Section V: Protein Function

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Section V Protein Function
- proteins perform nearly all of the functions
required in biological systems
2 principles of biochemistry
- Many proteins interact with other
macromolecule(s) or substrate(s) to carry out
their function(s)
Qualifying statement - all macromolecules in
cells (proteins, DNA, RNA, lipids, carbohydrates,
vitamins, etc.) as well as small molecules
(salts, etc.) are essential to life and required
for biological systems to function
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Protein Function
Some functions of proteins
1. Structural. collagen, elastin 2. Mobility.
actin/myosin,tubulin, flagella 3. Receptors.
insulin receptor 4. Ligands. insulin 5. Immune
system. (antibodies)... 6. housekeeping.
chaperones... 7. signalling. kinases... 5.
Enzymes. (proteases) 6. Storage and Transport.
(haemoglobin)
over the next several lectures we will examine
in some detail, examples of these two protein
functions
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We have already very briefly talked about enzymes
in sections II. These key points were made

• They are catalysts, that dramatically increase
  the rate of reaction. (106 or more)
• Highly specific in terms of substrate in terms
  of reaction
• Enzymes do not drive reactions. They allow
  favorable reactions to happen faster
• Enzymes (and other catalysts) increase the
  reaction RATE by decreasing ?G.

denotes transition state
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This rise is needed to ensure the reaction
doesn't always occur
- before we look more at catalysis, let us look
at a simple reaction, A ? B - in this example a
carbohydrate is altering its conformation to a
more energetically favorable chair conformation
from a boat conformation - during the reaction,
the molecule reaches a point of highest energy
and at this point the half-chair conformation
is exactly intermediate to the other two - in
chemical reactions this intermediate is called a
TRANSITION STATE
Additional free energy needed for reaction to
occur
Figure 11.2
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Transition State
uncatalyzed reaction
?G1(A?A)
Free energy, G
GA
?G2(A?B)
Intermediate State (A)
GB
Reaction Coordinate
A?A?B
- the activation energies for the formation of
the intermediate state, and its conversion to the
final product are each lower than the activation
energy for the uncatalyzed reaction
-intermediate state- resembles transition state
but with lower energy due to interaction with a
catalyst - transition state defines free energy
maximum state
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How Does an Enzyme Catalyze a Reaction?
The role of an enzyme is to lower ?G - this is
usually done by stabilizing the transition state
Emil Ficher (1894) The Lock Key hypothesis -
explains substrate specificity - says nothing
about why catalysis occurs Dan Koshland
(1958) Induced Fit hypothesis - enzymes prefer
to bind to a distortion of the substrate that
resembles the transition state - both enzyme and
substrate must adjust to one another - in
reality, enzyme is not distorted but has
evolved to bind in a certain way and sometimes
undergo conformational changes
TWO MODELS
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Figure 11.7 two models for enzyme-substrate
interactions
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Figure 11.8 Induced conformational change in
hexokinase
- catalyzes phosphorylation of glucose to glucose
6-phosphate during glycolysis - such a large
change in a proteins conformation is not
unusual BUT not all enzymes undergo such large
changes in conformation - not only enzymes
change their conformations many
structural/motor proteins undergo large
conformational changes during their cycles
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How enzymes aid in the catalytic process

• Bind substrates
• Lower the energy of the transition state
• Directly promote the catalytic event
• Either through acidic or basic side chains that
  promote addition or removal of proteons
• Or through holding ions in correct position to
  participate in the catalysis
• Release the products

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Transition State Analogue
Sometimes, it is possible to synthesize a stable
molecule that happens to resemble the (unstable)
transition state of a reaction - these
Transition State Analogues (TSA), as expected,
bind the enzyme active site tightly, and act as
potent inhibitors for the enzyme - TSAs also help
to confirm the structure of the natural
transition state - TSAs are sometimes
co-crystallized with the enzymes in order to
observe what the catalytic mechanism of the
enzyme is
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Entropic and enthalpic factors in catalysis
molecules often need to go through energy
demanding (strained/distorted) conformations
for reaction to take place
? G ? H - T ? S
activation energy is lowered during catalysis
energy required for the reaction
change in entropy (degree of conformational flexib
ility) during reaction
solved by having an intermediate state that
resembles the transition state but is of lower
energy because of favourable binding to the
catalyst
cannot be too large or else reaction will be slow
- see figure 11.6 in Mathews
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Cofactors
In many cases we find proteins use prosthetic
groups to aid in catalysis or some other role.
These groups are termed cofactors Cofactors form
an intricate part of the active site and play a
direct chemical role in the chemistry of the
reaction
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Bond polarization
An example of how an enzyme polarizes bonds in a
substrate to facilitate a chemical reaction
Zn2 is a Cofactor
Polarizes the CO bonds and also polarizes an O-H
bond in water, allowing for HO- formation.
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Allostery
When a small molecule can act as an effector or
regulator to activate or inactivate an action of
a protein - the protein is said to be under
allosteric control. The binding of the small
ligand is distant from the proteins active site
and regulation is a result of a conformational
change in the protein when the ligand is
bound Many types of proteins show allosteric
control - haemoglobin (NOT myoglobin) - various
enzymes - various gene-regulating proteins
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Lac Repressor as an Allosteric Protein
This is a protein that regulates the expression
of a few genes in E. coli - we will see this
protein as an example again when we talk about
prokaryote gene expression
Lactose
Lac operator DNA sequence
Active Repressor
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Enzyme catalyzed processes SERINE PROTEASES

• Have a Serine residue that is important for the
  catalysis
• Chymotrypsin Needed for protein digestion.
• - selectively cleaves at the carboxyl side of
  BULKY, HYDROPHOBIC amino acids.
• Tyr
• Trp
• Phe
• Met

Aromatic Side Chains
Large, hydrophobic side chain
N-term
H2O
C-Term
Hydrophobic
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Chymotrypsin can also cleave ESTER linkages.
H2O
P-nitrophenyl acetate (Colorless)
This is not biologically relevant but
a convenient way to study the reaction
P-nitrophenolate (Yellow)
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Catalysis of peptide bond hydrolysis by
chymotrypsin
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Other Serine Proteases

• Chymotrypsin (Cuts -COOH side of Hydrophobic
  Groups)
• Trypsin (Cuts -COOH side of Arg Lys)
• Elastase (Cuts -COOH side of Val Thr)

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Why these proteases recognize different amino
acids sequences
recognition, or specificity sites
Active site pockets -always close to the active
site serine -trypsin- long, narrow, negatively
charged (perfect for lys or arg side
chains) -chymotrypsin- wide hydrophobic pocket
(perfect for phe)
Figure 11.12
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carboxypeptidase A
Figure 11.27 Another type of peptidase
This is an example of a metallo-enzyme -
metallo-enzymes are a class of enzymes which
utilize metal cofactors
Zn2 stabilizes and helps promote the formation
of O- during the reaction
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Enzyme Kinetics
Now we will look at how enzymes are studied on
the level of function or catalysis (as apposed to
structure)
Vmax maximal rate- all active sites bound-
saturation - it is related to a constant for a
given enzyme kcat by the following relationship
VmaxkcatE - Km represents the substrate
concentration at which 1/2 of Vmax is achieved -
Km is also a constant for a given enzyme
Vmax
Rate (v)
Vmax/2
Substrate
Km
common abreviations E, enzyme S, substrate
P, product k, rate constant (measure of how
fast a reaction occurs) v, rate I, inhibitor
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What do these 3 quantities means in terms of
enzyme kinetics?
Km Relates to how strongly an enzyme binds its
substrate (constant) Kcat Relates to how rapid a
catalyst the enzyme is- measures product
formation (substrate turnover)(constant) Vmax Re
lated to kcat and E by VmaxkcatE
High Km means strength of binding is low- weak
affinity
High Kcat means high speed of catalysis
High Vmax means high rate of catalysis
kcat / Km is a measure of enzyme efficiency
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Michaelis Menton Kinetics
k1
k2
E S ES E P
k-1
(slow)
(fast)
Here, kcat k2
V kcat ES
Rate of Reaction By rearrangement we can
get Michaelis Menton rate equation
-For any reaction that follows M-M kinetics, Km
S where Vmax/2 -How do you find Vmax and Km?
Use double reciprocal plot
- derivation of this in Mathews pages 376-377
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Lineweaver Burke Plots
Here we plotted reaction rate vs. S for a
fixed amount of enzyme
How to derive Vmax and Km from experimental
observations- Plot the observed velocity for a
number of S
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Measuring Enzymatic Rates
- ideally done with a system where the product or
substrate absorb a particular wavelength of light
(i.e. P-nitrophenyl acetate) - this depends on
enzyme, though - reaction can be monitored with a
spectrophotometer by measuring the appearance of
product or disappearance of substrate
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Why does the enzymatic rate level off?
Vmax
Catalyzed
RATE
Uncatalyzed
Substrate
- at low substrate, the enzyme active site is
occupied at a frequency ? substrate - however,
at a certain point, saturation is reached -
enzyme active site is always occupied - it is
operating as maximum rate, Vmax
- most cellular enzymes do not operate at Vmax -
however, under certain conditions the cell needs
more of particular enzymes (e.g., more enzymes
for galactose metabolism are made in its presence)