1. Chapter 9
Catalytic Strategies

2. Structural and mechanistic comparisons of enzyme action
4 classes of enzymes.
Protease
Carbonic anhydrase
Restriction endonuclease
Myosins
Structural and mechanistic comparisons of enzyme action
are the sources of insight into the evolutionary history of enzymes.

3. 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.

4. - 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.

5. Chymotrypsin possesses a highly reactive serine redisue
- Chymotrypsin cleaves peptide bonds selectively on the carboxyl-terminal side of the large hydrophobic amino acids such as tryptophan, tyrosine, phenylalanine, and methionine.

6. 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
A nucleophile is a chemical species that donates an electron pair to an electrophile to form a chemical bond in relation to a reaction.
Because nucleophiles donate electrons, they are by definition Lewis bases.

7. 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)

8. 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

9. (acyl-enzyme intermediate)
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; it takes longer for the enzyme to be reset.

10. 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]

11. 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.

12. Burst phase Steady-state phase Tetrahedral intermediate (acyl-enzyme)
Nucleophilic attack
Amine component free
Burst phase
Substrate binding
Steady-state phase
Water mediated deacylation
Carboxylic acid product
OH- Attacks the carbonyl carbon

13. 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.

14. Preference for cleaving the peptide bonds just past residues with large, hydrophobic side chains.
Chymotrypsin has a deep, hydrophobic pocket, called the S1 pocket.

15. Other proteases have more complex specificity patterns.
Such enzymes have additional pockets on their surfaces for the recognition of their residues in the substrate.

16. 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

17. Similar structure Chymotrypsin
- Cleaves after residues with an aromatic or long nonpolar side chains.
Trypsin
Cleaves after residues with an long, positively charged side chains. (R.K)
Asp189 at bottom of the S1 pocket
Elastase
Cleaves after residues with a small side chains. (A.S)
Val190 and Val216 at bottom of the S1 pocket

18. 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.

19. 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.

20. 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

21. 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

22. 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

23. HIV protease : a dimeric aspartyl protease

24. Cysteine, Aspartyl, and Metalloproteases are other major classes of peptide-cleaving enzyme.
- 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
glutamate

25. 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.

26. - X-ray crystallography revealed that, in the active site, indinavir adopts a conformation that approximates the twofold symmetry of the enzyme.

27. (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

28. Activation strategies for three classes of proteases
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

29. 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

30. 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.

31. Metal ion 1/3 of all enzymes contain metal ion. Properties
positive charges,
ability to form relatively strong yet kinetically labile bonds,
more than one oxidation state; iron(II) sulphate, iron(III) chloride

32. 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=6)
<Effect of pH on carbonic anhydrase>

33. 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.

34. 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-

35. 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.

36. 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 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

37. The deprotonation of the zinc-bound water molecule in carbonic anhydrase is aided by buffer component B.

38. 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.

39. 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 the zinc-bound water molecule, generating a nucleophilic hydroxide ion and a protonated His.
② The buffer B removes a proton form the His, regenerating the unprotonated form.
Many biochemical reactions show the prominence of acid-base catalysis

40. 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.

41. 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.

42. 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

43. Mechanism 1 (covalent intermediate)
: analogy with chymotrypsin
: two displacement, retained configuration

44. Mechanism 2 (direct hydrolysis)
: analogy with asparty protease and metalloproteases
: one displacement, inverted configuration

45. How to figure out the stereochemistry of the product?
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?

46. - 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.

47. Water 18O labeled
The hydrolysis takes place by water’s direct attack at the phosphorus atom.

48. 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.

49. 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.

50. The structure of EcoRV (dimer) bound to a cognate DNA fragment.

51. 5’ – G A T A T C – 3’
3’ – C T A T A G – 5’

52. 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.

53. - The noncognate DNA conformation is not substantially distorted.
- 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

54. 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.

55. 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

56. 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

57. 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

58. 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

59. 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.

60. Water molecule attacks the g-phosphoryl group, with the hydroxyl group of Ser236, which is deprotonated by g-phosphate.
Mechanism of ATP hydrolysis

68. 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.

69. 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

70. - P loop interact with ATP
Phosphate

71. 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?

72. 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.

73. 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

74. 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.