Download Lectures 13-14 Enzymes: Catalytic Strategies

BIOC 460, Spring 2008
Lectures 13-14
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., 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
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
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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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
intermediate, acylation, deacylation, nucleophile, oxyanion hole
• 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 to nucleophilic 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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Strategies
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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.
General Catalytic Mechanisms
• Different enzymes use different combinations of mechanisms to reduce
activation energy 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,
(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're already in "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.
LECTURES 13-14, Enzymes - Catalytic
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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.
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.
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 on enzyme,
– promotes tighter transition state binding, and/or
– excludes H2O (obviously related to tight transition state binding, too )
• Example: hexokinase, binding of glucose (1st reaction in glycolysis)
– Ends of the U-shaped enzyme hexokinase pinch toward each other -conformational change induced by binding 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
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Strategies
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• 1st 4 concepts (above) in catalysis rather general, all related to BINDING of
substrate and/or transition state
• Reaction takes place in active site, not in bulk solution.
• 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), or
– accept a proton (act as a general base)
• helps enzyme avoid unstable charged intermediates in reaction
• Group that donates a proton (acts as a general acid) in catalysis has to then
accept a proton (act as a general base) later in catalytic mechanism for
catalyst to be regenerated in its original conjugate acid form.
• Likewise, general base that accepts a proton must give it up later.
• Amino acid functional groups that can act as general acids/general bases:
– His imidazole
– α-amino group
– thiol of Cys
– R group carboxyls of Glu, Asp
– ε-amino group of Lys
– aromatic OH of Tyr
– guanidino group of Arg
6. COVALENT CATALYSIS
• rate acceleration by transient formation of a COVALENT catalystsubstrate 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.
• Nucleophile: an electron-rich group that attacks nuclei
– examples of nucleophiles among protein functional groups:
• unprotonated His imidazole
• unprotonated α-amino group
• unprotonated ε-amino group of Lys
• unprotonated thiol (thiolate anion, -S–) of Cys
• aliphatic -OH of Ser
• unprotonated R group (carboxylates) of Glu, Asp
– some coenzymes, e.g., thiamine pyrophosphate (TPP) & pyridoxal
phosphate (PLP)
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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+...)
•
Functions of metal ions in catalysis:
A. Binding and orientation of substrate (ionic interactions with 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: Enzymes that bind ATP (adenosine triphosphate) require
Mg2+ to be bound to nucleotide (so ligand is actually Mg2+•ATP) in
order to
• shield negative charges, and
• orient the ATP substrate
–
Example: Kinases (enzymes catalyzing phosphoryl transfer involving
ATP or other nucleoside triphosphates)
–
All kinases require Mg2+ for activity, but it's actually in complex with
nucleotide (usually ATP).
–
example: hexokinase (transfer of terminal phosphate group from
ATP to glucose, producing glucose-6-P and ADP as products)
•
[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.
Enzyme Chemical Mechanisms (Chymotrypsin as an example)
• digestive protease
• synth. in mammalian pancreas
• secreted in inactive form as single
polypeptide chain (chymotrypsinogen)
• activated by proteolytic processing to
rearrange conformation to active enzyme,
which because of the activating peptide
bond "clips" has 3 chains
• structure stabilized by disulfide bonds
(true for many extracellular proteins)
LECTURES 13-14, Enzymes - Catalytic
Strategies
Berg et al., Fig. 9-6
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PROTEASES
•
•
•
•
Reaction catalyzed = hydrolysis of peptide bonds
in vivo functions
digestion of nutrient protein
protein turnover (degradation of proteins that are old or no longer needed as
conditions change)
• specific proteolytic “clips” for activation of enzymes (e.g., developmental
sequence, or signal for special event like blood clotting)
• Peptide bond hydrolysis (SN2 attack by :O of water on carbonyl C of the
peptide bond)
• Equilibrium (in 55.5 M H2O) lies FAR to the right, but in absence of catalyst,
reaction is extremely slow (fortunately -- or our bodies would all be puddles
of amino acids in solution!)
• Peptide bonds "kinetically stable"
Mechanism of Peptide Bond Hydrolysis
• Mechanism of uncatalyzed reaction:
simple nucleophilic attack by :O of H2O on carbonyl C of peptide bond,
forming tetrahedral intermediate
• Tetrahedral intermediate then breaks down as the amine "half" of original
peptide leaves.
• Reason uncatalyzed reaction is so slow: partial double bond character of
peptide bond makes carbonyl carbon much less reactive than carbonyl
carbons in carboxylate esters.
• Catalytic task of proteases is to make that normally unreactive
carbonyl group more susceptible to nucleophilic attack by H2O.
LECTURES 13-14, Enzymes - Catalytic
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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 potent nucleophile with assistance of nearby 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 made into a potent
nucleophile with assistance of nearby His imidazole that acts 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, but assisted by
binding to a metal (e.g. Zn2+) and by general base catalysis by
some enzyme base group, e.g. Glu-COO–.
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 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”?
LECTURES 13-14, Enzymes - Catalytic
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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 on substrate --> covalent intermediate, the acyl
enzyme intermediate (actually a carboxylate ester of carboxylate
"half" of original substrate, attached to enzyme's Ser R group that's
the alcohol component of the ester).
– Amine "half" of original peptide/protein released as product (P1) at
end of first phase.
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 intermediate is hydrolyzed to regenerate 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
LECTURES 13-14, Enzymes - Catalytic
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THE CATALYTIC TRIAD
• 3 amino acid residues in active site in a hydrogen-bonded network:
– Ser (residue #195)
– His (residue #57)
– Asp (residue #102)
• essential for effective catalytic activity in chymotrypsin
• 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)
LECTURES 13-14, Enzymes - Catalytic
Strategies
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FIRST PHASE OF CATALYSIS (PHASE I, ACYLATION)
• Formation of acyl-enzyme covalent intermediate and generation of
the amine product
1. Formation of ES Complex
• Enzyme binds substrate (peptide or
protein), with aromatic bulky
hydrophobic side chain "specificity
group" in "pocket”.
• Bound susbstrate is positioned for
the peptide bond on carbonyl side
(i.e., "carboxyl" side) of that residue
to be cleaved.
• Polypeptide chain of substrate also
forms a short β-sheet (hydrogen
bonds) with a β strand of enzyme in
binding site.
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
• His accepts proton from Ser-OH (general base catalysis), to become
HisH+
• Ser-O(–) (potent nucleophile) carries out nucleophilic attack on carbonyl C
of substrate (nucleophilic catalysis, i.e. covalent catalysis) --> COVALENT
bond to carbonyl C (1st tetrahedral intermediate).
[Proton transfer and nucleophilic attack may be concerted (occur at same
time), so Ser-O may not actually exist as an oxyanion/alkoxide ion (-O–)].
• 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
LECTURES 13-14, Enzymes - Catalytic
Strategies
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Transition State Stabilization, the “Oxyanion Hole”
• Product of step 2 (nucleophilic attack in acylation half-reaction) = FIRST
TETRAHEDRAL INTERMEDIATE
• structure presumed similar to that of transition state for its formation
and breakdown, with negatively charged "carbonyl" oxygen (not a
carbonyl group anymore), an OXYANION
•The "oxyanion hole", an area in the
active site of serine proteases that binds
the transition state particularly tightly.
•Active site binds oxyanion more tightly
than it bound original carbonyl group of
the substrate.
•An additional hydrogen bond forms
between tetrahedral oxyanion and
enzyme groups around it (which form
"oxyanion hole" portion of active site)
•That hydrogen bond couldn't form to
carbonyl oxygen (=O) but can form now,
because of structural change
(lengthening of C–O bond) on forming
tetrahedral intermediate.
•Also, hydrogen bonds to negatively
charged oxygen are stronger than to
neutral O.
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+
acts as general acid) to generate R2-NH2.
Breaking of amide bond (departure of amine product) --> conversion of
oxyanion back into a C=O, still covalently attached to Ser residue of
enzyme, forming acyl-enzyme intermediate.
Berg et al., Fig. 9-8, Step 3
LECTURES 13-14, Enzymes - Catalytic
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4. Amine Product (R2-NH2) dissociates from active site
(1st product leaves).
• Breaking of amide bond
(departure of amine product) -->
conversion of oxyanion back into
a C=O, still covalently attached to
Ser residue of enzyme, forming
acyl-enzyme intermediate.
• Original carbonyl group of peptide
bond is now a carbonyl group
again, but it's covalently attached
to the Ser-O in the acyl-enzyme
product of first half reaction
(acylation phase).
• What kind of linkage
does acyl-enzyme
have between
"carboxyl half"
of original peptide
substrate and O from
the Ser alcohol R
group?
Berg et al., Fig. 9-8, Step 4
SECOND PHASE OF CATALYSIS (PHASE II, DEACYLATION)
• Breakdown of acyl-enzyme (covalent intermediate) by reaction with
H2O (HYDROLYSIS) and release of the carboxylic acid product
• This phase (second "half reaction") is almost an exact repeat of the first in
terms of catalytic steps/mechanisms
5. Binding of Second Substrate,
H2O, in Active Site
• nucleophilic attack facilitated by HisN:
acting as general base (but
nucleophile is H2O, attacking
carbonyl C of acyl-enzyme)
• formation of second tetrahedral
intermediate with transition state
stabilization by binding of C-O– in
oxyanion hole
• breakdown of 2nd tetrahedral
intermediate (cleavage of what had
been ester bond in acyl-enzyme
intermediate, with HisH+ as general
acid catalyst) to regenerate enzyme
Ser-OH and release the carboxylic
acid product (from the original
peptide/protein substrate).
Berg et al., Fig. 9-8, Step 5
LECTURES 13-14, Enzymes - Catalytic
Strategies
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6. Formation of the Second Tetrahedral Intermediate
HOH forms hydrogen bond with HisN: in catalytic triad (just like Ser-OH did
in first phase).
• His again acts as a general base, to become HisH+, activating O from H2O
to make it a potent nucleophile, to attack carbonyl C of acyl-enzyme
intermediate (an ester).
• nucleophilic attack of HOH on carbonyl C of acyl-enzyme intermediate
(nucleophilic catalysis, i.e. covalent catalysis) → COVALENT bond
between OH of water and carbonyl C --> 2nd tetrhedral intermediate).
• Asp in catalytic triad:
a) helps maintain perfect orientation of catalytic triad, and
b) facilitates H+ transfer by electrostatic stabilization of HisH+ after it
has accepted the proton.
•
Berg et al., Fig. 9-8, Step 6
Transition State Stabilization 2, the “oxyanion hole” again
• Product of step 6 (nucleophilic attack in deacylation half-reaction) =
SECOND TETRAHEDRAL INTERMEDIATE
• structure presumed similar to that of transition state for its formation and
breakdown, with negatively charged "carbonyl" oxygen (not a carbonyl
group anymore), an OXYANION
• Note that SECOND tetrahedral intermediate has an -OH group on it (from
H2O) instead of amido group of amine "half" of original substrate as in
FIRST tetrahedral intermediate.
• Otherwise, 2nd TI binding in
oxyanion hole is similar to
binding of first TI, with extra
hydrogen bond, etc.
• Again, oxyanion hole is
presumed to be stabilizing
transition states for formation
and breakdown of 2nd
tetrahedral intermediate by
binding them tightly.
LECTURES 13-14, Enzymes - Catalytic
Strategies
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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 (R1-COOH) from original substrate.
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
LECTURES 13-14, Enzymes - Catalytic
Strategies
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BIOC 460, Spring 2008
Mammalian Serine Proteases are Homologous.
• 3-dimensional folds (tertiary structures) of chymotrypsin (red) and trypsin
(blue). (Only the backbone tracings of α carbon positions are shown.)
• family of mammalian serine proteases:
– e.g., chymotrypsin, trypsin & elastase
• obviously homologous (share a
common evolutionary ancestral
gene) -- primary structures about
40% identical and 3-dimensional
folds nearly identical
• suggests 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.)
• Family also includes many
proteolytic enzymes in the blood
clotting cascade.
Berg et al., Fig. 9-12
The hydrophobic “specificity pocket” of chymotrypsin
• area of active site responsible
for chymotrypsin’s substrate
specificity)
• Position of aromatic ring
bound in pocket is shown in
green in center.
• Note Gly residues in
“lining” of pocket (small, so
bulky, hydrophobic side chains
fit in binding site)
• Also note Ser 189 in bottom
of pocket. (This residue is
Asp in structure of trypsin.)
Berg et al., Fig. 9-10
LECTURES 13-14, Enzymes - Catalytic
Strategies
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The specificity pockets of chymotrypsin, trypsin, and elastase
• Substrate binding sites on enzymes 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+)
•specificity "assisted" by Asp– residue in bottom of S1 site.
• Pocket of elastase is partly closed off so only small side chains may enter (Val
residues in stead of Gly residues).
Elastase cleaves
after small neutral
residues (e.g., Gly
and Ala).
Berg et al., Fig. 9-13
Subtilisin: a bacterial protease
• no apparent evolutionary relationship to mammalian chymotrypsin/trypsin
family (no primary structure or tertiary structural resemblance)
• same catalytic mechanism as mammalian serine proteases: a catalytic Ser
assisted by a His and an Asp residue (catalytic triad), in same orientation,
and an oxyanion hole to stabilize oxyanion intermediates (transition states)
• example of convergent evolution: independent evolution of same catalytic
strategy
• (Chymotrypsin mechanism must be a very effective hydrolytic mechanism!)
Berg et al., Fig. 9-14
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Strategies
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site-directed mutagenesis studies on structural alterations
in subtilisin active site
• gene for subtilisin was cloned
• specific mutations in the catalytic triad residues (individually and in
combination) introduced by molecular biological methods
• mutant enzymes expressed, purified and studied
• Results: Mutations in catalytic triad residues have dramatic effects on
kcat (turnover number) for subtilisin.
• Note log scale for kcat;
mutation of Ser or His
reduces kcat by a factor of
about 106!
1st letter is normal (wild type) residue
in that (number) position; 2nd letter is
mutant amino acid residue replacing
normal residue at that position.
Berg et al., Fig. 9-16
• However, also note that
what might seem to be
"fatally" modified
(mutated) enzymes still
have higher kcat values
than kcat for uncatalyzed
reaction, by factor of ~103.
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
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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
LECTURES 13-14, Enzymes - Catalytic
Strategies
Berg et al., Fig. 9-21
19