Download Enzymes: Catalytic Strategies

BIOC 460 Summer 2010
Enzymes: Catalytic Strategies
Reading: Berg, Tymoczko & Stryer, 6th ed., Chapter 9, pp. 241-254
hexokinase conformational change (Jmol):
http://www.biochem.arizona.edu/classes/bioc462/462a/jmol/hexokinase/newhk.html
movie of chemical mechanism of serine proteases (from Voet & Voet,
Biochemistry 3rd ed
Biochemistry,
ed., 2004
2004, Wiley):
http://www.biochem.arizona.edu/classes/bioc460/spring/460web/lectures/LEC1314_EnzCatMech/15-3c_SerineProtease-b3/SerineProtease.htm
Serine proteases (Jmol)
http://www.biochem.arizona.edu/classes/bioc462/462a/jmol/serprot/serprot1.htm
Induced Fit: citrate synthase
open
form
closed
form
Berg et al.,
Fig. 17-20
Key Concepts
•
Mechanisms used by enzymes to enhance reaction rates include:
(1st 4 mechanisms based on BINDING of substrate and/or transition state)
1. Proximity & orientation
2. Desolvation (one type of electrostatic catalysis)
3. Preferential binding of the transition state
4 Induced fit
4.
5. General acid/base catalysis
6. Covalent (nucleophilic) catalysis
7. Metal ion catalysis
8. (Electrostatic catalysis)
•
The chemical mechanism of serine proteases like chymotrypsin illustrates:
– Proximity and orientation
– Transition state stabilization
– Covalent catalysis, involving a “catalytic triad” of Asp, His and Ser in
the active site
– general acid-base catalysis
– electrostatic catalysis
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General Catalytic Mechanisms
• Different enzymes use different combinations of mechanisms to reduce
activation energy (∆G‡) and thus increase rate of reaction.
• 7 (or 8) "types" of mechanisms below -- really "overlapping" concepts in
many cases.
• 1st 4 mechanisms related to BINDING of substrate and/or transition state
state,
(reaction takes place in active site, not in bulk solution)
1. PROXIMITY AND ORIENTATION (catalysis by “approximation”)
– Proximity
• Reaction between bound molecules doesn't require an improbable
collision of 2 molecules.
• They
They're
re already in "contact"
contact (increases local concentration of
reactants).
– Orientation
• Reactants are not only near each other on enzyme, they're oriented
in optimal position to react.
• The improbability of colliding in correct orientation is taken care of.
General Catalytic Mechanisms, continued
2. DESOLVATION
• Active site gets reactants out of H2O.
• Lower dielectric constant environment than H2O (more nonpolar
environment), so stronger electrostatic interactions (strength inversely
related to dielectric constant).
• Reactive groups of reactants are protected from H2O, so H2O doesn't
compete with reactants.
– H2O won't react to give unwanted byproducts, e.g., by hydrolysis of some
reactive intermediate in the reaction that was supposed to transfer its
reactive group to another substrate.
3. TIGHT TRANSITION STATE BINDING
• used to be called "strain and distortion"
• Enzyme binds transition state very tightly, tighter than substrate
(stabilizes T.S.)
• Free energy of transition state (peak of free energy barrier on reaction
diagram) is lowered because its "distortion" (electrostatic or structural) is
"paid for" by tighter binding of transition state than of substrate.
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4. INDUCED FIT
• Conformational change resulting from substrate binding
• Binding may stabilize different conformation of enzyme or substrate or both.
• Conformational change
– orients catalytic groups
– promotes tighter transition state binding, and/or
– excludes H2O
• Example: hexokinase, binding of glucose (1st reaction in glycolysis)
– closure of enzyme around upon binding of D-glucose (red)
– hexokinase conformational change (jmol)
“Open” conformation (no glucose)
“Closed” conformation (glucose bound)
Hexokinase,
induced fit
Nelson & Cox, Lehninger
Principles of Biochemistry,
4th ed. Fig. 8-21
“Open” conformation (no glucose)
Nelson & Cox, Lehninger
Principles of Biochemistry,
4th ed. Fig. 8-21
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“Closed” conformation (glucose bound)
Nelson & Cox, Lehninger
Principles of Biochemistry,
4th ed. Fig. 8-21
1st 4 concepts (above) in catalysis rather general, all related to BINDING of
substrate and/or transition state
Other catalytic mechanisms (below) involve specific groups and chemical
mechanisms that depend on the specific reaction.
5. GENERAL ACID-BASE CATALYSIS
• Specific functional groups in enzyme structure positioned to
– donate a proton (act as a general acid)
acid), or
– accept a proton (act as a general base)
• helps enzyme avoid unstable charged intermediates in reaction
• the general acid (H+ donor) has to then accept a proton (act as a general
base) later in catalytic mechanism to regenerate itself.
• Likewise, general base that accepts a proton must give it up later.
• Amino acid functional groups that can act as general acids/general bases:
– thiol of Cys
– R group carboxyls of Glu
Glu, Asp
– ROH of Ser, Thr, and Tyr
− α-amino group
– His imidazole
– guanidino group of Arg
− ε-amino group of Lys
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6. COVALENT CATALYSIS
• rate acceleration by transient formation of a COVALENT enzymesubstrate bond
• Covalent intermediate is more reactive in next step in reaction, so
that step has lower activation energy than it would have for a noncovalent catalytic mechanism -- enzyme alters pathway to get to product.
• Nucleophilic attack often involved in covalent catalysis
• Nucleophile: an electron-rich group that attacks nuclei
• unprotonated His imidazole
• unprotonated α-amino group
• unprotonated ε-amino group of Lys
• unprotonated thiol of Cys (thiolate anion, RS–)
• Unprotonated alcohol of Ser
Ser,Thr,
Thr and Tyr (alkoxide
(alkoxide, RO-)
• unprotonated R group of Glu, Asp (carboxylates, COO- )
– some coenzymes, e.g., thiamine pyrophosphate (TPP) & pyridoxal
phosphate (PLP)
7. METAL ION CATALYSIS (several catalytic roles)
Metal ions can be
–
tightly bound (metalloenzymes), i.e., as a prosthetic group
(usually transition metal ions, e.g., Fe2+ or Fe3+, Zn2+, Cu2+, Mn2+....)
–
loosely bound, binding reversibly and dissociating from enzyme
(usually Na+, K+, Mg2+, Ca2+, Zn2+...)
• Functions of metal ions in catalysis:
A Binding and orientation of substrate (ionic interactions with
A.
negatively charged substrate)
B. Redox reactions (e.g., Fe2+ / Fe3+ in some enzymes)
C. Shielding or stabilizing negative charges on substrate or on
transition state (electrophilic catalysis)
–
example: Kinases (e.g., hexokinase) bind ATP (adenosine
triphosphate) and require Mg2+ to be bound to nucleotide (so ligand
is actually Mg2+•ATP) in order to
g
charges,
g
and
• shield negative
• orient the ATP substrate
–
All kinases require Mg2+ for activity.
•
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[8. ELECTROSTATIC EFFECTS]
•
concept not always "listed" separately because it’s involved in many other
aspects of catalytic mechanisms
•
Some examples:
–
providing lower dielectric constant of environment in active site
(hydrophobic environment)
–
altering pK values of specific functional groups
–
stabilizing a particular conformation of critical groups in active site by
electrostatic interactions
–
stabilizing (binding) a charged intermediate or transition state by
providing an oppositely charged enzyme group nearby.
PROTEASES
• Reaction catalyzed = hydrolysis of peptide bonds
• Functions in vivo:
– digestion of nutrient protein
– degradation of unwanted proteins
– specific proteolytic cleavage for activation of specific (developmental
processes; blood clotting)
• Peptide bond hydrolysis (SN2 attack by :O of water on carbonyl C of
the peptide bond)
+ H+
• Equilibrium (in 55.5 M H2O) lies FAR to the right, but in absence of catalyst,
reaction is extremely slow; t1/2 = 10 – 1000 yrs.
• Peptide bonds "kinetically stable"
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Mammalian Serine Proteases are Homologous.
• 3-dimensional folds (tertiary structures) of chymotrypsin (red) and trypsin
(blue).
• Mammalian serine proteases: chymotrypsin, trypsin & elastase
• Vary with respect to Substrate specificity
• Homologous (share a common
evolutionary ancestral gene) -primary structures about 40%
identical and 3-dimensional folds
nearly identical
• Common evolutionary origin with
a single ancestral gene that
duplicated a number of times, after
which sequences and substrate
specificities diverged (example of
divergent evolution.)
• Also includes many proteolytic
enzymes in the blood clotting
cascade.
Berg et al., Fig. 9-12
Substrate Binding: the hydrophobic “specificity pocket” of
chymotrypsin
• area of substrate recognition
site responsible for specificity
• Position of aromatic ring of S
bound in pocket is shown.
• Note small Gly residues in
“lining” of pocket
• Also note Ser 189 in bottom
of pocket. (This residue is
Asp in structure of trypsin)
Berg et al., Fig. 9-10
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Specificity pockets of chymotrypsin, trypsin, and elastase
• Substrate binding sites where "R1" group of the substrate binds
("R1" = R group of the amino acid residue contributing the carbonyl group of
the peptide bond to be cleaved).
• Trypsin cleaves peptide bonds on carbonyl side ("after") long + charged
residues (R1 = Lys+ or Arg+) facilitated by Asp– residue in bottom of S1 site.
• Pocket of elastase is occluded so only small side chains may enter.
Elastase cleaves
after small neutral
residues (e.g., Gly
and Ala).
Berg et al., Fig. 9-13
Mechanism of Peptide Bond Hydrolysis
• Mechanism of uncatalyzed reaction:
+ H+
• SN2 Nucleophilic attack by :O of H2O on carbonyl C
• Formation of tetrahedral intermediate
• Tetrahedral intermediate then breaks down
• Partial double bond character of peptide bond makes carbonyl carbon a
poor nucleophilic site.
• Catalytic task of proteases is to make that normally unreactive
carbonyl group more susceptible to nucleophilic attack by H2O.
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4 Classes of Proteases
• 4 classes of proteases based on different mechanisms to enhance the
susceptibility of the carbonyl group to nucleophilic attack
1. Serine proteases (e.g., chymotrypsin) -- covalent catalysis, with
initial nucleophilic attack carried out by enzyme Ser-O(H) group made
into a p
potent nucleophile
p
with assistance of nearby
y His imidazole
that acts as a general base
2. Cys proteases -- again, covalent catalysis, with initial nucleophilic
attack carried out by an enzyme Cys-S(H) group using nearby His
imidazole as a general base
3. Asp proteases -- nucleophile is HOH itself, assisted by 2 Asp
residues, general base catalysis by 1st Asp carboxyl group and
orientation/polarization of substrate carbonyl by 2nd Asp residue
4. Metalloproteases -- again, nucleophile is HOH assisted by metal
(e.g. Zn2+) and by general base catalysis due to Glu-COO–.
The mechanism of protease catalysis is similar to that
found in many classes of other enzymes: kinases,
phosphatases, transferases.
Detailed look at a serine protease, chymotrypsin
• Chymotrypsin makes carbonyl C of peptide bond more reactive by
changing pathway of reaction.
• Covalent catalysis by Ser residue, with assistance of a general base (His)
• Overall reaction: 2 separate "half reactions" (2 "phases" of catalysis),
with a metastable covalent intermediate (("acyl-enzyme
y
y
intermediate"))
between the 2 half reactions.
• Overall chemical steps in the 2nd phase are almost an exact repeat of
steps in the first phase.
What’s an “acyl group”?
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Overview of chymotrypsin mechanism: 2 half reactions
• First step/phase ("acylation")
– Enzyme provides potent nucleophile, a specific Ser O(H) group.
– Ser OH made more nucleophilic than usual with assistance of nearby
His residue as general base
– Nucleophilic attack --> the acyl enzyme intermediate (covalent)
– Amine "half" of original peptide/protein released as product (P1) at
end of first phase.
phase
S1
P1
S2
P2
Berg et al., Fig. 9-5
Overview of chymotrypsin mechanism: 2 half reactions
• Second phase ("deacylation")
– 2nd substrate, H2O, is nucleophile, attacking carbonyl C of the
carboxylate ester of acyl enzyme, again with assistance of active site
His residue as general base.
– Ester bond of acyl-intermediate
acyl intermediate hydrolyzed regenerating alcohol
component (the enzyme chymotrypsin, with its Ser-OH free again) and
carboxylic acid component, the 2nd product (P2) (carboxyl "half" of
original substrate peptide/protein).
Berg et al., Fig. 9-5
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THE CATALYTIC TRIAD
• 3 amino acid residues in active site in a hydrogen-bonded network:
– Ser 195
– His 57
– Asp 102
• essential
ti l for
f effective
ff ti catalytic
t l ti activity
ti it in
i chymotrypsin
h
t
i
• Catalytic triad action converts OH group of Ser 195 into a potent
nucleophile.
Berg et al., Fig. 9-7
Movie of chemical mechanism of chymotrypsin:
http://www.biochem.arizona.edu/classes/bioc460/spring/460web/lectures/LEC1314_EnzCatMech/15-3c_SerineProtease-b3/SerineProtease.htm
Whole Chymotrypsin Mechanism (Berg et al., Fig. 9-8)
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FIRST PHASE: ACYLATION of Enzyme
• Formation of acyl-enzyme covalent intermediate and generation of
the amine product
1. Formation of ES Complex
• Enzyme binds substrate with bulky
aromatic side chain in "specificity
pocket”.
• Bound substrate is positioned for
the peptide bond on carbonyl side
(i.e., "carboxyl" side) of that residue
to be cleaved.
Berg et al., Fig. 9-8, Step 1
2. Formation of 1st Tetrahedral Intermediate (the chemistry begins)
•
•
•
Oxygen atom of active site Ser-OH activated by hydrogen bond to His
(imidazole ring N:) in catalytic triad
Ser-O(–) carries out nucleophilic attack on carbonyl C of substrate (i.e.
covalent catalysis) --> COVALENT bond to carbonyl C (1st tetrahedral
intermediate).
Asp in catalytic triad:
a) helps maintain perfect orientation of His and Ser residues in hydrogen
bonded network, and
b) facilitates H+ transfer by electrostatic stabilization of HisH+ after it
has accepted the proton.
Berg et al., Fig. 9-8, Step 2
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Transition State Stabilization, the “Oxyanion Hole”
• FIRST TETRAHEDRAL INTERMEDIATE:
similar to that of transition state for its formation and breakdown, with
negatively charged OXYANION
•The "oxyanion hole", an area in the
active site of serine proteases that binds
the transition state particularly tightly
tightly.
•Active site binds oxyanion more tightly
than it bound original carbonyl group of
the substrate.
•C–O- bond longer than C=O
•Additional hydrogen bond forms
with peptide NH groups
•Net result: Stabilization of tetrahedral
intermediate
Berg et al., Fig. 9.9
3. Formation of Acyl-Enzyme Intermediate
•
•
•
First tetrahedral intermediate breaks down -- original amide (peptide)
bond cleaves
HisH+ donates a proton to the amino "half" of the original substrate (HisH+
now a general acid) to generate R2-NH2.
Acyl-enzyme intermediate: Peptide covalently attached to Ser residue of
enzyme.
Berg et al., Fig. 9-8, Step 3
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4. Amine Product (R2-NH2) dissociates from active site
(1st product leaves).
• Original carbonyl group of peptide
b d iis now a carbonyl
bond
b
l group
again, but it's covalently attached
to the Ser-O in the acyl-enzyme
product of first half reaction
(acylation phase).
Berg et al., Fig. 9-8, Step 4
SECOND PHASE OF CATALYSIS: DEACYLATION
• Breakdown of acyl-enzyme (covalent intermediate) by reaction with
H2O (HYDROLYSIS) and release of the carboxylic acid product
• Almost an exact repeat of the first steps in terms of catalytic
steps/mechanisms
5. Binding of Second Substrate,
H2O, in Active Site
• H2O activated by His acting as general
base
Berg et al., Fig. 9-8, Step 5
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•
6. Formation of the Second Tetrahedral Intermediate
Nucleophilic attack of HO- on carbonyl C of acyl-enzyme intermediate
→ COVALENT bond between OH and carbonyl C --> 2nd tetrhedral
intermediate.
Berg et al., Fig. 9-8, Step 6
Transition State Stabilization 2, the “oxyanion hole” again
• 2nd TETRAHEDRAL INTERMEDIATE with negatively charged
oxyanion.
• 2nd TI binding
g in oxyanion
y
hole
is similar to binding of first TI.
• Oxyanion hole is presumed to
be stabilizing transition states
for formation and breakdown of
2nd tetrahedral intermediate by
binding them tightly.
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7. Breakdown of 2nd Tetrahedral Intermediate:
Original ester bond (from acyl-enzyme) CLEAVES.
•
•
HisH+ (general acid) donates proton back to Ser O, generating alcohol
product of hydrolysis of acyl-enzyme, Ser-OH
Ester bond from acyl-enzyme intermediate breaks --> carboxylic acid
product
d t (R1-COOH)
COOH) ffrom original
i i l substrate.
b t t
Berg et al., Fig. 9-8, Step 7
8. Carboxylic acid product dissociates from active site.
• Enzyme molecule now in its
original state, with His imidazole in
neutral form, catalytic triad
appropriately hydrogen-bonded,
and active site ready to bind
another molecule of substrate and
do it all again.
Does hydrolysis occur in the
acylation or deacylation half
reaction of serine proteases?
What is the nucleophile in the
acylation half reaction?
What is the nucleophile in the
deacylation half reaction?
Berg et al., Fig. 9-8, Step 8
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Activation strategies for 3 more classes of proteases
(besides Ser proteases)
• problem faced by proteases: activation of carbonyl C of peptide bond for
attack by a nucleophile
• All generate a potent nucleophile to attack peptide carbonyl group.
– Cys proteases: nucleophile a Cys thiol activated by His (gen. base)
– Asp proteases: nucleophile is HOH itself assisted by 2 Asp residues:
general base catalysis by 1 Asp carboxyl group and
orientation/polarization of substrate carbonyl by 2nd Asp residue
– Metalloproteases: nucleophile is HOH assisted by binding to a metal
(e.g. Zn2+) and by general base catalysis by some enzyme base
group, e.g. Glu-COO–.
Berg et al., Fig. 9-18
HIV protease: an Asp protease
• Homodimer: 2 identical subunits, each contributing an Asp to active site.
• 2 catalytic Asp residues, 1 from each subunit, on opposite sides of 2-fold
axis of symmetry (below the bound crixivan in Fig. 9-21).
• Structure in Fig. 9.19 has substrate binding pocket indicated, with the 2
catalytic Asp residues in ball-and-stick structures.
• "Flaps" (a portion of each polypeptide chain, labeled) close down after
)
substrate binds ((induced fit).
• Structure shown in Fig. 9.21 is in complex with an inhibitor, crixivan, which
has a conformation that approximates the 2-fold symmetry of the enzyme.
• Crixivan thus inhibits HIV protease without affecting normal cellular Asp
proteases, which don't have the 2-fold symmetry that HIV protease has.
• Crixivan designed to mimic tetrahedral intermediate (transition state) -- it's a
transition state analog, with groups to bind various sub-pockets in substrate
binding site.
Berg et al., Fig. 9-19
Enzymes: Catalytic Strategies
Berg et al., Fig. 9-21
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Learning Objectives
• Discuss (briefly explain): 8 general catalytic mechanisms used by enzymes
to increase the rates of chemical reactions. (You won't be asked on an
exam to simply LIST them, but you could be expected to explain any one -or 2 or 3 or 4 -- of them.)
• Terminology: proteolysis, serine protease, general acid, general base,
catalytic triad, acyl group, tetrahedral intermediate, acyl-enzyme
i t
intermediate,
di t acylation,
l ti
d
deacylation,
l ti
nucleophile,
l
hil oxyanion
i h
hole
l
• Explain why peptide bonds are kinetically stable in the absence of a catalyst,
given that equilibrium lies far in the direction of hydrolysis in 55.5 M H2O.
(Why is any specific reaction a slow reaction?)
• Describe the chemical mechanism of hydrolysis of peptide bonds by
chymotrypsin, including the following:
– What is the "job" of the catalyst (the protease), i.e., what group needs to
be made more susceptible
p
to nucleophilic
p
attack?
– Describe substrate binding, including the role and chemical nature of the
"specificity pocket" in chymotrypsin, and which peptide bond in the
substrate (relative to the specificity group) will be cleaved.
– Draw the structure of the catalytic triad at the beginning of the reaction,
and explain how the states of ionization and hydrogen bonding pattern of
those 3 groups change step by step during catalysis.
Learning Objectives, continued
•
(chemical mechanism of chymotrypsin, continued)
–
Explain the role of each member of the catalytic triad in the reaction.
–
Identify the nucleophile that attacks the carbonyl carbon in acylation;
identify the nucleophile that attacks the carbonyl carbon in
deacylation.
–
Describe the acyl-enzyme intermediate, including identifying the
type of bond attaching the acyl group to the enzyme (Is it an amide
linkage? anhydride? ester? etc.) and how that acyl group relates to
the structure of the original substrate.
–
Draw the structures of each of the tetrahedral intermediates in the
reaction. (If you can do this, you understand the chemistry by which
they formed.)
–
Identify the leaving group coming from each of the tetrahedral
intermediates as the intermediate breaks down.
–
State what is being acylated and deacylated in the chymotrypsin
reaction (be specific about the functional group involved).
–
Explain the role of the "oxyanion hole" in the mechanism.
–
Describe which type(s) of general catalytic mechanisms (first
learning objective above) are used by chymotrypsin, and how.
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Learning Objectives, continued
•
•
•
Compare (very briefly, just the “bottom line”) the overall 3-dimensional
structures of chymotrypsin, trypsin, and elastase, and compare the
substrate binding specificities of those 3 enzymes, explaining the
relationship of the “specificity site/pocket” structure to the differences in
substrate specificity..
How do 3 other classes of proteases (besides the serine proteases)
generate nucleophiles potent enough to attack a peptide carbonyl group?
To which protease class does HIV protease belong? Describe the
quaternary structure and symmetry of the HIV protease and where in the
quaternary structure the active site residues are located.
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