Transcript Catalytic Strategies

Mechanisms of Enzyme Action
What You Need to Know
• Understand the importance of and need for enzymes in
biological reactions.
• Understand how an enzyme’s effect on the transition state and
destabilization of the enzyme-substrate complex can affect
reaction rates.
• What role does transition-state stabilization play in enzyme
catalysis?
• Why is protein motion important in enzyme catalysis?
• Understand importance of near attack conformations in
catalysis.
• How does destabilization of ES affect enzyme catalysis?
• What factors influence enzymatic activity?
• Have a general understanding of the 4 basic types of enzyme
catalysis discussed in this lecture.
In chemical reactions all reacting atoms or molecules pass through a
state that is intermediate in structure between the reactant and product.
An Enzyme accelerates the reaction rate of a chemical reaction.
• For the rate of a reaction to be accelerated:
1. Rate acceleration by an enzyme means that the
energy barrier between ES and EX ‡ must be smaller
than the barrier between S and X‡
2. The enzyme must stabilize the transition-state
complex (EX‡)
ΔGe
‡
<
‡
ΔGu
• KS=[E][S]/[ES]; KT=[E][X++]/[EX++] KT<KS
• Enzymatic rate enhancement is approximately equal to:
ke/ku ~ KS/KT
How Does Destabilization of ES Affect Enzyme Catalysis?
Raising the energy of ES increases the rate
• For a given energy of EX‡, raising the energy
of ES will increase the catalyzed rate
• This is accomplished by
a) loss of entropy due to formation of ES
b) destabilization of ES by
• strain
• distortion
• desolvation
How Does Destabilization of ES Affect Enzyme
Catalysis?
Due to favorable interactions
of S and AAs on enzyme
The intrinsic binding energy of ES is compensated by entropy
loss due to binding of E and S and by destabilization due to
strain, distortion & desolvation.
How Does Destabilization of ES Affect Enzyme Catalysis?
Favorable interactions of S and
AAs on enzyme
Due to 1) Loss of entropy binding of S to E
and 2) destabilization of ES due to: strain,
distortion , desolvation
(a) Catalysis does not occur if ES and X‡ are equally stabilized.
(b) Catalysis will occur if X‡ is stabilized more than ES.
How Does Destabilization of ES Affect Enzyme
Catalysis?
(a) Formation of the ES complex results in entropy loss. The ES
complex is a more highly ordered, low-entropy state for the substrate.
How Does Destabilization of ES Affect Enzyme
Catalysis?
(b) Substrates typically lose waters of hydration in the formation in
the formation of the ES complex. Desolvation raises the energy of the
ES complex, making it more reactive.
How Does Destabilization of ES Affect Enzyme Catalysis?
(c) Electrostatic destabilization of a substrate may arise from juxtaposition of
like charges in the active site. If charge repulsion is relieved in the reaction,
electrostatic destabilization can result in a rate increase.
How Tightly Do Transition-State Analogs Bind to the
Active Site?
• The affinity of the enzyme for the transition state
may be 10 -20 to 10-26 M!
• Can we see anything like that with stable molecules?
• Transition state analogs (TSAs) are stable molecules
that are chemically and structurally similar to the
transition state
• They bind do the enzyme more tightly than does the
substrate but not as tightly as the natural substrate
already in the transition state
Purine riboside inhibits adenosine deaminase. The hydrated
form is an analog of the transition state of the reaction.
Transition-State Analogs and Drug Discovery
• Enzymes are often
targets for drugs and
other beneficial
agents
• Transition state
analogs often make
ideal enzyme
inhibitors
Enalapril and Aliskiren lower blood pressure
Statins lower serum cholesterol
Protease inhibitors are AIDS drugs
Juvenile hormone esterase is pesticide target
Tamiflu is a viral neuraminidase inhibitor
USP2a
ubiquitin
Mdm2
p53 ubiquitin Ligase
Mdm2
p53 ubiquitin Ligase
P53
Ubiquitination
p53
Tumor Suppressor
ubiquitin
ubiquitin
Degradation of p53
Tumor Suppressor
USP2a expression in Cancer cells
USP2a
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Expression Purification and Crystallization of USP2a Core Domain
 Expression: preformed in Ecoli strain Rosetta
(DE3) pLysS
 Purification:
– Affinity chromatography (Ni column)
– Size exclusion (Superdex 75)
– Total yield (2 liters)≈ 60 mg USP2a.
 Crystallization:

Screened with/without Ubiquitin
195
USP2a 40 kDa
142
96
71
48
33
28
22
12
7.6
(8.5 kDa Boston Biochem)
100mM HEPES pH7.8
5mM DTT
5% PEG 4000
100mM Pipes pH7.8
5% PEG 4000
100mM HEPES pH7.8
5mM DTT, 10% Isopropanol
5% PEG 4000
Catalytic triad:
214-229 Cys-box;
292-305 QDE-box;
446-520 His-box
218 oxyanion hole
•USP2a and ubiquitin
have complementary
shapes
• Of the 20 USP
residues within 4
angstroms of the Cterminal pentapeptide 14 are
conserved
• However, USP
residues interacting
with the ubiquitin core
are poorly conserved
1.26Å Structure of USP2-ubiquitin Complex: Active Site and Vicinity
Active site residues shown on left (colored as in previous slide)
•
Two sites where interruption of ubiquitinin – USP2a interaction disrupts binding:
1. Large pocket in proximity of active site (C-terminus of ubiquitinin inserts here)
2. Second large pocket is visible on the right (area of Lys48 [colored in magenta] of
ubiquitinin)
USP2 is shown in green, ubiquitin is shown in cyan, water molecules are shown as blue spheres
How many other drug targets might there be?
• The human genome contains approximately
20,000 genes
• How many might be targets for drug therapy?
• More than 3000 experimental drugs are
presently under study and testing
• These and many future drugs will be
designed as transition-state analog inhibitors
How to read and write mechanisms
• In written mechanisms, a curved arrow
shows the movement of an electron pair
• And thus the movement of a pair of electrons
from a filled orbital to an empty one
• A full arrowhead represents an electron pair
• A half arrowhead represents a single electron
• For a bond-breaking event, the arrow begins
in the middle of the bond
How to read and write mechanisms
How to read and write mechanisms
• It is important to appreciate that a proton
transfer can change a nucleophile into an
electrophile, and vice versa.
• Thus, it is necessary to consider:
– The protonation states of substrate and active-site
residues
– How pKa values can change in the environment of
the active site
• For example, an active-site histidine, which
might normally be protonated, can be
How to read and write mechanisms
Proton transfer can change a
nucleophile into an electrophile,
and vice versa.
Example: An active-site histidine,
which might normally be protonated,
can be deprotonated by another
group and then act as a base,
accepting a proton from the
substrate.
How to read and write mechanisms
Water can often act as an acid or base at the active
site through proton transfer with an assisting activesite residue.
Mechanisms of Catalysis
• Enzymes facilitate formation of near-attack
complexes
• Protein motions are essential to enzyme
catalysis
• Covalent catalysis
• General acid-base catalysis
• Low-barrier hydrogen bonds
• Metal ion catalysis
How Do Active-Site Residues Interact to
Support Catalysis?
• About half of the amino acids engage directly
in catalytic effects in enzyme active sites
• Other residues may function in secondary
roles in the active site:
– Raising or lowering catalytic residue pKa values
– Orientation of catalytic residues
– Charge stabilization
– Proton transfers via hydrogen tunneling
Enzymes facilitate formation of near-attack complexes
The energy separation between the NAC and the transition
state is approximately the same in the presence and
absence of the enzyme.
Enzymes Facilitate Formation of NAC
• Precise positioning
– Reacting atoms are in van der Waals contact
– Angle resembling bond to be formed in
T-state
• NACs are precursors to reaction transition states
• Without enzyme, molecules adopt NAC ~0.0001%
of the time
• NACs form in enzyme active sites 1%-70% of the
time
The active site of liver alcohol dehydrogenase – a
near-attack complex.
Protein Motions Are Essential to Enzyme Catalysis
• Proteins are constantly moving
• Enzymes depend on such motions to provoke and
direct catalytic events
• Protein motions support catalysis in several ways.
Active site conformation changes can:
– Assist substrate binding
– Bring catalytic groups into position
– Induce formation of NACs
– Assist in bond making and bond breaking
– Facilitate conversion of substrate to product
Protein Motions Are Essential to Enzyme Catalysis
The active site of cyclophilin with a bound peptide
containing proline in cis and trans conformations. Motion by
active site residues promote catalysis in cyclophilin.
Proteins are on the Move
Covalent Catalysis
• Some enzymes derive much of their rate
acceleration from formation of covalent bonds
between enzyme and substrate
• Amino acids side chains offer variety of
nucleophilic centers for catalysis
• These groups readily attack electrophilic centers of
substrates, forming covalent enzyme-substrate
complexes
• The covalent intermediate can be attacked in a
second step by water or by a second substrate,
forming the desired product
Covalent Catalysis
Example of covalent bond formation between
enzyme and substrate. A nucleophilic center X: on
an enzyme attacks a phosphorus atom to form a
phosphoryl enzyme intermediate.
General Acid-base Catalysis
Catalysis in which a proton is transferred in the
transition state
• "Specific" acid-base catalysis involves H+ or OH- that
diffuses into the catalytic center
• "General" acid-base catalysis involves acids and
bases other than H+ and OH• These other acids and bases facilitate transfer of H+
in the transition state
General Acid-base Catalysis
Catalysis of p-nitrophenylacetate hydrolysis can occur either by
specific acid hydrolysis or by general base catalysis.