Transcript Chapter 14 - Richsingiser.com

Reginald H. Garrett
Charles M. Grisham
www.cengage.com/chemistry/garrett
Chapter 14
Mechanisms of Enzyme Action
Reginald Garrett & Charles Grisham • University of Virginia
Outline
• What are the magnitudes of enzyme-induced rate
accelerations?
• What role does transition-state stabilization play in enzyme
catalysis?
• How does destabilization of ES affect enzyme catalysis?
• How tightly do transition-state analogs bind to the active
site?
• What are the mechanisms of catalysis?
• What can be learned from typical enzyme mechanisms?
14.1 What Are the Magnitudes of EnzymeInduced Rate Accelerations?
• Enzymes are powerful catalysts
• The large rate accelerations of enzymes (107 to 1015)
correspond to large changes in the free energy of
activation for the reaction
• All reactions pass through a transition state on the
reaction pathway
• The active sites of enzymes bind the transition state
of the reaction more tightly than the substrate
• By doing so, enzymes stabilize the transition state
and lower the activation energy of the reaction
14.2 What Role Does Transition-State
Stabilization Play in Enzyme Catalysis?
• The catalytic role of an enzyme is to reduce the
energy barrier between substrate S and transition
state X‡
• Rate acceleration by an enzyme means that the
energy barrier between ES and EX‡ must be
smaller than the barrier between S and X‡
• This means that the enzyme must stabilize the
EX‡ transition state more than it stabilizes ES
14.2 What Role Does Transition-State Stabilization
Play in Enzyme Catalysis?
Enzymes catalyze reactions by lowering the activation energy.
Here the free energy of activation for (a) the uncatalyzed reaction
is larger than that of the enzyme-catalyzed reaction.
14.2 What Role Does Transition-State
Stabilization Play in Enzyme Catalysis?
Competing effects determine the position of ES
on the energy scale
• Try to mentally decompose the binding effects
at the active site into favorable and unfavorable
• The binding of S to E must be favorable
• But not too favorable!
• Km cannot be "too tight" - goal is to make the
energy barrier between ES and EX‡ small
14.3 How Does Destabilization of ES Affect
Enzyme Catalysis?
Raising the energy of ES raises 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
14.3 How Does Destabilization of ES Affect
Enzyme Catalysis?
(a) Catalysis does not occur if ES and X‡ are equally stabilized.
(b) Catalysis will occur if X‡ is stabilized more than ES.
14.3 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.
14.3 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.
14.3 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.
14.4 How Tightly Do Transition-State Analogs
Bind to the Active Site?
•
•
•
•
Very tight binding 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
Proline racemase was the first case
14.4 How Tightly Do Transition-State Analogs
Bind to the Active Site?
The proline racemase reaction. Pyrrole-2-carboxylate and Δ-1pyrroline-2-carboxylate mimic the planar transition state of the
reaction.
Transition-State Analogs Make Our World
Better
• Enzymes are often targets for drugs and other
beneficial agents
• Transition-state analogs often make ideal enzyme
inhibitors
• Enalapril lowers blood pressure
• Statins lower serum cholesterol
• Protease inhibitors are AIDS drugs
• Juvenile hormone esterase is a pesticide target
• Tamiflu is a viral neuraminidase inhibitor
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
• See the DrugBank: http://drugbank.ca
14.5 What Are the 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
Enzymes facilitate formation of near-attack
complexes
• X-ray crystal structure studies and computer modeling
have shown that the reacting atoms and catalytic
groups are precisely positioned for their roles
• Such preorganization selects substrate conformations
in which the reacting atoms are in van der Waals
contact and at an angle resembling the bond to be
formed in the transition state
• Thomas Bruice has termed such arrangements nearattack conformations (NACs)
• NACs are precursors to reaction transition states
Enzymes facilitate formation of near-attack
complexes
• Thomas Bruice has proposed that near-attack
conformations are precursors to transition states
• In the absence of an enzyme, potential reactant
molecules adopt a NAC only about 0.0001% of the
time
• On the other hand, NACs have been shown to form
in enzyme active sites from 1% to 70% of the time
Enzymes facilitate formation of near-attack
complexes
Figure 14.7 NACs are
characterized as having
reacting atoms within 3.2 Å
and an approach angle of
±15° of the bonding angle in
the transition state.
Figure 14.8 The active site of liver alcohol
dehydrogenase – a near-attack complex.
Protein Motions Are Essential to Enzyme Catalysis
• Proteins are constantly moving – bonds vibrate, side
chains bend and rotate, backbone loops wiggle and
sway, and whole domains move as a unit
• 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
Covalent Catalysis
• Some enzymes derive much of their rate
acceleration from formation of covalent bonds
between enzyme and substrate
• The side chains of amino acids in proteins offer a
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
Examples of
covalent
enzymesubstrate
intermediates.
Covalent Catalysis
General Acid-Base Catalysis
Catalysis in which a proton is transferred in the
transition state
• "Specific" acid-base catalysis involves H+ or OHthat 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.
Low-Barrier Hydrogen Bonds (LBHBs)
• The typical H-bond strength is 10-30 kJ/mol, and
the O-O separation is typically 0.28 nm
• As distance between heteroatoms becomes
smaller (<0.25 nm), H bonds become stronger
• Stabilization energies can approach 60 kJ/mol in
solution
• pKa values of the two electronegative atoms must
be similar
• Energy released in forming an LBHB can assist
catalysis
Low-Barrier Hydrogen Bonds (LBHBs)
Energy diagrams for conventional H bonds (a), and low-barrier
hydrogen bonds (b and c). In (c), the O-O distance is 0.23 to
0.24 nm, and bond order for each O-H interaction is 0.5.
Quantum Mechanical Tunneling
• Tunneling provides a path “around” the usual energy
of activation for steps in chemical reactions
• Many enzymes exploit this
• According to quantum theory, there is a finite
probability that any particle can appear on the other
side of an activation barrier for a reaction step
• The likelihood of tunneling depends on the distance
over which a particle must move
• Only protons and electrons have a significant
probability of tunneling
Quantum Mechanical Tunneling
• The de Broglie equation relates the “de Broglie
wavelength” to the mass and energy of a particle
h
l=
2mE
• Tunneling can only play a significant role in a
reaction when the wavelength of the transferring
particle is similar to the distance over which it is
transferred
• de Broglie wavelengths for protons and electrons are
0.9Å and 38Å, respectively
• Tunneling probably contributes to most, if not all,
hydrogen transfer reactions
Tunneling between donor and acceptor
If the distance for particle transfer is sufficiently
small, overlap of probability functions (red) permit
efficient quantum mechanical tunneling between
donor (D) and acceptor (A)
Metal Ion Catalysis
Thermolysin is an endoprotease with a catalytic Zn2+ ion in the
active site. The Zn2+ ion stabilizes the buildup of negative charge
on the peptide carbonyl oxygen, as a glutamate residue
deprotonates water, promoting hydroxide attack on the carbonyl
carbon.
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
14.5 What Can Be Learned From Typical Enzyme
Mechanisms?
First Example: the serine proteases
• Enzyme and substrate become linked in a
covalent bond at one or more points in the
reaction pathway
• The formation of the covalent bond provides
chemistry that speeds the reaction
• Serine proteases also employ general acid-base
catalysis
The Serine Proteases
Trypsin, chymotrypsin, elastase, thrombin,
subtilisin, plasmin, TPA
• All involve a serine in catalysis - thus the name
• Ser is part of a "catalytic triad" of Ser, His, Asp
• Serine proteases are homologous, but locations
of the three crucial residues differ somewhat
• Enzymologists agree, however, to number them
always as His57, Asp102, Ser195
• Burst kinetics yield a hint of how they work
The Catalytic Triad of the Serine Proteases
Structure of chymotrypsin
(white) in a complex with eglin
C (blue ribbon structure), a
target substrate. His57 (red) is
flanked by Asp102 (gold) and
Ser195 (green). The catalytic
site is filled by a peptide
segment of eglin. Note how
close Ser195 is to the peptide
that would be cleaved in the
reaction.
The Catalytic Triad of the Serine Proteases
The catalytic triad at
the active site of
chymotrypsin (and the
other serine
proteases.)
Serine Protease Binding Pockets are Adapted to
Particular Substrates
The substrate-binding pockets of trypsin, chymotrypsin, and
elastase. Asp189 (aqua) coordinates Arg and Lys residues of
substrates in the trypsin pocket. Val216 (purple) and Thr226 (green)
make the elastase pocket shallow and able to accommodate only
small, nonbulky residues. The chymotrypsin pocket is
hydrophobic.
Serine Proteases Cleave Simple Organic Esters,
such as p-Nitrophenylacetate
Chymotrypsin cleaves simple esters, in addition to peptide
bonds. p-Nitrophenylacetate has been used in studies of the
chymotrypsin mechanism.
Serine Protease Mechanism
•
•
•
•
•
A mixture of covalent and general acid-base
catalysis
Asp102 functions only to orient His57
His57 acts as a general acid and base
Ser195 forms a covalent bond with peptide to be
cleaved
Covalent bond formation turns a trigonal C into
a tetrahedral C
The tetrahedral oxyanion intermediate is
stabilized by the backbone N-H groups of Gly193
and Ser195
The Serine Protease Mechanism in Detail
Figure 14.21 The chymotrypsin mechanism: binding of a
model substrate.
The Serine Protease Mechanism in Detail
Figure 14.21 The chymotrypsin mechanism: the formation
of the covalent ES complex (E-Ser195–S complex) involves
general base catalysis by His57
The Serine Protease Mechanism in Detail
Figure 14.21 The chymotrypsin mechanism: His57 stabilized by
a LBHB.
The Serine Protease Mechanism in Detail
Figure 14.21 The chymotrypsin mechanism: collapse of the
tetrahedral intermediate releases the first product.
The Serine Protease Mechanism in Detail
Figure 14.21 The chymotrypsin mechanism: The amino
product departs, making room for an entering water molecule.
The Serine Protease Mechanism in Detail
Figure 14.21 The chymotrypsin mechanism: Nucleophilic
attack by water is facilitated by His57, acting as a general base.
The Serine Protease Mechanism in Detail
Figure 14.21 The chymotrypsin mechanism: Collapse of the
tetrahedral intermediate cleaves the covalent intermediate,
releasing the second product.
The Serine Protease Mechanism in Detail
Figure 14.21 The chymotrypsin mechanism: Carboxyl product
release completes the serine protease mechanism.
The Serine Protease Mechanism in Detail
Figure 14.21 The chymotrypsin mechanism: At the completion
of the reaction, the side chains of the catalytic triad are
restored to their original states.
Transition-State Stabilization in the Serine
Proteases
• The chymotrypsin mechanism involves two
tetrahedral oxyanion transition states
• These transition states are stabilized by a pair of
amide groups that is termed the “oxyanion hole”
• The amide N-H groups of Ser195 and Gly193 provide
primary stabilization of the tetrahedral oxyanion
The “oxyanion hole”
The oxyanion hole of
chymotrypsin stabilizes the
tetrahedral oxyanion transition
state seen in the mechanism
of Figure 14.21.