the reacEon in one direcEon
by pulling the product of one reacEon
forward through the pathway.

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Thermal moEon allows enzymes to nd their

substrates
Diusion of molecules within a cell is extremely
rapid due to their kineEc energy but is
proporEonal to their size.
As a result diusion of small molecular substrates
is one of the rate limiEng factors of enzymaEc
reacEons and is proporEonal to the substrate
concentraEon.
Ex. a high concentraEon of substrate (0.5mM)
leads to an approximate 500,000 enzyme
substrate collision per second, lowering the
concentraEon 10-fold leads to an equal decrease
in E S interacEons.

Thermal moEon allows enzymes to nd their

substrates
Even then, if the molecules are not well
matched, their interacEon is transient.
The power of enzymes comes from their
complementary shape to the conforma0ons of
their substrates and the large number of
electrostaEc interacEons that are formed upon
contact.
Therefore, in order to catalyze a reacEon, an
enzyme must nd and bind its substrate

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Enzyme performance
1. Find and bind
2. Catalyze reacEon
3. Release substrate
The rates of each of these steps varies from enzyme
to enzyme and are quanEable value which well
explore now

How we know: Measuring Enzyme performance

Determining reacEon speed: Max velocity

Rxns under standard condiEon with:
Fixed concentraEon of enzyme, and
Increasing substrate concentraEon

Monitor either the appearance of product

or disappearance of reactant (mol/Eme)
Number of was to measure: Textbook
describes spectrophotometry

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How we know: Measuring Enzyme performance

These values are then plo`ed, generaEng a
hyperbolic curve that reaches a plateau
where velocity (v) no longer increases with
the addiEon of substrate.

This value is called the Vmax or Maximum
velocity

At this value the enzyme has become

saturated (fully occupied) by substrate
The number of molecules an enzyme

can process per second is its turnover
And the rate of product formaEon is
number
dependent enErely on the intrinsic kineEcs
of the enzyme

How we know: Measuring Enzyme performance

Because reacEons taper o as
substrate is added, it is dicult to
get a precise measure of Vmax
By plocng the inverse of v and [S]
produces a plot known as the
Double-reciprocal or Lineweaver-
Burk Plot, a straight line with two
important points

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How we know: Measuring Enzyme performance

1. A precise measurement of Kmax: The
Y intercept of the line is 1/Vmax
2. The Michaelis constant (KM) is
dened by the X-intercept (-1/KM)
and equals the substrate
concentraEon where the enzyme
works at half its maximal speed.
KM is an inverse measure of an
enzymes anity for its substrate.
Small KM = robust binding
Large KM = weak binding

Whats the value in determining KM?

Helps researchers studying enzymaEc pathways determine
order of interacEons, as well as potenEal bo`lenecks in
pathways, where within a pathway a new drug might aide
in speeding up or slowing down producEon of a specic
metabolite.
Also explains much of the physiological diversity observed
within a populaEon.

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Whats the value in determining KM?

For example:

alcohol
dehydrogenase

CH3CH2OH + NAD+ ------------------------------- > CH3CHO + NADH

acetylaldehyde

dehydrogenase
CH3CHO + NAD+ -------------------------------- > CH3COO- + NADH
Two forms of acetylaldehyde dehydrogenase:

A low KM mitochondrial form (which promotes rapid turnover of acetylaldehyde)
A high KM cytosolic form, that oxidizes acetylaldehyde very slowly.

Some people inherit a mutaEon in the mitochondrial acetylaldehyde gene that subsEtutes
a glutamic acid (acidic side chain) with a lysine (basic).

EnzymaEcally catalyzed reacEons can be inhibited

Enzyme acEvity can be altered through binding of molecules
other than substrate: small molecules, substrate analogs,
products, inhibitors and acEvators.

Cells use such regulaEon to control when and how much
product is generated

Determining how an inhibitor decreases enzyme acEvity can
elucidate how cells regulate specic metabolic pathways

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CompeEEve vs non-compeEEve
inhibiEon

Compe66ve inhibitors
Resemble substrate in structure and bind the enzymes acEve site.
The maximum velocity of enzymaEc reacEon is reached, but KM is
larger than uninhibited rxn
Can be overcome with high substrate/inhibitor raEo.

CompeEEve vs non-compeEEve
inhibiEon

Noncompe66ve inhibitors
Bind to sites other than acEve site.
Alter enzyme conformaEon reducing binding eciency
The maximum velocity of enzymaEc reacEon cannot be reached, but
binding anity (KM) is not altered.
Cannot be overcome with high substrate/inhibitor raEos.

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AcEvated carriers and biosynthesis

Energy released by favorable reacEons of most metabolic processes (like
cellular respiraEon) is not immediately used, but stored in acEvated
carriers: Small organic molecules with high-energy covalent bonds.
Small size allows rapid diusion and distribuEon throughout the
cytoplasm.
AcEvated carriers store energy as
transferrable high-energy chemical
groups or electrons. The transfer of
these groups releases energy in

coupled reacEons.

Adenosine triphosphate
Highly versaEle acEvated carrier
Hydrolysis of ATP to ADP and
inorganic orthophosphate is a
highly favored reacEon with a
G=-7.3kcal/mol

Conversely, the condensaEon reacEon of ADP + Pi to form ATP is
highly unfavored and requires am equal amount of energy.

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Adenosine triphosphate
EnzymaEc hydrolysis of ATP involves
transfer of terminal 1 or 2 phosphate
groups to a substrate.
This hydrolysis releases energy as
highly stable covalent bonds are
formed between the phosphate and
substrate.
Energy released by ATP hydrolysis comes not from breaking phosphate
bonds, but from forma0on of more stable bonds in inorganic phosphate.
Such phosphorylaEon reacEons serve to acEvate substrates, mediate
exchange of chemical energy and phosphorylated molecules serve diverse
roles especially in cell signaling.

QuesEon 3-8
The phosphoanhydride bond that links two
phosphate groups in ATP in a high-energy linkage
has a G of -7.3kcal/mol. Hydrolysis of this bond in
a cell liberates from 11 to 13 kcal/mol of useable
energy. How can this be? Why do you think a range
of energies is given, rather than a precise number
as for G?

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Energy stored in ATP is osen harnessed to join

two molecules together
CondensaEon reacEons are highly
unfavorable, but can be powered by
enzymaEc coupling with ATP hydrolysis.
In the simplest two step reacEon the OH
of one reactant is replaced by the
terminal phosphate group of ATP
This high-energy intermediate then
readily reacts with the second substrate,
loosing the phosphate and forming a new
covalent bond

NADH and NADPH are acEvated carriers of electrons

NicoEnamide adenine
dinucleoEde
NicoEnamide adenine
dinucleoEde phosphate
NADH and NADPH are
reduced electron carriers
The nicoEnamide ring is
reduced by 2 electrons and
a proton (a hydride ion
collecEvely) in diverse
cellular processes

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NADH and NADPH are acEvated carriers of electrons

When the acEvated
(reduced) electron carriers
pass their electrons to an
acceptor they are oxidized
to NAD+ and NADP+
respecEvely
OxidaEon of NADH/NADPH and hydrolysis of ATP occur because in
both reacEons there is a negaEve free energy change from
reactants to products.

NADH and NADPH have dierent cellular roles

NADH and NADPH have equivalent Redox
potenEal
But, the phosphate group alters
conformaEon, so they are not recognized
by same enzymes
NADH is used exclusively in ATP synthesis
and there is a high NAD+ to NADH raEo
NADPH is used as a reducing agent for
anabolic processes and is kept in high
concentraEon compared to the oxidized
form NADP+

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Range of acEvated carriers in cell processes

Flavine adenine dinucleoEde (FADH2) another electron carrier
used in ATP biosynthesis
Other energy carriers transfer a range of groups during
biosynthesis
InteresEngly, most carrier



molecules are build around





a nucleoEde. This supports





a widely accepted




hypothesis that early life




was RNA based and RNA and its nucleoEdes served most
biological roles from geneEc storage, energy transfers and
enzymaEc processes.

GeneraEon of acEvated carrier

Usually coupled to ATP hydrolysis in mul0step enzyma0c reac0ons
Ex. Pyruvate carboxylase: BioEn is a vitamin used by many
enzymes like PC. It is charged with a carboxyl group which is then
transferred to a substrate molecule
Hydrolysis of ATP drives this reacEon
by splicng the bicarbonate and
joining CO2 to Pi before it is released
The freed CO2 is then a`acked by
bioEn forming a high-energy bond
Which allows its transfer to pyruvate

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Synthesis of biological polymers Energy Input

DisEnct enzyme catalyzed pathways regulate the polymerizaEon
(unfavorable condensaEon reacEons) and catabolism (favorable
hydrolysis reacEons) of cells macromolecules
Though catalyzed by dierent enzymes, the chemistry is similar
for polysaccharides, pepEdes and nucleic acids.
The condensaEon step in each reacEon depends on the
hydrolysis of a nucleoEde triphosphate.

Synthesis of biological polymers Energy Input

CondensaEon reacEons:

As was the case for conversion of
glutamic acid to glutamine, the OH
group to be removed is rst
acEvated by forming a high-energy
bond to a second molecule
For pepEdes and polysaccharides
addiEonal intermediate molecules
are generated during the process
(pepEde synthesis CH7).

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LimitaEons to ATP hydrolysis

Hydrolysis of the terminal () phosphate group from ATP yields
G=-7.3kcal/mol.
Under cellular physiology this number is closer to -11-13 kcal/mol
How is this possible?
SEll many reacEons have a higher + G than this
Some reacEons are driven by an alternate route to ATP hydrolysis

Pyrophosphate hydrolysis
DNA polymerase catalyzes the
hydrolysis of ATP cleaving the
terminal 2 phosphates a
molecule called pyrophosphate.

Wikibooks

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Pyrophosphate hydrolysis
This reacEon yields an
approximate - 26kcal/mol due to
the resulEng pyrophosphate.
How? Another enzyme rapidly
catalyzes cleavage of
pyrophosphate to 2
orthophosphates (Pi). Because
each yields approximately 13 kcal/
mol, this reacEon is very
exergonic.

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