Protein Solubility

The solubility of proteins in aqueous buffers depends on the

distribution of hydrophilic and hydrophobic amino acid residues on
the proteins surface.

Hydrophobic residues predominantly occur in the globular protein
core, but some exist in patches on the surface. Proteins that have
high hydrophobic amino acid content on the surface have low
solubility in an aqueous solvent

Charged and polar surface residues interact with ionic groups in the
solvent and increase solubility
Protein precipitation using neutral-salt
Salting out

Addition of a neutral salt, such as
ammonium sulfate, compresses the
solvation layer and increases protein-
protein interactions. As the salt
concentration of a solution is
increased, more of the bulk water
becomes associated with the ions. As a
result, less water is available to partake
in the solvation layer around the
protein, which exposes hydrophobic
patches on the protein surface. Proteins
may then exhibit hydrophobic
interactions, aggregate and precipitate
from solution.
Protein precipitation at Isoelectric point
The isoelectric point (pI) is the pH of a solution at which the net
primary charge of a protein becomes zero. At a solution pH that is
above the pI the surface of the protein is predominantly negatively
charged and therefore like-charged molecules will exhibit repulsive
forces.

Likewise, at a solution pH that is below the pI, the surface of the
protein is predominantly positively charged and repulsion between
proteins occurs.

However, at the pI the negative and positive charges cancel, repulsive
electrostatic forces are reduced and the attraction forces predominate.
The attraction forces will cause aggregation and precipitation.

The greatest disadvantage to isoelectric point precipitation is the
irreversible denaturation caused by the mineral acids
Protein denaturation
Denaturation of proteins involves
the disruption and possible
destruction of both the secondary
and tertiary structures. Since
denaturation reactions are not
strong enough to break the peptide
bonds, the primary structure
(sequence of amino acids) remains
the same after a denaturation
process. Denaturation disrupts the
normal alpha-helix and beta sheets
in a protein and uncoils it into a
random shape.
Heat:
Heat can be used to disrupt hydrogen bonds and non-polar
hydrophobic interactions. This occurs because heat increases
the kinetic energy and causes the molecules to vibrate so
rapidly and violently that the bonds are disrupted. The proteins
in eggs denature and coagulate during cooking. Other foods are
cooked to denature the proteins to make it easier for enzymes to
digest them. Medical supplies and instruments are sterilized by
heating to denature proteins in bacteria and thus destroy the
bacteria.
Alcohol (or organic solvents) Disrupts Hydrogen Bonding
Hydrogen bonding occurs between amide groups in the secondary
protein structure. Hydrogen bonding between "side chains" occurs in
tertiary protein structure in a variety of amino acid combinations. All
of these are disrupted by the addition of another alcohol. Alcohol
denatures proteins by disrupting the side chain intramolecular
hydrogen bonding. New hydrogen bonds are formed instead between
the new alcohol molecule and the protein side chains.
Acids and Bases
Disrupt Salt Bridges:
Salt bridges result from the neutralization of an acid and amine on
side chains. Review reaction. The final interaction is ionic between the
positive ammonium group and the negative acid group. Any
combination of the various acidic or amine amino acid side chains will
have this effect. As might be expected, acids and bases disrupt salt
bridges held together by ionic charges. A type of double replacement
reaction occurs where the positive and negative ions in the salt change
partners with the positive and negative ions in the new acid or base
added. This reaction occurs in the digestive system, when the acidic
gastric juices cause the curdling (coagulating) of milk.
Heavy Metal Salts:
Heavy metal salts act to denature proteins in much the same manner
as acids and bases. Heavy metal salts usually contain Hg
+2
, Pb
+2
,
Ag
+1
Tl
+1
, Cd
+2
and other metals with high atomic weights. Since
salts are ionic they disrupt salt bridges in proteins. The reaction of a
heavy metal salt with a protein usually leads to an insoluble metal
protein salt

Reducing Agents Disrupt Disulfide Bonds:
Disulfide bonds are formed by oxidation of the sulfhydryl groups on
cysteine. Review reaction. Different protein chains or loops within a
single chain are held together by the strong covalent disulfide bonds.
Both of these examples are exhibited by the insulin in the graphic on the
left.
If oxidizing agents cause the formation of a disulfide bond, then reducing
agents, of course, act on any disulfide bonds to split it apart. Reducing
agents add hydrogen atoms to make the thiol group, -SH. The reaction i
Your turn !
1. Find out what is the differences between coagulation and
denaturation of protein.
2. Why do we use 70% ethanol for disenfectant rather than 95% ?
LEHNINGER
PRINCIPLES OF BIOCHEMISTRY
Fifth Edition
David L. Nelson and Michael M. Cox
2008 W. H. Freeman and Company
CHAPTER 6
Enzymes
A living system controls its activity through enzymes

An enzyme is a protein molecule that is a biological catalyst
with three characteristics :
the basic function of an enzyme is to increase the rate of a
reaction

most enzymes act specifically with only one reactant
(called a substrate) to produce products.

enzymes are regulated from a state of low activity to high
activity and vice versa

The individuality of a living cell is due in large part to the
unique set of some 3,000 enzymes that it is genetically
programmed to produce. If even one enzyme is missing or
defective, the results can be disastrous.

The activity of an enzyme depends, at the minimum, on a specific
protein chain.

In many cases, the enzyme consists of the protein and a combination
of one or more parts called cofactors.

Apoenzyme: The polypeptide or protein part of the enzyme is called
and may be inactive in its original synthesized structure (The inactive
form of the apoenzyme is known as a proenzyme or zymogen)

An apoenzyme together with its cofactor(s) is called a holoenzyme


substrate
Binding site
Reaction coordinate diagram.
Complementary shapes of a substrate and its binding site
on an enzyme.
Lysozyme reaction
Derivation of Michaelis Menten Equation
We can now express V0 in terms of [ES].
Substituting the right side of Equation 619 for
[ES] in Equation 611 gives
A double-reciprocal of Michaelis Menten
equation or Lineweaver-Burk plot
Enzyme inhibitions
Reversible
Competitive (used for drug binding/medication technique)
Uncompetitive
Mixed

Irreversible (used for drug design)
Ex. Ethanol dehydrogenase
Irreversible inhibition
Factor affecting Enzymes
substrate concentration
pH
temperature
inhibitors
The effect of change in concentration
(think about Michaelis-Menten Curve)
Enzyme concentration: at low enzyme concentration there is
great competition for the active sites and the rate of
reaction is low. As the enzyme concentration increases,
there are more active sites and the reaction can proceed at
a faster rate.
Eventually, increasing the enzyme concentration beyond a
certain point has no effect because the substrate
concentration becomes the limiting factor.


Substrate concentration: at a low substrate concentration
there are many active sites that are not occupied. This
means that the reaction rate is low.
When more substrate molecules are added, more enzyme-
substrate complexes can be formed. As there are more
active sites, and the rate of reaction increases.
Eventually, increasing the substrate concentration yet
further will have no effect. The active sites will be
saturated so no more enzyme-substrate complexes can be
formed
.


Properties of Enzymes relating to
their tertiary structure.
The activity of enzymes is strongly affected by
changes in pH and temperature. Each enzyme
works best at a certain pH and temperature,its
activity decreasing at values above and below
that point. This is because of the importance of
tertiary structure (i.e. shape) in enzyme function
and forces, e.g., ionic interactions and hydrogen
bonds, in determining that shape.

The pH dependency of enzyme
The effect of pH
Extreme pH levels will produce denaturation
The structure of the enzyme is changed
The active site is distorted and the substrate
molecules will no longer fit in it
At pH values slightly different from the enzymes
optimum value, small changes in the charges of
the enzyme and its substrate molecules will
occur
This change in ionisation will affect the binding of
the substrate with the active site.
The effects of change in
temperature.

Temperature: enzymes work best at an optimum
temperature.

Below this, an increase in temperature provides more
kinetic energy to the molecules involved. The numbers of
collisions between enzyme and substrate will increase so
the rate will too.

Above the optimum temperature, and the enzymes are
denatured. Bonds holding the structure together will be
broken and the active site loses its shape and will no
longer work
The effect of Inhibitors
Look back to enzyme inhibitions !!!
Allosteric enzymes are enzymes that change their
conformational ensemble upon binding of an effector, which
results in an apparent change in binding affinity at a different
ligand binding site.

This "action at a distance" through binding of one ligand
affecting the binding of another at a distinctly different site, is
the essence of the allosteric concept.

Allostery plays a crucial role in many fundamental biological
processes, including but not limited to cell signaling and the
regulation of metabolism

Allosteric enzymes
Whereas enzymes without coupled domains/subunits display
normal Michaelis-Menten kinetics, most allosteric enzymes
have multiple coupled domains/subunits and show cooperative
binding. Generally speaking, such cooperativity results in
allosteric enzymes displaying a sigmoidal dependence on the
concentration of their substrates in positively cooperative
system
This allows most allosteric
enzymes to greatly vary catalytic
output in response to small
changes in effector concentration.
Subunit interactions in an allosteric enzyme, and
interactions with inhibitors and activators.
Feedback inhibition
(important for controlling metabolims)
The inhibition itself can be reversible
inhibitions or through allosteric
modulation
Regulation of muscle glycogen phosphorylase
activity by multiple mechanisms
It involves allosteric regulation
And cascade mechanism regulated by hormone
phosphoprotein phosphatase 1 (PP1)
phosphoprotein phosphatase inhibitor 1 (PPI-1)
phosphoprotein phosphatase 2B (PP2B)
cAMP-dependent protein kinase (PKA).

Enzymes are the tools of life
End of Enzymes lecture