Transcript Biochemistry 6/e

Chapter 9
Catalytic Strategies
4 classes of enzymes.
1. Protease
2. Carbonic anhydrase
3. Restriction endonuclease
4. Myosins
Structural and mechanistic comparisons of enzyme action
are the sources of insight into the evolutionary history of enzymes.
9.1 Proteases facilitate a fundamentally
difficult reaction
- Proteins must be degraded to that their constituent amino acids
can be recycled for the synthesis of new proteins.
- In the absence of a catalyst, the half-life for the hydrolysis of a peptide
at neutral pH is estimated to be between 10 and 1000 years.
- But, peptide bonds must be hydrolyzed within milliseconds in some
biochemical processes.
Chymotrypsin possesses a highly reactive serine redisue
- Chymotrypsin cleaves peptide bonds selectively on the carboxylterminal side of the large hydrophobic amino acids such as
tryptophan, tyrosine, phenylalanine, and methionine.
-Chymotrypsin is a good example of the use of “covalent catalysis”
-Employs a powerful nucleophile to attack the unreactive carbonyl carbon
atom of the substrate.
- DIPF(diisopropylphosphofluoridate) treatment → Serine 195
modification → activity irreversibly
Chymotrypsin action proceeds in two steps linked by a covalently bound
intermediate
Substrate
Ester bond
Product
- N-Acetyl-L-phenylalanine p-nitrophenyl ester : chromogenic substrate of
Chymotrypsin.
- Product : Yellow colored nitrophenolate(by Chymotrypsin cleavage)
-Product measurement : absorbance of light(amount of p-nitrophenolate)
-Under steady-state conditions, Km = 20μM, kcat = 77s-1
-Hydrolysis (by Chymotrypsin) proceeds in two steps.
-1). Burst phase (rapid)
-2). Steady-state phase
Burst phase
Steady-state phase
-Burst phase : the acyl group of the substrate becomes covalently
attached to the enzyme as p-nitrophenolate.
(acyl-enzyme intermediate)
-Steady-state phase : acyl-enzyme intermediate is hydrolyzed to
release the carboxylic acid component of the substrate and regenerate
the free enzyme.
Serine is part of a catalytic triad that also includes histidine and aspartate
-The three-dimensional structure
of chymotrypsin was solved by
David Blow in 1967.
-It is synthesized as a single
polypeptide, termed
Chymotrypsinogen, which is
activated by the proteolytic
cleavage to yield the three chains.
[1GCT.pdb]
cleft
-The active site of chymotrypsin, marked by serine 195, lies in a
cleft on the surface of the enzyme.
-This side chain of serine 195 is hydrogen bonded to the imidazole
ring of histidine 57.
Tetrahedral
intermediate
(acyl-enzyme)
Nucleophilic attack
Amine is free
Burst phase
Substrate binding
Steady-state phase
Carboxylic acid
product
Water mediated
deacylation
OH- Attacks the
carbonyl carbon
-Four atoms bounded to the carbonyl
carbon, arranged as a tetrahedron.
-Tetrahedron intermediate formed bears a
formal negative charge .
-This charge is stabilized by interactions
with NH groups from the protein in a site
termed the oxyanion hole.
-Preference for cleaving the
peptide bonds just past residues
with large, hydrophobic side
chains.
-Chymotrypsin has a deep,
hydrophobic pocket, called the S1
pocket.
-Other proteases have more complex specificity patterns.
-Such enzymes have additional pockets on their surfaces for the
recognition of their residues in the substrate.
Catalytic triads are found in other hydrolytic enzymes
- Trypsin and elastase are obvious
homologs of chymotrypsin.
- 40% sequence identity with
chymotrypsin.
Red : chymotrypsin
Blue : trypsin
Similar structure
Chymotrypsin
Trypsin
- Cleaves after residues with -Cleaves after residues with
Elastase
an aromatic or long nonpolar
small side chains. (A.S)
-Val190 and Val216 at bottom
of the S1 pocket
side chains.
an long, positively charged
side chains. (R.K)
-Asp189 at bottom of the S1
pocket
-Cleaves after residues with a
Subtilisin : a protease in bacteria(Bacillus amyloliquefaciens).
not homologs of chymotrypsin, but similar active site.
-The active site of this enzyme includes both the catalytic triad and
the oxyanion hole.
-NH groups, side chain from Asn located in the oxylanion hole rather
than from the peptide backbone.
Carboxypeptidase
: from wheat. not similar to either chymotrypsin or subtilisin structures.
- Catalytic triad of carboxypeptidase is composed of the same amino
acids as those in chymotrypsin.
The catalytic triad has been dissected by site-directed mutagenesis
- Each of the residues within the catalytic triad of Subtilisin,
(Aspartate32, Histidine64, and Serine221) has been individually converted into
alanine.
S221A dramatically reduced
catalytic power by 106.
H64A reduced catalytic power
to a similar degree.
N155G in subtilisin reduced by
50 fold
Cysteine, Aspartyl, and Metalloproteases are other major classes of
peptide-cleaving enzyme.
- A cys residue, activated by a his,
plays the role of the nucleophile that
attacks the peptide bond.
-Ex). Papain : purified from the fruit
of the papaya.
-Caspases
Cysteine, Aspartyl, and Metalloproteases are other major classes of
peptide-cleaving enzyme.
- A pair of aspartic acid residues that act
together to allow a water molecule to attack
the peptide bond. One aspartic acid activates
the attacking water molecule. The other
polarizes the peptide carbonyl group.
- Ex). Renin : having a role in the regulation of blood
pressure.
Pepsin : the digestive enzyme.
HIV protease
HIV protease : a dimeric aspartyl protease
Cysteine, Aspartyl, and Metalloproteases are other major classes of
peptide-cleaving enzyme.
glutamate
- The active site of such a protein contains a
bound metal ion, almost always zinc, that
activates a water molecule to act as a
nucleophile to attack the peptide carbonyl group.
- Ex). Thermolysin : bacterial enzyme
Carboxypeptidase : digesitve enzyme
Protease inhibitors are important drugs
-Several important drugs are protease inhibitors.
-Ex). Indinavir : Inhibitor of HIV protease, which is an aspartyl protease.
Mimics the tetrahedral intermediate.
-To prevent unwanted side effects, protease inhibitors used as drugs must
be specific.
- X-ray crystallography revealed that, in the active site, indinavir
adopts a conformation that approximates the twofold symmetry of
the enzyme.
(1) Cysteine proteases:
• quite analogous to serine protease
• histidine-activated cysteine residue as a nucleophile
e.g., papain, cathepsin, caspases
(2) Aspartyl proteases:
• approximate two-fold symmetry
• a pair of Asp at active site; asymmetric mode of action
• aspartate-activated water molecule as a nucleophile
• general acid-base catalyst
e.g., renin, pepsin, dimeric aspartyl protease of HIV
(3) Metalloproteases:
• almost always Zn++ at the active site
• metal-activated water molecule as a nucleophile
• base (often, Glu) helps deprotonate the metal bound
water
• No covalent intermediate
• e.g., matrix metalloproteases
In each of these classes of enzymes,
the active site includes features that allow for
(1) the activation of water or another nucleophile
(2) the polarization of the peptide carbonyl group
(3) subsequent stabilization of a tetrahedral intermediate
Activation strategies for three classes of proteases
9.2 Carbonic anhydrases make a fast
reaction faster
CO2 = end product of
metabolism
Strong acid (pKa = 3.5)
Even in the absence of a catalyst, this hydration reaction proceeds at a moderate rate.
But carbonic anhydrase speeds up carbon dioxide hydration and HCO3- dehydration!
(Kcat = 106 s-1)
In red blood cell (carbon dioxide hydration), Blood  lungs (HCO3- dehydration)
Concentration of H2O = 55.5 M
Carbonic anhydrase contains a bound zinc ion essential for
catalytic activity
- Carbonic anhydrase contains Zinc ion; first known zinc-containing enzyme.
- Zinc ion is necessary for catalytic activity.
- At least 7 carbonic anhydrase in human.
- Carbonic anhydrase Ⅱ is the most extensively studied.
- Three coordination sites are occupied by the imidazole rings of three His.
Metal ion
• 1/3 of all enzymes contain metal ion.
• Properties
1. positive charges,
2. ability to form relatively strong yet kinetically labile bonds,
3. more than one oxidation state; iron(II) sulphate, iron(III) chloride
How does this zinc complex facilitate carbon dioxide
hydration?
Major clue from pH profile
-At pH8, the reaction proceeds near
its maximal rate.
-The midpoint of this transition is
near pH7.
-Loss of proton at pH7 is important
 histidine (pK a=7)
<Effect of pH on carbonic anhydrase>
Nucleophile attack
- The binding of a water molecule to the positively charged zinc center reduces the pKa
of the water molecule from 15.7 to 7.
- A zinc-bound hydroxide ion (OH-) is a potent nucleophile able to attack carbon
dioxide.
Release of a proton from water
Generate a hydroxide ion
Catalytic site is regenerated with the
release of HCO3- and binding another
water.
CO2 bond to hydrophobic active site
HO- attacks CO2
converting it into HCO3-
Synthetic analog model system.
An organic compound, capable of binding zinc(like His in enzyme).
Water-zinc-compound complex accelerates the hydration of CO2.
The model system strongly suggests that the zinc-bound
hydroxide mechanism is correct.
A proton shuttle facilitates rapid regeneration of the active
form of the enzyme
Equilibrium constant
K-1 = 1011 M-1s-1 because proton diffusion rate 10-11 M-1s-1
So, K1 = 104 M-1s-1 then, How carbon dioxide is hydrated at 106s-1 ?
In the first step of a CO2 hydration reaction, the zinc-bound water molecule
must lose a proton to regenerate the active form of the enzyme.
The rate of reverse reaction, the protonation of the zinc-bound hydroxide
ion, is limited by the rate of proton diffusion
The deprotonation of the zinc-bound water molecule in carbonic
anhydrase is aided by buffer component B.
The rate of carbon dioxide hydration increases with the concentration of
the buffer 1,2-dimethylbenzimidazole.
The buffer enables the enzyme to achieve its high catalytic rates.
Many buffers are too large to reach the active site of carbonic
anhydrase. So proton shuttle is necessary (carbonic anhydrase II)
① His 64 abstracts a proton from ② The buffer B removes a proton
the zinc-bound water molecule,
form the His, regenerating the
generating a nucleophilic hydroxide unprotonated form.
ion and a protonated His.
Many biochemical reactions show the prominence of acid-base catalysis
Convergent evolution has generated zinc-based active sites in
different carbonic anhydrases
- In addition to a-carbonic anhydrases, two other families of carbonic
anhydrases have been discovered.
- β-carbonic anhydrases : found in higher plants and in many bacteria. Zinc
ion is bound by one His and two Cys.
- γ-carbonic anhydrases : identified in the archaeon Methanosarcina thermophila.
Three zinc sites similar to α-carbonic anhydrase. But zinc sites lie at the interfaces
between the three subunits of a trimeric enzyme.
9.3 Restriction enzymes perform highly
specific DNA-cleavage reactions
-Bacteria evolved mechanism to protect themselves from viral
infections; restriction endonucleases
Methylated
Viral
Host
-EcoRV cleaves double-stranded viral DNA molecules that contain
the seq.5’ GATATC 3’ but leaves host DNA.
-The host DNA is protected by other enzymes called
methylases, which methylate adenine bases within host recognition
seq.
-For each restriction enzymes, corresponding methylases exist.
Cleavage is by in-line displacement of 3’-oxygen from
phosphorus by magnesium-activated water
-Restriction endonuclease catalyzes the hydrolysis of the phosphodiester
backbone of DNA.
-Product : free 3’-hydroxyl group + 5’ phosphoryl group
-Hydrolysis of a phosphodiester bond
-Possible two mechanisms
Mechanism 1 (covalent intermediate)
: analogy with chymotrypsin
: two displacement, retained configuration
Mechanism 2 (direct hydrolysis)
: analogy with asparty protease and metalloproteases
: one displacement, inverted configuration
Each mechanism postulates a different nucleophile to attack the
phosphorus.
In either case, each reaction takes place by in-line displacement inducing
the interconversion of the configuration.
Trigonal bipyramidal
geometry
How to figure out the stereochemistry of the product?
- In case of EcoRV, this enzyme cleaves the phosphodiester bond
between the T and the A at the center of the recognition seq.
- Oxygen atom in the cleavage site is replaced by a sulfur.
Water 18O labeled
The hydrolysis takes place by water’s direct attack at the
phosphorus atom.
Restriction enzymes require magnesium for catalytic activity
-One or more Mg2+ cations are essential to the function of restriction
endonucleases.
H2O
-This metal ion is coordinated to the protein through two aspartate.
-The Magnesium ion helps to activate a water molecule and positions it so
that it can attack the phosphorus.
The complete catalytic apparatus is assembled only within
complexes of cognate DNA molecules, ensuring specificity
- The recognition seq. for restriction endonucleases are inverted
repeats.
- This arrangement gives the three-dimensional structure of the
recognition site a twofold rotational symmetry.
The structure of EcoRV (dimer)
bound to a cognate DNA
fragment.
5’ – G A T A T C – 3’
3’ – C T A T A G – 5’
-The most striking feature of this complex is the distortion of the
DNA, which is kinked in the center.
-The central two TA base pairs in the recognition seq. play a key
role in producing the kink even no contact with enzyme.
- The noncognate DNA
conformation is not
substantially distorted.
cognate
non-cognate
- This lack of distortion has important consequences with regard to
catalysis.
-No phosphate is positioned sufficiently close to the active-site
aspartate residues to complete a Mg2+ binding site.
-DNA distortion and subsequent Mg2+ binding account for the
catalytic specificity
-The distorted DNA makes
additional contacts with the enzyme,
increasing the binding energy
- The increase is canceled by the
energetic cost of DNA distortions.
- Interactions that take place within the distorted substrate
complex stabilize the transition state leading to DNA hydrolysis.
-The distortion in the DNA explains
how methylation blocks catalysis and
protects host DNA.
-The presence of the methyl group
blocks the formation of a hydrogen
bond between the amino group and
the side-chain carbonyl group of
Asn185.
-Asn185 is closely linked to the other
residues of specific contact with the
DNA
-Leading to the no distortion of DNA
TypeⅡ restriction enzymes have a catalytic core in common
and are probably related by horizontal gene transfer
No significant seq. similarity. However, a core structures are
conserved. It includes b strands (blue) that contain the aspartate
residues of the Mg-binding sites.
Horizontal gene transfer: passing between species of pieces of
DNA (plasmid).
Representative example: antibiotics
9.4 Myosins Harness Changes in EnzymeConformation to
Couple ATP Hydrolysis to Mechanical Work
- Myosins catalyze the hydrolysis of ATP
to form ADP & Pi
- Thermodynamically favorable reaction
ATP hydrolysis proceeds by the attack of water on the
gamma phosphoryl group
• Myosin structure revealed a water-filled pocket.
• Myosin crystal soaked with ATP; No conformational change.
• Divalent metal ions (Mg2+ , Mn2+ ) are required for activity.
Myosin-ATP complex structure with or without ATP & Mg
-Myosin are essentially inactive in the absence of metal ions (Mg2+,
Mn2+).
-Nucleotides bind these ions, and it is the metal ion-nucleotide complex
that is the true substrate for the enzymes.
-Kd of ATP-Mg2+ complex ~0.1mM, Mg2+ conc in cytosol is mM range. So?
• Nucleophilic attack by a water requires a basic residue or bound
metal to activate the water
• ATP hydrolysis includes a pentacoordinate transition state.
• Water molecule attacks the g-phosphoryl group, with the
hydroxyl group of Ser236, which is deprotonated by gphosphate.
Mechanism of ATP hydrolysis
NMP kinases are a family of enzymes containing P-loop
structures
Core domain
-Comparison of NMP kinase structures reveals that these enzymes form
a family of homologous proteins.
-The nucleoside triphosphate binding domain is a common feature in
these homologous nucleotide kinases and others.
-Core domain of NMP kinases
-Consist of a central β sheet,
surrounded on both sides by α
helices.
-P loop : between the first β
strand and the first α helix.
Seq. : Gly-X-X-X-X-Gly-Lys
Interact with NTP
- P loop interact with ATP
Phosphate
Magnesium or manganese complexes of nucleoside
triphosphates are the true substrates for essentially all
NTP-dependent enzymes.
-NMP kinases are essentially inactive in the absence of metal ions
(Mg2+, Mn2+).
-Nucleotides bind these ions, and it is the metal ion-nucleotide complex
that is the true substrate for the enzymes.
-Kd of ATP-Mg2+ complex ~0.1mM, Mg2+ conc in cytosol is mM range. So?
-Magnesium ion is bound to the β and γ phosphoryl groups and to four
water molecules at the remaining coordination position.
→ The magnesium ion provides additional points of interaction between
the ATP-Mg2+ complex and the enzyme, thus increasing the binding
energy; coordinated to six groups in an octahedral arrangement.
-Mg2+ binding hold the nucleotide in a well-defined conformation that
can be bound by an enzyme.
ATP binding induces large conformational changes
-P loop closes down on top of the polyphosphate chain
-This bring down the lid domain to bind to the nucleotide.
→ Substrate binding induces large structural changes in the kinase ;
prototype of induced fit
P-loop NTPase domains are present in a range of important
proteins.
ATP synthase, myosin, G proteins, helicase and elongation factor
Tu contain P loop NTPase domain.
Similar structure.