CHAPTER 9: CATALYTIC STRATEGIES
Chess vs Enzymes
King vs Substrate
INTRODUCTION
What are the sources of the catalytic power and specificity of
enzymes?
Problems in reactions in cells
• Neutral pH , water as a solvent and 37 ˚C
• Hard to achieve optimal reaction rate at these conditions
• Need a special strategy to achieve specificity
Enzyme classes discussed in this chapter
• Serine proteases
• Carbonic anhydrases
• Restriction endonucleases
• Myosins
CHAPTER 9
INTRODUCTION
Comparison between class members reveals how enzyme
active sites have evolved and been refined
Our knowledge of catalytic strategies has been used to
develop practical applications
• Potential drugs
• Enzyme inhibitors
• Other catalytic molecules (catalytic RNA)
CHAPTER 9
A FEW BASIC CATALYTIC PRINCIPLES
Substrate binding
• Establishes substrate specificity
• Stabilizes the transition state (lowers the activation E)
• Increases effective concentration
• Induced fit : the binding promotes structural changes that
facilitate catalysis
Covalent catalysis
• The active site contains a reactive group (nucleophile)
• The nucleophile becomes temporarily attached to a part of
the substrate in the course of catalysis
• Proteolytic enzymes
CHAPTER 9
A FEW BASIC CATALYTIC PRINCIPLES
General Acid-Base Catalysis
• A molecule other than water plays the role of a proton donor
or acceptor
• Histidines in chymotrypsin and carbonic anhydrase,
phosphate group of ATP in myosins
Catalysis by Approximation
• Reaction rate enhancement by proximity effect
Metal Ion Catalysis
• Nucleophile activation (carbonic anhydrase)
• Stabilizing reaction intermediate (EcoRV)
• Increasing binding affinity of substrates (myosin)
CHAPTER 9
9.1 CATALYSIS OF PROTEASES
Proteases facilitate a fundamentally difficult reaction
Involved in protein turnover
Important in regulating the activity of enzymes and proteins
Perform a hydrolysis reaction
• The addition of a water molecule to a peptide bond
• Thermodynamically favored reaction
• Half-life ~ 10 – 1000 yrs at physiological condition
• Peptide bonds need to be hydrolyzed within miliseconds in
some biochemical processes
CHAPTER 9
9.1 CATALYSIS OF PROTEASES
Kinetic stability of peptide bonds
• The resonance structure of a peptide bond
- Planar conformation
- Partial double bond character
- The amide carbon is less electrophilic than the ester carbon
• These properties make the peptide bond stable
CHAPTER 9
CHYMOTRYPSIN POSSESSES
A HIGHLY REACTIVE SERINE RESIDUE
Chymotrypsin cleaves peptide bonds selectively on the
carboxyl side of the large hydrophobic AA
• C-term of Trp, Phe, Tyr and Met
• A good example of the use of covalent catalysis
- A powerful Nu: attacks the unreactive carbonyl carbon of
the substrate
- The Nu: becomes covalently attached to the substrate
CHAPTER 9.1 PROTEASES
Fig 9.1 Specificity of chymotrypsin
Chymotrypsin cleaves the amide bond
of the carboxyl side of hydrophobic
amino acid.
CHYMOTRYPSIN POSSESSES
A HIGHLY REACTIVE SERINE RESIDUE
Identification of the reactive Nu: in chymotrypsin
• Treatment of chymotrypsin with diisopropylphospho-fluoridate
(DIPF) makes the enzyme irreversibly inactive
• Only a single residue, Ser195 was modified
- The residue plays a central role in the catalytic mechanism
of chymotrypsin
CHAPTER 9.1 PROTEASES
Fig 9.2 An unusually reactive serine
residue in chymotrypsin.
Chymotrypsin is inactivated by
treatment with DIPF, which reacts only
with Ser195 among 28 possible serine
residues
TWO-STEP ACTION OF CHYMOTRYPSIN
Kinetic study of chymotrypsin
• N-acetyl-L-phenylalanine p-nitrophenyl ester was used to
monitor the reaction by a colored product.
CHAPTER 9.1 PROTEASES
Fig 9.3 Chromogenic substrate.
N-acetyl-L-phenylalanine p-nitrophenyl ester
yields a yellow product, p-nitrophenolate, on
cleavage by chymotrypsin.
TWO-STEP ACTION OF CHYMOTRYPSIN
Kinetic study of chymotrypsin
• Michealis-Menten kinetics with a KM of 20 mM and a kcat of
77 s-1.
• Hydrolysis proceeds in two steps: an initial rapid burst
followed by a steady-state
CHAPTER 9.1 PROTEASES
Fig 9.4 Kinetics of chymotrypsin
catalysis. Two phases are evident in
the cleaving of N-acetyl-L-phenylalanine
p-nitrophenyl ester by chymotrypsin.
TWO-STEP ACTION OF CHYMOTRYPSIN
Explanation of the two-step hydrolysis
• The first step: acyl-enzyme intermediate formation
- Ser195 attacks the carbonyl carbon of the substrate
- p-nitrophenolate is released
• The second step: Hydrolysis of the acyl-enzyme intermediate
CHAPTER 9.1 PROTEASES
Fig 9.5 Covalent Catalysis. Hydrolysis by chymotrypsin takes place in two steps: (A) acylation to
form the acyl-enzyme intermediate followed by (B) deacylation to regenerate the free enzyme.
CATALYTIC TRIAD
The 3-D structure of chymotrypsin was solved in 1967
Comprises three polypeptide chains linked by disulfide bonds
CHAPTER 9.1 PROTEASES
Fig 9.6 The 3-D structure of chymotrypsin.
Synthesized as a
single polypeptide
(chymotrypsinogen),
which is activated by
the proteolytic
cleavage to yield the
three chains
CATALYTIC TRIAD
The active site lies in a left on the surface of the enzyme
Two hydrogen bonds in the active site
• Between Ser195 and His57
• Between His57 and Asp102
These three residues are referred to as the catalytic triad
CHAPTER 9.1 PROTEASES
Fig 9.7 The catalytic triad. The catalytic triad, shown on the left, converts serine 195
into a potent nucleophile, as illustrated on the right
CATALYTIC TRIAD
CHAPTER 9.1 PROTEASES
Fig 9.8 Peptide hydrolysis by chymotrypsin.
Substrate binding
Nucleophilic attack
Cleavage of the amide bond
Release of the amino
component
Water binding
Nucleophilic attack
Cleavage of the ester bond
Release of the carboxylic
acid component
Stabilizes the (-) charge of the
intermediate
Activate the Nu:
CATALYTIC TRIAD
CHAPTER 9.1 PROTEASES
Fig 9.11 Specificity nomenclature for protease-substrate interactions.
Fig 9.9 The oxyanion hole.Fig 9.10 Specificity pocket of
chymotrypsin
Positioning of Ser195
Hydrophobic environment
CATALYTIC TRIADS FOUND IN OTHER ENZYMES
Catalytic triads are found in other
hydrolytic enzymes
• Trypsin and elastase are obvious
homologs of chymotrypsin
- 40% identical sequence
- overall structures are quite similar
- remarkable difference in substrate
specificity: aromatic/hydrophobic,
long/(+) charged, and small (Fig
9.13)
CHAPTER 9.1 PROTEASES
Fig 9.12 Structural similarity of
trypsin and chymotrypsin.
Chymotrypsin (red); trypsin (blue)
CATALYTIC TRIADS FOUND IN OTHER ENZYMES
CHAPTER 9.1 PROTEASES
Fig 9.13 The S1 pockets of chymotrypsin, trypsin, and elastase.
CATALYTIC TRIADS FOUND IN OTHER ENZYMES
CHAPTER 9.1 PROTEASES
Fig 9.14 The catalytic triad and oxyanion hole of subtilisin.
Catalytic triads are found in many other hydrolytic enzymes
This catalytic strategy must be an especially effective approach
to the hydrolysis of peptides and related bonds
SITE-DIRECTED MUTAGENESIS STUDY
CHAPTER 9.1 PROTEASES
Fig 9.15 Site-directed mutagenesis of subtilisin.
How can we prove that the proposed mechanism is correct?
One way is to test the contribution of individual AA
Each residues within the catalytic triad in subtilisin are individually
converted into Ala
• Asp32, His64, and Ser221
• By site-directed mutagenesis
Oxyanion hole
• Asn155 to Gly
• kcat reduces to 0.2%
• Shows the significant role
of the amide proton
OTHER PEPTIDE-CLEAVING ENZYMES
CHAPTER 9.1 PROTEASES
Fig 9.16 Three classes of proteases
and their active sites.
Three alternative approaches to peptide-bond hydrolysis
OTHER PEPTIDE-CLEAVING ENZYMES
CHAPTER 9.1 PROTEASES
Fig 9.17A The activation strategy for
cysteine proteases.
Cysteine proteases
• Their catalytic strategy is similar to the chymotrypsin family
• Cysteine plays the role of serine in chymotrypsin
• The cysteine is activated by a histidine residue
• Asp in the catalytic triad does not exist. (better nucleophilicity
of Cys)
OTHER PEPTIDE-CLEAVING ENZYMES
CHAPTER 9.1 PROTEASES
Fig 9.17B The activation strategy for
aspartyl proteases.
Aspartyl proteases
• There are two aspartic acid residues in the active site
• One Asp activates the attacking water molecule
• The other polarizes the peptide carbonyl group
• Examples are renin and pepsin
OTHER PEPTIDE-CLEAVING ENZYMES
CHAPTER 9.1 PROTEASES
Fig 9.17C The activation strategy for
metalloproteases.
Metalloproteases
• The active site contains a metal ion (zinc in most cases)
• The metal ion activates the attacking water molecule and
polarizes the peptide carbonyl group
• Examples are thermolysin and
carboxylpeptidase A
PROTEASE INHIBITORS
CHAPTER 9.1 PROTEASES
Figure Structures of captopril
Captopril
• The first angiotensin-converting enzyme inhibitor (ACE
inhibitor)
• Developed in 1975 by three researchers at the U.S. drug
company Squibb (now Bristol-Myers Squibb)
• Used for the treatment of
hypertension and some types of
congestive heart failure ( )
PROTEASE INHIBITORS
CHAPTER 9.1 PROTEASES
Indinavir
• A protease inhibitor used to treat HIV infection and AIDS
• FDA-approved in 1996
• Aspartyl protease
• Non-peptidic substrate analog
▲Fig 9.18 HIV protease, a dimeric
aspartyl protease.
◄Fig 9.19 Indinavir, an HIV protease
inhibitor.
9.2 CATALYSIS OF CARBONIC ANHYDRASES
Hydration of carbon dioxide (CO2)
• CO2 is a major end product of aerobic metabolism and
released into the blood and transported to the lungs
• In the red blood cells, it reacts with water.
CHAPTER 9
pKa = 3.5
k1 = 0.0027 M-1s-1
k-1 = 50 s-1
K1 = 5.4 X 10-5
Carbonic anhydrases (CAs) accelerate CO2 hydration
• The most active CAs hydrate CO2 at kcat = 106 M-1s-1.
ZINC ION IN CARBONIC ANHYDRASE (CA)
9.2 CATALYSIS OF CARBONIC ANHYDRASES
CA was discovered in 1932
In 10 years after the discovery, the enzyme was found to
contain a bound zinc ion
• Appeared to be necessary for catalytic activity
• It made CA the first known zinc-containing enzyme
Hundreds of enzymes are known to contain zinc.
One-third of all enzymes contain or require metal ions for
activity
• Metal ions have several properties that increase chemical
reactivity: (+) charges, strong yet kinetically labile bonds, and
several oxidation states
• The chemical properties explain why they are important for
enzyme activity
ZINC ION IN CARBONIC ANHYDRASE (CA)
9.2 CATALYSIS OF CARBONIC ANHYDRASES
At least seven CAs are present in human beings
• They are all clearly homologous
• CA II is a major protein component of red blood cells and one
of the most active CAs
Fig 9.21 The structure of human CA II
and its zinc site.
CATALYSIS ENTAILS
ZINC ACTIVATION OF A WATER MOLECULE
9.2 CATALYSIS OF CARBONIC ANHYDRASES
pH dependence of enzymatic
CO2 hydration
• kcat increases with increasing pH
• The midpoint is near pH 7
Fig 9.22 Effect of pH on CA activity.
Fig 9.23 The pKa of zinc-bound water. Binding to zinc lowers the pKa of water form
15.7 to 7.
CATALYSIS ENTAILS
ZINC ACTIVATION OF A WATER MOLECULE
9.2 CATALYSIS OF CARBONIC ANHYDRASES
Zinc ion acts as a Lewis acid
and lowers the pKa of the bound
water
The zinc-bound OH- is a potent
nucleophile
CA also possesses a
hydrophobic patch
• Serves as a binding site for
carbon dioxide Fig 9.24 Carbon dioxide binding site.
CATALYSIS ENTAILS
ZINC ACTIVATION OF A WATER MOLECULE
9.2 CATALYSIS OF CARBONIC ANHYDRASES
Mechanism of CA
• Water deprotonation
• CO2 binding
• Nucleophilic attack by HO-
• Displacement of HCO3- by
water
Fig 9.25 Mechanism of CA.
CATALYSIS ENTAILS
ZINC ACTIVATION OF A WATER MOLECULE
9.2 CATALYSIS OF CARBONIC ANHYDRASES
Fig 9.26 A synthetic analog model system for carbonic anhydrase.
Studies of a synthetic analog model system
• Provide evidence for the mechanism’s plausibility
• The pKa of the bound water is 8.7
• At pH 9.2, this complex accelerates the hydration of CO2
more than 100-fold
• This experiment supports the proposed mechanism is
correct
PROTON TRANSFER IN CA CATALYSIS
9.2 CATALYSIS OF CARBONIC ANHYDRASES
Fig 9.27 Kinetics of water deprotonation.
Protons diffuse very rapidly with 2nd order rate constants
near 1011 M-1s-1.
k-1 ≤ 1011 M-1s-1 and K = 10-7 M, then k1 ≤ 104 s-1
If CO2 is hydrated at a rate of 106 s-1, then every step in the
mechanism must take place at least this fast!!
(pKa of the bound water is 7)
PROTON TRANSFER IN CA CATALYSIS
9.2 CATALYSIS OF CARBONIC ANHYDRASES
Fig 9.28 The effect of buffer on deprotonation.
The highest rates of carbon dioxide hydration require the
presence of buffer.
k1′ and k-1′ will be limited by buffer diffusion, ≤ 109 M-1s-1
At [B] = 1 mM, the rate of proton abstraction becomes
106 M-1s-1
K = k1′/k-1′ ≈ 1
if pKa of BH+ is 7.
PROTON TRANSFER IN CA CATALYSIS
9.2 CATALYSIS OF CARBONIC ANHYDRASES
Fig 9.29 Histidine proton shuttle.
His64 in CA II functions as the buffer
His64 abstracts a proton from the zinc-bound water and then
transfer the proton to the buffer
In many enzymatic reactions, the proton transfer is crucial to
the function
9.3 CATALYSIS OF RESTRICTION ENZYMES
Bacteria and archaea have mechanisms to protect
themselves from viral infections
• A major protective strategy for the host is to use restriction
endonucleases (REs)
REs recognize particular base sequences
REs must show tremendous specificity at two levels
• They must not degrade host DNA containing the recognition
sequences
• They must cleave only DNA molecules containing
recognition sites
For example, a recognition site, 5′-GATATC-3′, requires more
than 46 (4096) times more efficient activity
How do REs achieve these specificity?
CHAPTER 9
CLEAVAGE MECHANISM
9.3 CATALYSIS OF RESTRICTION ENZYMES
Fig 9.32 Hydrolysis of a phosphodiester bond.
REs catalyze the hydrolysis of the phosphodiester backbone
of DNA
• Generate a free 3′-hydroxyl group and a 5′-phosphoryl
group
TWO PROPOSED MECHANISMS
9.3 CATALYSIS OF RESTRICTION ENZYMES
Mechanism 1 (covalent intermediate)
Mechanism 2 (direct hydrolysis)
TWO PROPOSED MECHANISMS
9.3 CATALYSIS OF RESTRICTION ENZYMES
How can we figure out which mechanism is correct?
In-line displacement (SN2)
• Interconversion of the R and S configurations
Comparison of the two mechanisms
• In both cases, the reaction takes place by in-line displacement
• Two conversions occur in mechanism 1
• Only one conversion occurs in mechanism 2
TWO PROPOSED MECHANISMS
9.3 CATALYSIS OF RESTRICTION ENZYMES
How can we figure out which mechanism is correct?
Analysis of the product configuration
• A problem is that the product is not chiral !
Fig 9.33 Labeling with phosphorothioates.
Designing a special substrate
• Phosphorothioate
• Water containing 18O
TWO PROPOSED MECHANISMS
9.3 CATALYSIS OF RESTRICTION ENZYMES
Fig 9.34 Stereochemistry of cleaved DNA.
The analysis revealed that the stereochemical configuration
at the phosphorus atom was inverted only once with
cleavage
→ Mechanism 2
MAGNESIUM FOR CATALYTIC ACTIVITY
9.3 CATALYSIS OF RESTRICTION ENZYMES
One or more Mg2+ are essential to the function of RE
• As many as three metal ions have been found to be present
per active site
• The roles of the multiple metal ions is still under investigation
Fig 9.35 A magnesium ion-binding site in EcoRV.
• One ion-binding site
appears in all RE
structures
• In the EcoRV structure,
the Mg2+ activates and
positions a water
molecule to attack the
phosphorus atom
THE ORIGIN OF THE SEQUENCE-SPECIFICITY
9.3 CATALYSIS OF RESTRICTION ENZYMES
The recognition sequences for most REs are inverted repeats
• Palindromic sequence / twofold rotational symmetry
• Most REs functions as a dimer
Fig 9.36 Structure of the recognition site of EcoRV.
THE ORIGIN OF THE SEQUENCE-SPECIFICITY
9.3 CATALYSIS OF RESTRICTION ENZYMES
The binding affinity of EcoRV to the cognate DNA
• The enzyme can bind DNA in the absence of Mg2+
• Almost no affinity difference between the cognate and
noncognate !
Fig 9.37 Structure of EcoRV embracing a cognate
DNA molecule.
THE ORIGIN OF THE SEQUENCE-SPECIFICITY
9.3 CATALYSIS OF RESTRICTION ENZYMES
A unique set of interactions between the enzyme and the
cognate DNA
• Direct interaction of GA in 5′-GATATC-3′ with the enzyme
Fig 9.37 Structure of EcoRV embracing a cognate
DNA molecule.
THE ORIGIN OF THE SEQUENCE-SPECIFICITY
9.3 CATALYSIS OF RESTRICTION ENZYMES
The most striking feature is the distortion of the DNA
• The central TA in 5′-GATATC-3′ is distorted to be positioned for
cleavage, which results in the specificity
• Catalytic activity difference is > 106-fold
Fig 9.38 Distortion of the recognition site. Fig 9.39 Nonspecific and cognate DNA
within EcoRV.
PROTECTION OF THE HOST-CELL DNA
9.3 CATALYSIS OF RESTRICTION ENZYMES
Restriction-modification system
• The host DNA is methylated by methylases for protection
• REs cannot cleave methylated DNA
• For each RE, the host cell produces a corresponding
methylase to methylate the cognate sequence
Fig 9.41 Protection by methylation.
9.4 ATP HYDROLYSIS OF MYOSINS
Myosins comprise a family of ATP-dependent motor proteins
Involved in muscle contraction and a wide range of other
eukaryotic motility processes
Found in all eukaryotes and the human
genome encodes more than 40 different myosins
Catalyze the hydrolysis of ATP
• Produce ADP and inorganic phosphate
• Thermodynamically favorable reaction
• Use the energy to drive the motion of molecules
CHAPTER 9
MYOSIN-ATP COMPLEX STRUCTURE
9.4 ATP HYDROLYSIS OF MYOSINS
The ATPase domain structure of the myosin from the soil-living
amoeba Dictyostelium discoideum
• Approximately 750 amino acids
• No significant structural change between the apo form and
complexed form
Fig 9.45 Myosin-ATP complex structure. blue,
no ligands bound; purple, complexed with ATP.
• No hydrolysis observed in the
complexed structure
• Mg2+ is not present in the
enzyme
• All NTPs are present as
NTP-Mg2+ complex
MYOSIN-ATP COMPLEX STRUCTURE
9.4 ATP HYDROLYSIS OF MYOSINS
How does the hydrolysis occur?
• Water needs to be activated
• Requires a basic residue or activation by a metal ion
Fig 9.45 Myosin-ATP complex structure. blue,
no ligands bound; purple, complexed with ATP.
The enzyme-ATP complex is
stable
• No basic residue and
nucleophilic water observed
• Conformational change
required for catalysis
THE COMPLEX STRUCTURE WITH A TS-ANALOG
9.4 ATP HYDROLYSIS OF MYOSINS
For catalysis, ATPase must stabilize the TS of the reaction
Expected that ATP hydrolysis includes a pentacoordinate TS
The complex structure of the ATPase with VO43-, ADP and Mg2+
• The vanadium atom is coordinated to five oxygen atoms
• Ser236 is positioned to play a role in catalysis
Pentacoordinated transition state of ATPFig 9.46 Myosin ATPase transition state analog.
THE COMPLEX STRUCTURE WITH A TS-ANALOG
9.4 ATP HYDROLYSIS OF MYOSINS
The proposed mechanism of ATP hydrolysis
• The water molecule attacks the γ-phosphoryl group
• The hydroxyl group of Ser236 mediates the proton transfer
from the water molecule to γ-phosphoryl group
• The ATP serves as a base to promote its own hydrolysis
Fig 9.47 Facilitating water attack.
THE COMPLEX STRUCTURE WITH A TS-ANALOG
9.4 ATP HYDROLYSIS OF MYOSINS
Conformational change of myosin
• Some residues in the active site moves by ~2 Å
- This helps facilitating the hydrolysis by stabilizing the TS
Fig 9.48 Myosin conformational changes. red,
ATP-bound; blue, TS analog-bound.
• 60 amino acids at the C-
terminus moves by ~25 Å
- This motion is amplified
even more as the C-terminal
domain is connected to
other structures.
THE RATE LIMITING STEP
9.4 ATP HYDROLYSIS OF MYOSINS
Slow turnover rate of myosin
• Once per second
• What steps limit the rate of turnover?
Experiment with H218O
THE RATE LIMITING STEP
9.4 ATP HYDROLYSIS OF MYOSINS
Experiment with H218O
• Two or three 18O were observed
→ the hydrolysis reaction is reversible
→ the release of the products (Pi) is rate limiting
Fig 9.48 Reversible hydrolysis of ATP within the myosin active site.
THE RATE LIMITING STEP
9.4 ATP HYDROLYSIS OF MYOSINS
Myosins are examples of P-loop NTPase enzymes
• P-loop is named because it interacts with phosphoryl groups
• P-loop is found in many enzymes involved in ATP-mediated
conformation change
Fig 9.51 Three proteins containing P-loop NTPase domains.