METABOLISM and ENZYMES

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METABOLISM and ENZYMES Metabolism management of
materials and energy resources of the cell.
Two basic kinds of reactions

• 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
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Energy in endergonic and exergonic reactions
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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.
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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
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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
glucose glucose
maltase
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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.
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• How do enzymes lower the energy of activation ?
• 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.
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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.
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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?

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Variables that affect enzyme activity

• Temperature
• pH
• Substrate/enzyme concentration

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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. Butsince we are
talking about reactions in cells we have to
consider the environment inside a living organism
and the effect that heat has on enzymes. At a
certain point the temperature will become too
great and the enzyme will loose its shape and no
longer be able to function. This is called
denaturing.
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Each enzyme has a temperature range in which a
maximal rate of reaction is achieved. This
maximum is known as the temperature optimum of
the enzyme.                                     
                   
Enzyme in cold water shrimp
Enzyme in a bacteria living in a hot spring
Digestive enzyme in human
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Substrate concentration
The amount of substrate will affect the rate of
reaction- as substrate concentration increases,
rate of reaction will increase proportionally
until all the enzymes are saturated, then it will
level off.
Increasing the amount of substrate increases the
number of collisions, assuming there is enough
enzyme present.
Increasing the amount of enzyme will also
increase the rate of reaction, but this is
limited by the amount of substrate. Enzyme is
reused, substrate is not.
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Graph showing effect of increasing substrate
concentration
Once all enzymes are occupied, increasing
substrate will no longer have an effect on rate
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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.
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Extremes in pHs 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 an enzyme will tend to gain H
ions, and eventually enough side chains will be
affected so the enzyme's shape is disrupted.
Likewise, as the pH is raised, the enzymes will
lose H ions and eventually lose its active
shape. Many of the enzymes function properly in
the neutral pH range and are denatured at either
an extremely high or low pH.
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Measuring the rate of reaction of catalase

• Catalase is present in most living cells.
• It catalyzes the breakdown of hydrogen peroxide
  into water and oxygen
• 2H2O2 O2 2H20

catalase

• There are several ways to measure this reaction
  in the lab
• Research and present one of these protocols
• -volume of gas produced
• - amount of hydrogen peroxide reacted by
  titrating with potassium permanganate
• - time taken for catalase soaked disc to float
  to top of liquid

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Enzymes in Biotechnology 1. Washing powders that
have enzymes are called biological washing
powders. They act on certain stains such as
blood, grass stainsproteases, oils, fats
lipases. They make the detergent more effective
(gets stains out better) and more efficient (use
less)
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2. Use of lactase in producing lactose-free milk
The disaccharide lactose is present in milk and
milk products.
70 of adults cant 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).
When the bacteria use the lactose it produces
hydrogen gas and the person has intestinal
symptoms.
It is therefore benificial to have lactose-free
milk available.
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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
Most milk is low in lactose but can also be
made with no lactose at all (these come from
plant sources like soy)
Milk can also be treated with a bacterium like
acidopholus, which is used in yogurt making.
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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.
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• 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

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• Instructions for catalase lab
• Fill burette with ml of potassium permanganate
• Measure 10 ml of H2O2, 1 ml of yeast suspension
  and 10 ml of H2SO4
• Mix the yeast with the H2O2 and start timing.
  Baseline group add 1 ml of water instead of yeast
  
• Add the H2O2 at YOUR time interval. Remove 5 ml
  of that solution
• Note the volume of the burette at the beginning
• Titrate until you have a persistent pink or brown
  color
• Note the end volume and subtract to find the
  volume used

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Enzyme review
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Control and inhibition of enzyme reactions
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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 non-competitive
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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 cant bind to the enzyme. A
competitive inhibitors 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.
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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
• an important enzyme in the Krebs cycle (in
  cellular respiration) is succinate, it can be
  inhibited by malonate, which has a similar
  structure
• Sulfa antibiotics inhibit folic acid synthesis in
  bacteria

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The antibiotic sulfanilamide is similar in
structure to para-aminobenzoic acid (PABA), an
intermediate in the biosynthetic pathway for
folic acid. Sulfanilamide can competitively
inhibit the enzyme that has PABA as it's normal
substrate by competitively occupying the active
site of the enzyme.
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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.
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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 non-competitive inhibitor acts on a
site other than the active site increasing the
substrate concentration will not affect the rate
of reaction
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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

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Control of Metabolism There has to be a system
for shitting 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.
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Allosteric control of metabolism by allosteric
enzymes
Molecules that regulate metabolic pathways act
like reversible, non-competitive inhibitors.
They bind to a specific site on the enzyme which
is remote from the active site.
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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
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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--gt excess product
  pathway shut down, product in short supply,
  pathway back on.

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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 is 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.
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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.
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Design Catalase Lab What factors could affect the
rate of reaction in the breakdown of hydrogen
peroxide? Possibly research inhibitors of
catalase. Catalse from different sources Consider
levels of IV i.e, if you choose Temp, how many
different temps will you use? Consider sample
size and replicates Method of data collection for
DV, other ways of measuring DV Data
analysis Design due next class
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Enzyme question on protease
Decrease in mass vs pH
Mass dec. (mg)
pH