Transcript Ch 11!! Cont. Classification of Enzyme • Commonly, enzymes were named by identifying the type of reaction and adding –ase. • As more enzymes.

Ch 11!! Cont.
Classification of Enzyme
• Commonly, enzymes were named by
identifying the type of reaction and adding
–ase.
• As more enzymes were discovered, modifyers
were placed before the name
Problems with Modifiers
• Modifiers came from different things, such as:
– Specific substrate
– Enzyme source
– Mode of regulation
– Distinguishing feature of mechanism
• Even then, more enzymes were discovered
including multiple forms.
• To handle this, alphanumeric designations
were made such as Roman Numerals and
greek alphabet
New System
• To clear up the confusion, and address the
increasing amount of new enzymes being
discovered, the IUB system was created
• The IUB system is very complex and some
what mirrors species identification
• It gives each enzyme a unique name and a
code number that reflects the type of reaction
catalyzed and the substrate involved.
IUB naming system
• As a result, enzymes are now grouped into 6
classes by reaction type and each class has
multiple subclasses, etc
• Example:
• ATP: D-Hexose-6-phosphatetransferase
E.C.2.7.1.1
• 2= transferase, 7=transfer of phophoral group,
1=alcohol in phosphoral acceptor
IUB cont
• Parts of the name provide information as well
• In the last case, the hexose-6 tells us the
alcohol phosphorylated is on carbon 6 of
hexose
Six major classes
•
•
•
•
•
•
•
1- Oxidoreductases
2- Transferases
3- Hydrolases
4-Lyases
5- Isomerases
6- Ligases
Examples on page 393.
Non Protein Material
• Enzymes often contain non-protein materials
as well
• These are termed prosthetic groups, cofactors,
or coenzymes
• All basically expand the enzymes capabilities
beyond the traditional functionality found in
proteins
Definitions
• Prosthetic groups- groups that are tightly
bound to the enzyme either by covalent or
non-covalent interactions
• Metal are the most common
• About 1/3 of all enzymes are termed
metalloenzymes because they contain metals
Definitions
• Cofactors- differ from prosthetic groups in that
they are not permanently attached.
• They bind to either the substrate or enzyme to
perform the reaction the move on
• Again, metals are most common
• Difference is they are not bound!!!
• The term metal-activated enzymes refers to
enzymes that need metals as cofactors.
Definitions
• Coenzymes- transporters. They don’t catalyze
a reaction, just move things from where they
were made to where they are used.
• Coenzymes can protect and stabilize
substrates until they are needed.
• The B vitamins supply components for
numerous coenzymes, Table 11.5 page 389
The Active Site
• The Active Site in enzymes is the specific location
of action. It is usually a small portion of the
enzyme.
• The Active site:
– Gives high selectivity
– Usually forms a cleft or pocket
– Involves groups from multi-monomer units
– Binds to non-reacting portion of substrate
– Also orientates/binds cofactors/prosthetic groups that
are needed
Enzymatic Mechanisms
• There are 4 general mechanisms that account
for an enzymes ability to catalyze reactions
1) Catalysis by Proximity
-molecules have to come together to react!
- Typically done by increasing the local concentration
- Binding increases the local concentration
Enzymatic Mechanisms
2) Acid/Base Catalysis
- The amino acid side groups and/or prosthetic
groups can act as acids or bases
- There are two types:
a) specific acid/base catalysis
b) general acid/base catalysis
Enzymatic Mechanisms
3) Catalysis by Strain
-Usually the mechanism for breaking bonds
-typically bends or orientates bonds into
unfavorable conformations
4) Covalent Catalysis
-a covalent bond is created between the enzyme
and substrate
-usually present in group transfers
-the covalent bond is temporary
Conformational Change in Enzyme
• Early on, the lock and key analogy was used to
explain enzyme activity
• But this inferred rigidity in the enzyme that
experimental evidence didn’t support
• Later, the hand and glove analogy was used
• When the substrate binds, enzymes change
slightly which allows for catalytic acitivity
• Figure 11.7 and 11.8, page 369
Examples of Acid/Base Catalysis
• Board
Example of Covalent Catalysis
• Board and Figure 11.13, page 374
Catalytic Residues are highly
conserved
• Most enzymes in particular family’s use the
same mechanism on different substrates
• It is believed that genes are duplicated to
create the individual proteins
• Use of more than one gene to encode proteins
allows for each to form independently which
accounts for different binding properties
• When different proteins have similar residues
in the same position, the residues are said to
be conserved residues
• Proteins with a large number of conserved
residues are said to be homologous
Isozymes
• Isozymes are different enzymes that catalyze
the same reaction
• The difference may be in certain properties
that adapt them to specific tissues or locations
• Other differences may be higher selectivity
– Ex. Hexokinase vs glucokinase
• Could also just supply back up copies of
essential enzymes
Detection of enzymes
• Enzymes are often present in very small
amounts
• This make detection and quantification
difficult
• Instead of trying to isolate and identify the
actual enzyme, often we just look for evidence
of their presence
• Amount of enzyme present can sometimes be
determined by the rate of the catalyzed
reaction.
• Detection of proteins that lack catalytic
activity is a little more complicated
• For these cases, Enzyme-Linked
ImmunoAssays (ELISA’s) are used
• There are two ways to do ELISA’s with the
difference being what is bound.
ELISA’s
• Step 1- adhere the protein
• Step 2- add an antibody with a reporter
enzyme
• Step 3- antibody binds to immobilized protein
• Step 4- use the catalytic activity of the
reporter enzyme to determine the presence
and quantity of the original protein
• Other methods are also available depending
on the properties of the reactants and
products of the reaction being catalyzed.
• This method is used to assay NAD(P)+
dependant dehydrogenases.
– Example:
Other Alternatives
• If your reactant/product is not accompanied
by a change in absorption or fluorescence, the
assay is generally more difficult
• Often, you are required to separate the
products from the substrates prior to
measuring
• Another option is to create a synthetic
substrate whose product can be detected
• Lastly, in some instances, the product of the
reaction of interest can be transformed into
something that is readily detectible by
coupling processes.
Why do we quantify/detect enzymes
• There are thousands of different enzymes
• Some of them function all the time and are
essential in the vitality of the cell and are
present throughout the body
• Other enzymes or isozymes are used only in
specific cells, or during specific times of
development, or in response to specific
physiologic or pathologic changes
• Analysis of these enzymes often aids in
diagnosis.
– Example: Non-functional plasma enzymes
• In addition, different forms of the same
enzymes can differentiate between multiple
problems
– Example- Isozymes of Lactate Dehydrogenase
Enzyme Kinetics
• Enzyme Kinetics- the quantitative
measurement of the rates of enzyme
catalyzed reactions and the systematic study
of factors that affect rates
• By studying the kinetics of a process we can
deduce the mechanism
• Knowing the mechanism helps us find ways to
either promote or prevent the process.
Temperature
• Raising the temperature increases the kinetic energy of
molecules.
• The total number of molecules whose kinetic energy
exceeds the energy barrier for the formation of
products increases as we go from low temp to medium
temp to high temp.
• Increasing the KE of molecules also increases their
motion and therefore the frequency with which they
collide
• This combination of more frequent and more highly
energetic and productive collisions increases the
reaction rate.
Reactant Concentration
• The frequency with which molecules collide is
directly proportionate to their concentration
• At constant temperature, the number of
molecules with enough energy to overcome
the energy barrier is constant but the rate is
proportionate to the number of collisions,
therefore, to the molar concentration.
Reactant Concentration
• The coefficients therefore becomes a power in
the rate equation.
• In general:
nA + mB → P
Rate ∞ [A]n[B]m
By including a rate constant, the proportion sign
is replaced by an equal sign
Rate = k[A]n[B]m
Rate Equations
• Remember, most reactions are reversible, so
the reverse equation would be:
Rate = k-1[P]
• At equilibrium, the total concentration of
reactants and products is constant
• Another way to state this is that the rate of
the forward reaction is equal to the rate of the
reverse reaction.
Equilibrium Constant
• The ratio of k1 to k-1 is termed the equilibrium
constant with the symbol keq
• There are four important properties of
systems at equilibrium which must be
considered:
1) keq is a ratio of rate constants, not rates!
2) The reaction rates are equal, not the rate
constants
3) Equilibrium is a dynamic state. There is no
net change in concentration, but continual
interconversion
4) The numeric value for keq can be calculated
from either the ratio of k1 to k-1 or from the
concentration of reactants and products
How enzymes work
• We have said before that enzymes speed up
reactions by lowering the activation energy
• They do this by lowering the energy of the
transition state
• When the mechanism is the same as the
uncatalyzed reaction, the environment of the
active site lowers ∆GF by stabilizing the
intermediates
Stabilization
• There are 3 ways for this to occur:
1) acid/base groups assist in transfer of H+ to or
from intermediates
2) Positioned charged groups or metal ions
stabilize developing charges
3) Creating or relieving steric strain
Stabilization
• If the reaction proceeds via a different
mechanism than the typical reaction, it is
usually covalent catalysis!
Enzymes and keq
• Enzymes DO NOT effect keq
• Enzymes increase rates only be lowering
activation energies
• They do no effect ∆G, keq, etc
Measuring Enzyme Catalyzed Reaction
Rates
• These are measured over very short periods of time
• There is a huge excess of substrate compared to
enzyme
• Under these conditions, only small amounts of
product are produced
• But by doing this, the reverse reaction is negligible,
so the initial velocity (vi) is essentially that of the
desired forward reaction
• One important factor is that vi is
proportionate to the enzyme concentration
• This provides two things:
1) In lab, we can increase or decrease the
reaction rate by altering the enzyme
concentration
2) By determining a concentration that gives a
rate comparable to in the cell, we can
determine enzyme concentration in the cell
Substrate Concentration
• Substrate concentration can also affect
reaction rates
• As you increase the substrate concentration, vi
increases until it reaches a maximum value,
vmax
• Once vmax is reached, increasing substrate
concentration has no affect
• The reasoning is the relationship between the
substrate and enzymes
Vmax
• The substrate must bind to the enzyme to
form the ES complex
• Only substrate in the ES complex can be
converted to product
• The equilibrium constant for forming the ES
complex is not infinitely large so even if the
substrate is in excess, only a fraction will form
the ES complex
Vmax
• So as you increase the substrate
concentration, you are also increasing that
fraction forming the ES complex
• However, once Vmax is reached, all enzymes are
in ES complexes and increasing concentration
of substrate will not increase the rate because
no more enzyme is available for binding.
• At this point, vi depends solely on the ability
of free enzyme to be released.
Inhibitors
• Inhibitors are classified by:
– The site of action on the enzyme
– Whether or not they chemically modify the
enzyme
– Kinetic parameters they influence
• There are two major Classes: Reversible
Inhibition and Irreversible Inhibition
Reversible Inhibition
• Kinetically, there are two classes based on
whether raising the substrate concentration
does or does not overcome inhibition
• Competitive Inhibitors- typically resemble the
substrate, bond to the active site, and acts by
decreasing the number of free enzymes
available to bind substrates
• A double reciprocal plot helps evaluate
inhibitors.
Kinetically
• Noncompetitive Inhibitors- binding of the
inhibitor does not effect binding of the
substrate.
• The inhibitor binds to a second site (not the
active site)
• By binding at the second site, it somehow
lowers the efficiency in which the enzyme
converts the substrate to product.
Regulation of Enzyme Activity
• Homeostasis is very important
• This is our ability to adapt to both changes in
the internal and external environment
• We respond to these changes by balanced,
coordinated changes in the rates of specific
metabolic reactions
• Many diseases are characterized by creating
certain dysfunctions in this regulatory
procedure
• If enzymes operated at their saturated levels,
they would not be able to adjust to more
substrate
• For this reason, most substrate concentrations
are maintained near the Km value.
Passive vs Active Regulation
• Passive Regulation refers to the ability to only
adapt to internal changes
– For example, substrate concentration
• Active Regulation responds to internal and
external signals.
Little backgroud
• Metabolic flow tends to be unidirectional!
• Despite short-term oscillations, living cells
exists in a dynamic, steady state in which the
average concentration of metabolic
intermediates remain relatively constant
• Linking of potentially reversible reactions in
this manner creates a unidirectional flow with
an overall -∆G!
Compartmentalization
• Not all reactions are occurring in the presence
of others
• Instead, some reactions take place in specific
sub-cellular compartments
• Other reactions only occur in specialized cells
• In cases with no physical barriers, one or more
unique intermediates are needed
Controlling Metabolic Pathways
• Active control of homeostasis is achieved by
regulating the enzyme or enzymes of the RDS!
• These enzymes constitute efficient targets for
regulatory intervention by drugs as well
Regulation of Enzyme Quantity
• The rate limiting reactions can be controlled
by two methods, the concentration of the
enzyme or the catalytic efficiency of the
enzyme
• The way to control the concentration or
quantity of the enzyme depends on one of
two factors, the synthesis or degradation of
the enzyme.
Control of Enzyme Synthesis
• Constitutive enzymes are enzymes that have a
constant concentration over time
• Other enzymes require the presence of inducers
• These inducers are typically substrates or
structurally similar compounds that initiate their
synthesis
• On the other hand when an excess of a
metabolite is present, it may prevent the
synthesis of an enzyme via repression
Control of Enzyme Degradation
• The absolute quantity of an enzyme depends
on both the synthesis of the enzyme and the
degradation of the enzyme
• Both these processes have equilibrium
constants
• Changes in one or both of these will effect the
quantity of enzyme
Control of Enzyme Degradation
• Susceptibility to degradation can be
influenced by the presence of ligands such as
substrates, coenzymes, or metal ions
• Enzyme levels in mammalian tissues respond
to a wide range of physiologic, hormonal, or
dietary factors.
Options for Regulating Catalytic
Activity
• Changes in catalytic efficiency can be done
much quicker
• There are two ways in which catalytic activity
can be altered:
1) Allosteric regulation- non-covalent binding of
dissociable ligands
2) Covalent Modifications
Allosteric effectors regulate certain
enzymes
• Feedback inhibition deals with the inhibition
of an enzyme by an end product of a pathway
• This is not caused by a backing up of the
process
• Instead, the final product begins to bind to an
enzyme involved in an earlier step, reducing
its catalytic capacity
• This binding does not occur at the active site,
but at an allosteric site
• Usually, the final product and the allosteric
site are completely different structurally from
the original substrate and active site
• This is an example of a negative allosteric
feedback
• This process does not completely shut down
the enzymatic activity
• This would be bad for processes that yield
multiple products
• Each product is typically only partially
inhibited by the reduced catalytic activity
• Most processes contain multiple feedbacks
which could be additive or greater than an
individual feedback
Covalent Modifications
• There are 2 types of covalent modifications:
– Partial proteolysis- the actual breaking of a
peptide bond to split the protein
– Phosphorylation- addition of a phosphoryl group
• Because cells do not have the ability to put
two pieces of a protein back together, partial
proteolysis is an example of an irreversible
covalent modification
• Both the addition and removal of the
phosphoryl group is thermodynamically
spontaneous, therefore it is considered a
reversible modification