Download Chapter 8 Enzymes

Chapter 8 Enzymes
Dr. Jeffrey P. Thompson bio350
Dr. Jeffrey P. Thompson bio350
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Dr. Jeffrey P. Thompson bio350
Modern day catalysis
• Catalysis (reaction promotion) may
have gotten its beginning in an RNAdominated world.
• Most catalysis today has evolved into
using PROTEIN enzymes to do the job.
– The capacity to specifically bind to a very
wide range of molecules.
Dr. Jeffrey P. Thompson bio350
What do enzymes do…
• Catalyze (accelerate) chemical
reactions
– Maybe by a factor of more than a million
– Biological reactions require fast
responses…in the absence of enzymes,
these reactions don’t proceed at a
perceivable rate.
– Carbonic anhydrase
• Hydrates 1 million CO2/sec
• 7,00,000 + times faster than the uncatalyzed
rate!
Dr. Jeffrey P. Thompson bio350
Dr. Jeffrey P. Thompson bio350
Dr. Jeffrey P. Thompson bio350
Protein break down is catalyzed by
enzymes called PROTEASES.
Dr. Jeffrey P. Thompson bio350
Many proteases are also ESTERASES.
Dr. Jeffrey P. Thompson bio350
Trypsin specificity
Thrombin specificity
Dr. Jeffrey P. Thompson bio350
Dr. Jeffrey P. Thompson bio350
Dr. Jeffrey P. Thompson bio350
What is so special about
enzymes?
• The are specific….
• They enhance reactions….
• They minimize unwanted side
reactions…
• The specificity of an enzyme is due to
the precise interaction of the substrate
with the enzyme. This precision is a
result of the intricate three-dimensional
structure of the enzyme protein.
Dr. Jeffrey P. Thompson bio350
Many enzymes require Cofactors
for activity…
• Our discussion of the RNA-world
mentioned that accessory molecules
(peptides)
(p
p
) may
y have enhance RNA’s
ability to do its many functions…
• Today…Some enzymes require
accessory molecules to enhance their
activity.
– cofactors
• Apoenzyme + cofactor = holoenzyme
Dr. Jeffrey P. Thompson bio350
Types of cofactors
• Metal ions
– Zn, Mg, Cu, Fe
• Small organic molecules
– Coenzymes (separate “helpers”)
• vitamins
– Prosthetic groups (physically attached
“helpers”)
Dr. Jeffrey P. Thompson bio350
Dr. Jeffrey P. Thompson bio350
Enzyme classification…
• Based on the TYPE OF REACTION
THEY CATALYZE
• 1964 International Union of
Biochemistry established an Enzyme
Commission for categorization of
enzymes.
– Like taxonomy for organisms…
– A systematic way of identifying enzymes…
Dr. Jeffrey P. Thompson bio350
Here are the major groups
This is what they do
Dr. Jeffrey P. Thompson bio350
Thermodynamics and
Enzymes
• Remember spontaneous reactions occur
when G changes are negative.
– Spontaneous, products have less energy than
reactants,
t t EXERGONIC.
EXERGONIC
– Positive G changes are nonspontaneous,
products have more energy than reactants,
ENDERGONIC
• THE G OF A REACTION IS INDEPENDENT
OF THE PATH OF THE
TRANSFORMATION!
Dr. Jeffrey P. Thompson bio350
Thermodynamics and
Enzymes
• The Standard free energy change of a
reaction is related to the equilibrium constant
Go
• Huh?
• In biology, reactions occur in different
environments
G OF A REACTION DEPENDS ON THE
NATURE AND CONCENTRATIONS OF THE
MOLECULES INVOLVED!!!!!
Dr. Jeffrey P. Thompson bio350
What do enzymes do?
• Important to note…..
• Enzymes alter only the REACTION
RATE and not the Reaction equilibrium
• A B
– Forward rate constant is 100 time that of
reverse reaction
– At equilibrium there is always going to be
100 times more “B” than “A”
– Enzymes only help to reach equilibrium
faster!
Dr. Jeffrey P. Thompson bio350
How do enzymes do this?
• Accelerate reactions by facilitating the
formation of the transition state of the
reaction.
• Enzymes decrease the Activation
Energy , aka the free energy of
activation.
• The essence of catalysis is SPECIFIC
BINDING of the transition state inside
the active site of the enzyme!
Dr. Jeffrey P. Thompson bio350
Dr. Jeffrey P. Thompson bio350
First step…
• Formation of the Enzyme-substrate
complex
• What is the evidence?
– SATURATION CURVES SUGGEST
PHYSICAL CONTACT BETWEEN
ENZYME AND SUBSTRATE.
– BIOPHYSICAL EVIDENCE
• FLUORESCENCE QUENCHING
EXPERIMENTS
• NMR analysis
Dr. Jeffrey P. Thompson bio350
Dr. Jeffrey P. Thompson bio350
Dr. Jeffrey P. Thompson bio350
Dr. Jeffrey P. Thompson bio350
Common feature of Active
Sites..
• Catalytic groups (atoms from amino
acids or cofactors in the active site that
promote the reaction))
p
• Interaction of the enzyme and substrate
at the active site promotes the
FORMATION OF THE TRANSITION
STATE!
Dr. Jeffrey P. Thompson bio350
Common features of Active
Sites..
• 1. 3-D cleft that is formed from groups that
come from different parts of the amino acid
sequence.
• 2. active sites take up a relativelyy small part
of the total volume of the enzyme.
• 3. Active sites are clefts or crevices.
• 4. Substrates are bound to enzymes by
multiple weak attractions.
– Why not covalent (strong) bonding?
• 5. Atoms in the active site give the specific
binding.
Dr. Jeffrey P. Thompson bio350
Lysozyme active
Site amino acids
Dr. Jeffrey P. Thompson bio350
Dr. Jeffrey P. Thompson bio350
Lock and Key
Dr. Jeffrey P. Thompson bio350
Induced fit
Dr. Jeffrey P. Thompson bio350
The observable kinetic properties
can be described mathematically!
• Kinetic math models can explain the
data
• Michaelis
Michaelis-Menten
Menten Model explains the
data.
• Their model will describe the current
model of enzyme reactions:
• E + SESE + P
• Reaction velocity vs. Substrate conc.
– Vmax and Km values for enzymes
Dr. Jeffrey P. Thompson bio350
Dr. Jeffrey P. Thompson bio350
Dr. Jeffrey P. Thompson bio350
Dr. Jeffrey P. Thompson bio350
Km values reflect efficiency
• To get a reaction to proceed, substrate
must bind…
• Km values are the concentration of the
give yyou half of the
substrate to g
maximum velocity
• Smaller the Km value (concentration)
the quicker the enzyme binds to the
substrate
• Equals best efficiency (even binding
efficiency of Enzyme to substrate)
Dr. Jeffrey P. Thompson bio350
Dr. Jeffrey P. Thompson bio350
Vmax values indicate speed
• Also called the Turnover number
– How many products are produced per
second.
• Which enzyme on the next page has the
greatest productivity?
Dr. Jeffrey P. Thompson bio350
A.K.A
Kcat
Dr. Jeffrey P. Thompson bio350
Probing for information…
• Testing the SAME ENZYME with
DIFFERENT SUBSTRATES can give
you some insight to the preferences of
th active
the
ti site.
it
• Calculate Km values for the different
experiments and compare…
• Look at the protease chymotrypsin for
example.
Dr. Jeffrey P. Thompson bio350
Dr. Jeffrey P. Thompson bio350
Enzymatic Perfection
Dr. Jeffrey P. Thompson bio350
Many Biochemical Reactions
Include Multiple Substrates…
• 2 classes of multiple substrate reactions
– Sequential Displacement reactions
• Ordered sequential
q
– substrates bind in a
defined sequence
• Random sequential – No defined sequence.
– Double Displacement reactions
• Also called a “ping pong” reaction
Dr. Jeffrey P. Thompson bio350
Ordered SequentialDisplacement Rx
LDH
(the enzyme)
Ternary Complex forms =
Enzyme+ substrate 1+ substrate 2
Dr. Jeffrey P. Thompson bio350
Ordered Sequential Displacement Rx
Cofactor adds first and leaves last
Cleland Notation- method to represent
An enzymatic reaction.
Dr. Jeffrey P. Thompson bio350
Random Sequential Displacement Rx
Addition of substrates and leaving of products shows no order.
Creatine kinase
(the enzyme)
Dr. Jeffrey P. Thompson bio350
Sequential Random Displacement Rx
Cleland Notation – diagram is a little
“messier” reflecting the different possibilities.
Dr. Jeffrey P. Thompson bio350
Double Displacement Rx
AKA “Ping-Pong reactions”
common for transferase enzymes
Marked by a substituted enzyme complex
(the enzyme is modified in the middle of the reactions)
The two substrates take turns binding to active site-like watching
A ping-pong match.
Dr. Jeffrey P. Thompson bio350
Double Displacement Rx
Steps for this example:
1. enzyme binds aspartate (S)
2. enzyme removes amino group (holds onto it)
3. enzyme releases product (oxaloacetate)(P)
4. enzyme binds -ketoglutarate (S)
5. enzyme transfers amino group to -ketoglutarate
y releases pproduct gglutamate(P)
( )
6. enzyme
Marked by a substituted enzyme complex
Dr. Jeffrey P. Thompson bio350
Allosteric Enzymes don’t follow
Michaelis-Menten Kinetics
Key Regulatory Enzymes
Dr. Jeffrey P. Thompson bio350
Enzyme Inhibitors
• Molecules that prevent the enzyme from
working as normal
y can cause changes
g in Vmax and
• They
Km
• Types
– Reversible inhibitors
• Noncovalently binds enzyme (bind and release)
– Irreversible inhibitors
• Covalently bind enzyme (permanently
attached)
Dr. Jeffrey P. Thompson bio350
Reversible
R
ibl inhibitorsi hibit
Bound by noncovalent
attachment
Like the desired substrate both types of inhibitors can
“bind and release”
Dr. Jeffrey P. Thompson bio350
These types of inhibitors
Are Kinetically Discernable!
They affect the kinetics curves
Differently.
The difference can be a
Diagnostic tool to determine
What kind of inhibitor is
at work!
Competitive=cause Km values
To increase, Vmax changes very
Little!
Noncompetitive=Km changes
Very little but Vmax decreases
Significantly!
Dr. Jeffrey P. Thompson bio350
A competitive
Inhibitor…it
Resembles the
Natural cofactor so it
Can take its place!
A cofactor
For many
enzymes
Dr. Jeffrey P. Thompson bio350
Lineweaver - Burk Plot
Dr. Jeffrey P. Thompson bio350
Dr. Jeffrey P. Thompson bio350
Dr. Jeffrey P. Thompson bio350
Irreversible inhibitors bind covalently to functional groups
in enzymes.
If the modified functional group is in an active site, the
enzyme is permanently inactivated.
Dr. Jeffrey P. Thompson bio350
Another example….
Dr. Jeffrey P. Thompson bio350
Suicide Inhibition- inhibitor irreversibly binds to enzyme
Only after being “worked on” by the enzyme!
The enzyme is responsible for its own activation!
Dr. Jeffrey P. Thompson bio350
Remembering Transition
States…
Dr. Jeffrey P. Thompson bio350
Transition state analogs are
potent inhibitors of enzymes.
1. Remember what a transition state is?
2. Remember that enzymes catalyze reactions by promoting
the formation of the transition state?
Dr. Jeffrey P. Thompson bio350
Transition state analogs (molecules that resemble the transition
State structure…as the substrate is turning into product) are
POTENT inhibitors….
Do you think they would be competitive or noncompetitive?
This is the hypothetical
Transition state structure.
This molecule, that resembles
The transition state structure,
Is a potent inhibitor of the
enzyme used to turn L-proline
Into its D form!
Dr. Jeffrey P. Thompson bio350
Generating a Man-made
“enzyme”
• Proteins can be developed to catalyze
reactions (like an enzyme)
• REMEMBER!! Enzyme function by
promoting the formation of the transition
state (this enhances the rate)
• If other proteins can be developed to
bind to and stabilize a reaction’s
transition state, then they should serve
as catalysts as well!
– ABZYMES DO THIS!
Dr. Jeffrey P. Thompson bio350
ABZYMES - “CATALYTIC
ANTIBODIES”
• How do antibodies work?
– Proteins in the immune system that naturally bind
g y to antigens
g
to clear them from yyour body.
y
tightly
– Inject antigen in an animal = antibody production
• flu shot = anti-flu antibodies = protection against flu
• anti-flu antibodies bind to flu virus to immobilize it
– Inject a reaction’s transition state analog (the
antigen)
• develop antibodies that bind to the transition state
• These antibodies now CATALYZE THE REACTION!!
Dr. Jeffrey P. Thompson bio350
An Example…
Ferrochelatase – enzyme needed to add metal ions into
Prosthetic groups…needs to bend the group in the transition state.
Use this antigen
instead
Dr. Jeffrey P. Thompson bio350
Dr. Jeffrey P. Thompson bio350
Dr. Jeffrey P. Thompson bio350
Dr. Jeffrey P. Thompson bio350