METABOLISM and ENZYMES
Metabolism: management of materials and energy resources
of the cell.
Two basic kinds of reactions:
1. Catabolic- complex molecules are broken down into simpler ones.
Energy is released (an exergonic reaction) ex: cellular respiration
2. Anabolic- complex molecules are built up from simpler ones. Energy
is required (an endergonic reaction) ex: protein synthesis
Energy in endergonic and exergonic reactions
For most reactions in the cell ATP is the immediate energy
source.
When ATP is broken down into ADP by hydrolysis, energy is
released. When ATP is formed energy is required.
Exergonic reactions occur spontaneously, but may take a long time.
A catalyst is a chemical that increases the rate of the reaction without
taking part in the reaction. An enzyme is a biological catalyst.
Exergonic reactions also require some energy to get started, even though
the net energy will be greater. Enzymes also lower the energy of
activation which is usually provided in the form of heat.
Without enzymes, considering the conditions in the cell (moderate
temperature, pH, pressure), reactions would be too slow to support life.
Endergonic reactions do not happen spontaneously and require energy,
again the amount of energy required is less with an enzyme
Enzymes are specific to the reactant they act on called the
substrate. So we say that enzymes are substrate specific. The
specificity is due to the shape of the enzyme (remember protein
structure- this is the tertiary structure of the protein).
Enzymes bind to the substrate at a specific place on the enzyme molecule
called the active site. This forms an enzyme-substrate complex.
While they are joined the catalytic action of the enzyme converts the
reactant/s to the product/s.Example:
Maltose + water maltase
glucose + glucose
Enzymes have an active site where the catalytic activity takes place.
Most often the substrate and enzyme are held together by Hydrogen
bonds. The active site is made up of the R groups (providing the
specificity). Once the reaction has happened, the bond is broken and the
enzyme can go on to catalyze another reaction.
How do enzymes lower the energy of activation ?
1. In a reaction involving two or more reactants the enzyme
provides a template for them to come together in the correct
orientation.
2. The active site holds the substrates, stretching and bending critical
chemical bonds that must be broken
3. The active site may provide a microenvironment that is conductive to
the reaction. Ex: an aa with an acidic R group would provide a small
acid pocket- this would facilitate the transfer of H+ ions to the
substrate catalyzing the reaction
4. There may be a brief bonding of the R group to the active site which
causes a chemical change in the substrate, inducing a reaction- the
bond would break after the reaction started, making the enzyme the
same again.
Factors that Determine the
Activity of Enzymes
There are two theories about how the enzyme and substrate
interact:
1.The Lock and Key theory: this theory proposes that the enzyme is
like a key fitting a lock- the shapes are fixed, neither the lock or the key
change their shape.
2. Induced Fit: In this hypothesis, the substrate does not simply bind
with the active site. It has to bring about changes to the shape of the
active site to activate the enzyme and make the reaction possible.
The hypothesis suggests that when the enzyme's active site comes into
contact with the right substrate, the active site slightly changes or moulds
itself around the substrate for an effective fit. This shape adjustment
triggers catalysis and helps to explain why enzymes only catalyse
specific reactions.
Rate of Reaction in an Enzyme controlled reaction
•The rate of reaction is measured either by the amount of reactant used
up or the amount of product formed
•Under ideal conditions (which are variable) there is a maximum rate of
reaction, called V max
•With constant enzyme and substrate the rate of reaction is highest at
the beginning, Why?
Variables that affect enzyme activity:
1. Temperature
2. pH
3. Substrate/enzyme concentration
TEMPERATURE
Increase in temperature causes
1) More energetic collisions
2) The number of collisions per unit time will increase.
3) The heat of the molecules in the system will increase.
These will all decrease the energy of activation and speed up the
reaction.
denaturing.
the temperature optimum of the enzyme.
Enzyme in cold water
shrimp
Digestive enzyme in human
Enzyme in a bacteria
living in a hot spring
Graph showing effect of increasing substrate concentration
Once all enzymes are occupied,
increasing substrate will no
longer have an effect on rate
Effect of pH.
Each enzyme has an optimal pH, so the effect of pH will depend on the
particular enzyme. Below are two proteases that work in different parts
of the body. Pepsin works in the stomach, which is very acidic, Trypsin
works in the small intestine where conditions are slightly alkaline.
Extremes in pH’s can also denature enzymes because their tertiary
bonds will change.
Amino acid side chains contain groups such as - COOH and NH2
that readily gain or lose H+ ions.
As the pH is lowered…… what happens
Measuring the rate of reaction of catalase
•Catalase is present in most living cells.
•2H2O2
O2 + 2H20
catalase
•Design a lab!
Enzymes in Biotechnology
1. Washing powders that have enzymes are called biological
washing powders. They act on certain stains such as blood,
grass stains:proteases, oils, fats: lipases. They make the
detergent more effective (gets stains out better) and more
efficient (use less)
2. Use of lactase in producing lactose-free milk
* The disaccharide lactose is present in milk and milk products.
* 70% of adults can’t breakdown lactose and so it builds up in the
intestine (only monosaccharides can be absorbed)
* The bacteria that live in the gut can switch to lactose as their
energy source by “turning on” the gene for lactase (an example of
control of gene expression called the lac operon model).
.
.
Production of lactose-free milk
* Milk is passed over the enzyme lactase, which is bound to an
inert carrier.
* The lactose is converted to glucose and galactose, which can be
absorbed
3. Fruit juice production
Pectinases increase the yield of juice from fruit and make it clearer. Pectin is a
large polysaccharide found in the cell wall. The enzymes hydrolyze the pectins
and enable the easy extraction of larger volumes of clear fruit juice. Pectinase is an
enzyme that is extracted from a fungus (Aspergillus niger). This fungus grows
naturally on fruits and uses this enzyme to soften cell walls enabling its hyphae to
grow through them.
Other examples of enzymes in food technology:
• Tenderizing meat with papain (a protease extracted from
papaya
• Conversion of starch into sugar in brewing using
amyloglucosidase
ENZYME INHIBITION
Certain chemicals can inhibit the action of an enzyme.
Inhibitors work by attaching to the enzyme.
If it attaches by a covalent bond it will be a permanent
inhibition because this is an irreversible process.
In some cases it can be reversed, if the bond is a weak one.
There are two main kinds of inhibitors: competitive and noncompetitive
Competitive inhibitors compete for the active site on the
enzyme. They have a similar shape as the substrate and so block
the active site so that the substrate can’t bind to the enzyme. A
competitive inhibitor’s affect can be lessened if more substrate
is added. If there is more substrate than inhibitor the substrate will
have a better chance to gain entry to the site.
No inhibitor
Competitive inhibitor present
Graph showing affect of increasing the amount of
substrate on the rate of enzymatic reaction. Given enough
substrate the reaction can reach its maximum rate with a
competitive inhibitor.
Examples of competitive inhibitors:
1. an important enzyme in the Krebs cycle (in cellular respiration)
is succinate, it can be inhibited by malonate, which has a
similar structure
2. Sulfa antibiotics inhibit folic acid synthesis in bacteria:
A Non-competitive inhibitor binds somewhere other than
the active site and alters the shape of the enzyme. In this
case, adding more substrate will not affect the rate of
reaction.
Examples: metals such as mercury, copper, silver, inhibit
many enzymes because they break the disulfide bridges.
Poisons such as nerve gas, and snake venom which inhibits
cholinesterase, the enzyme that metabolizes ACH a
neurotransmitter
Because a noncompetitive inhibitor acts
on a site other than the
active site; increasing the
substrate concentration
will not affect the rate of
reaction
Metabolism in a cell occurs in metabolic pathways:
•series of chemical reactions
•requires a set of enzymes, a different one for each reaction
•each molecule that is produced is different
•each substrate is transformed into a product that serves as the substrate
for the next reaction until a final product is generated called an end
product
•the pathway is directional
A simple pathway
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Theodor Hanekamp © 2003
cat
26
Control of Metabolism
There has to be a system for shutting down a metabolic pathway or
the cell would not only be inefficient there would be chemical chaos.
The pathways must be tightly controlled so only substances that are
needed and the right amounts are produced.
This is accomplished by two ways: gene regulation and enzyme
regulation.
We will look at enzyme regulation through end product inhibition.
Allosteric control of metabolism by allosteric enzymes
Molecules that regulate metabolic pathways act like reversible, noncompetitive inhibitors.
They bind to a specific site on the enzyme which is remote from the
active site.
Example of an allosteric enzyme
with a negative effector site. When
the effector molecule binds to the
allosteric site, substrate binding and
catalytic activity of the enzyme are
inactivated. When the effector is
detached from the allosteric site the
enzyme is active.
Allosteric activators have the opposite
effect, they will activate an enzyme by
stabalizing the enzyme in the active
form
Example of allosteric inhibition by an
end product in a metabolic pathway
•Threonine Deaminase is the first enzyme in the metabolic
pathway of changing threonine to isoeucine.
•Isoleucine, the end product, can inhibit threonine
deaminase
•The inhibition occurs at an inhibition site on the enzyme
but not the active site
•An excess of end product switches off any more production
of that product.
•As the end product is used up it detaches from the
inhibitory site.
•The active site becomes active again and the pathway
switches back on. Similar to non-competitive inhibition.
•This mechanism makes the pathway self-regulating in
terms of product manufacture--> excess product pathway
shut down, product in short supply, pathway back on.
Example
In the metabolic pathway of glycolysis (the initial steps in cellular
respiration where glucose is split into 2, 3 carbon molecules), there is
inhibition provided by ATP (the end product).:
In one of the first steps in glycolysis
Phosphofructokinase (PFK) catalyzes a reaction. This enzyme is
allosteric and one of the main regulators of glycolysis in the cell.
PFK is inhibited by high levels of ATP. This will stop cellular
respiration if there is adequate ATP available in the cell. If there are
low levels of ATP and or high levels of ATP or AMP, then the
metabolic pathway is turned on.
Allosteric inhibition is an example of negative
feedback.
Negative feedback is a regulatory mechanism that
keeps an organism or system in dynamic balance. (like
a thermostat, keeping a constant temperature in a water
bath). Negative feedback will slow or stop a process,
positive feedback will speed up a process. NFB
maintains equilibrium, PFB causes disequilibrium.
There are few examples of positive feedback in an
organism but lots of negative feedback: control of
glucose, temperature, etc.
Describe the energy profile of a
chemical reaction including EA, ∆G,
and transition state.
• What do enzymes do in biological systems?
• Explain this idea of “structure” and
“specificity” to me.
• Draw the steps of an enzyme as it goes
through its catalytic cycle.
• Explain why the speed of a reaction is not
just controlled by the [enzyme], but by the
[substrate] as well.
• Define and tell me what these
terms/concepts have in common?
–
–
–
–
cofactors
environmental conditions
enzyme inhibitors
allosteric regulators
• Explain how metabolic pathways are
regulated.