1. Online Counseling Resource YCMOU ELearning Drive… School of Architecture, Science and Technology Yashwantrao Chavan Maharashtra Open University, Nashik – 422222, India

2. SEP-SBI091-CP1-05 Introduction Programmes and Courses SEP –SBI091- U01 -CP1_05

3. School of Science and Technology, Online Counseling Resource… Credits  Academic Inputs by Sonali Alkari Faculty YCMOU Nagpur Centre, Faculty LAD college P.G. D of Biotechnology Research officer Ankur Seeds Pvt Ltd sonalisa_alkari@yahoo.co.in Sonalisaal@rediffmail.com 3© 2008, YCMOU. All Rights Reserved.

4. School of Science and Technology, Online Counseling Resource… © 2008, YCMOU. All Rights Reserved.4 How to Use This Resource  Counselor at each study center should use this presentation to deliver lecture of 40-60 minutes during Face-To-Face counseling.  Discussion about students difficulties or tutorial with assignments should follow the lecture for about 40-60 minutes.  Handouts (with 6 slides on each A4 size page) of this presentation should be provided to each student.  Each student should discuss on the discussion forum all the terms which could not be understood. This will improve his writing skills and enhance knowledge level about topics, which shall be immensely useful for end exam.  Appear several times, for all the Self-Tests, available for this course.  Student can use handouts for last minutes preparation just before end exam.

5. School of Science and Technology, Online Counseling Resource… © 2008, YCMOU. All Rights Reserved.5 Learning Objectives  After studying this module, you should be able to : Describe 3-D structure of Chymotrypsin Describe 3-D structure of Lysozymes

6. School of Science and Technology, Online Counseling Resource… Chymotrypsin-1  Bovine pancreatic chymotrypsin (Mr 25,191) is a protease, an enzyme that catalyzes the hydrolytic cleavage of peptide bonds.  This protease is specific for peptide bonds adjacent to aromatic amino acid residues (Trp,Phe, Tyr).  The three-dimensional structure of chymotrypsinis shown in Figure, with functional groups in the active site emphasized.  The reaction catalyzed by this enzyme illustrates the principle of transition-statestabilization and also provides a classic example of general acid-base catalysis and covalent catalysis. © 2008, YCMOU. All Rights Reserved.6

7. School of Science and Technology, Online Counseling Resource… Chymotrypsin-2  A number of proteolytic enzymes participate in the breakdown of proteins in the digestive systems of mammals and other organisms.  One such enzyme, 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 modification as a catalytic strategy.  The enzyme employs a powerful nucleophile to attack the unreactive carbonyl group of the substrate. © 2008, YCMOU. All Rights Reserved.7

8. School of Science and Technology, Online Counseling Resource… Chymotrypsin-3 © 2008, YCMOU. All Rights Reserved.8 A depiction of the enzyme emphasizing its surface. The pocket in which the aromatic amino acid side chain of the substrate is bound is shown in green. Key active-site residues, including Ser195, His57, and Asp102 are red. The roles of these residues in catalysis are illustrated in this Figure.

9. School of Science and Technology, Online Counseling Resource… © 2008, YCMOU. All Rights Reserved.9 The polypeptide backbone as a ribbon structure. Disulfide bonds are yellow; the three chains are colored as in part Chymotrypsin-4

10. School of Science and Technology, Online Counseling Resource… Specificity of Chymotrypsin © 2008, YCMOU. All Rights Reserved. 10 Chymotrypsin cleaves proteins on the carboxyl side of aromatic or large hydrophobic amino acids (shaded yellow). The likely bonds cleaved by chymotrypsin are indicated in red.

11. School of Science and Technology, Online Counseling Resource… Chymotrypsin-5  This nucleophile becomes covalently attached to the substrate briefly in the course of catalysis.  The determination of the three-dimensional structure of chymotrypsin by David Blow in 1967 was a source of further insight into its mechanism of action.  Overall, chymotrypsin is roughly spherical and comprises three polypeptide chains, linked by disulfide bonds.  Chymotrypsin is a serine protease with a well understood mechanism, featuring general acid base catalysis, covalent catalysis, and transition-state stabilization. © 2008, YCMOU. All Rights Reserved.11

12. School of Science and Technology, Online Counseling Resource… Chymotrypsin-6  Chymotrypsin enhances the rate of peptide bond hydrolysis by a factor of at least 109.  It does not catalyze a direct attack of water on the peptide bond; instead, a transient covalent acyl-enzyme intermediate is formed.  The reaction thus has two distinct phases.  In the acylation phase, the peptide bond is cleaved and an ester linkage is formed between the peptide carbonyl carbon and the enzyme.  In the deacylation phase, the ester linkage is hydrolyzed and the nonacylated enzyme regenerated. © 2008, YCMOU. All Rights Reserved.12

13. School of Science and Technology, Online Counseling Resource… Chymotrypsin-7  It is synthesized as a single polypeptide, termed chymotrypsinogen, which is activated by the proteolytic cleavage of the polypeptide to yield the three chains.  The active site of chymotrypsin, marked by serine 195, lies in a cleft on the surface of the enzyme  The structural analysis revealed the chemical basis of the special reactivity of serine 195  The side chain of serine 195 is hydrogen bonded to the imidazole ring of histidine 57. © 2008, YCMOU. All Rights Reserved.13

14. School of Science and Technology, Online Counseling Resource… Chymotrypsin-8  The -NH group of this imidazole ring is, in turn, hydrogen bonded to the carboxylate group of aspartate 102.  This constellation of residues is referred to as the catalytic triad.  The histidine residue serves to position the serine side chain and to polarize its hydroxyl group.  In doing so, the residue acts as a general base catalyst, a hydrogen ion acceptor, because the polarized hydroxyl group of the serine residue is poised for deprotonation. © 2008, YCMOU. All Rights Reserved.14

15. School of Science and Technology, Online Counseling Resource… 3-D Structure of Chymotrypsin © 2008, YCMOU. All Rights Reserved.15 The three chains are in ribbon form in orange, blue and green colour. The side chains of the catalytic triad residues, including serine 195, are shown as ball-and-stick representations, as are two intrastrand and interstrand disulfide bonds.

16. School of Science and Technology, Online Counseling Resource… © 2008, YCMOU. All Rights Reserved.16 Chymotrypsin-9

17. School of Science and Technology, Online Counseling Resource… Chymotrypsin-8  A close-up of the active site with a substrate (mostly green) bound.  Two of the active-site residues, Ser195 and His57 (both red), are partly visible.  Ser195 attacks the carbonyl group of the substrate (the oxygenis purple); the developing negative charge on the oxygen is stabilized by the oxyanion hole (amide nitrogens in orange).  In the substrate, the aromatic amino acid side chain and the amide nitrogen of the peptide bond to be cleaved (protruding toward the viewer and projecting the path of the rest of the substrate polypeptide chain) are in blue. © 2008, YCMOU. All Rights Reserved.17

18. School of Science and Technology, Online Counseling Resource… Chymotrypsin-9  The withdrawal of the proton from the hydroxyl group generates an alkoxide ion, which is a much more powerful nucleophile than an alcohol is.  The aspartate residue helps orient the histidine residue and make it a better proton acceptor through electrostatic effects.  How does this arrangement of residues lead to the high reactivity of serine 195?  These observations suggest a mechanism for peptide hydrolysis. © 2008, YCMOU. All Rights Reserved.18

19. School of Science and Technology, Online Counseling Resource… Catalytic Mechanism of Chymotrypsin © 2008, YCMOU. All Rights Reserved.19

20. School of Science and Technology, Online Counseling Resource… Three-Dimensional Structure of Chymotrypsin. © 2008, YCMOU. All Rights Reserved.20 The catalytic triad, shown on the left, converts serine 195 into a potent nucleophile, as illustrated on the right.

21. School of Science and Technology, Online Counseling Resource… Chymotrypsin-10  After substrate binding the reaction begins with the hydroxyl group of serine 195 making a nucleophilic attack on the carbonyl carbon atom of the substrate.  The nucleophilic attack changes the geometry around this carbon atom from trigonal planar to tetrahedral.  The inherently unstable tetrahedral intermediate formed bears a formal negative charge on the oxygen atom derived from the carbonyl group.  This charge is stabilized by interactions with NH groups from the protein in a site termed the oxyanion hole. © 2008, YCMOU. All Rights Reserved.21

22. School of Science and Technology, Online Counseling Resource… Chymotrypsin-11  These interactions also help stabilize the transition state that precedes the formation of the tetrahedral intermediate.  This tetrahedral intermediate then collapses to generate the acyl-enzyme.  This step is facilitated by the transfer of a proton from the positively charged histidine residue to the amino group formed by cleavage of the peptide bond.  The amine component is now free to depart from the enzyme and is replaced by a water molecule. © 2008, YCMOU. All Rights Reserved.22

23. School of Science and Technology, Online Counseling Resource… Chymotrypsin-12  The ester group of the acyl-enzyme is now hydrolyzed by a process that is essentially a repeat of steps 2 through 4.  The water molecule attacks the carbonyl group while a proton is concomitantly removed by the histidine residue, which now acts as a general acid catalyst, forming a tetrahedral intermediate.  This structure breaks down to form the carboxylic acid product.  Finally, the release of the carboxylic acid product (step 8) readies the enzyme for another round of catalysis. © 2008, YCMOU. All Rights Reserved.23

24. School of Science and Technology, Online Counseling Resource… Chymotrypsin-13  The mechanism of peptide hydrolysis illustrates the principles of covalent and acid-base catalysis.  The dashed green lines indicate favorable interactions between the negatively charge aspartate residue and the positively charged histidine residue, which make the histidine residue a more powerful base. © 2008, YCMOU. All Rights Reserved.24

25. School of Science and Technology, Online Counseling Resource… Lysozyme-1  Lysozyme is a natural antibacterial agent found in tears and egg whites.  The hen egg white lysozyme (Mr 14,296) is a monomer with 129 amino acid residues.  This was the first enzyme to have its three- dimensional structure determined, by David Phillips and colleagues in 1965.  The structure revealed four stabilizing disulfide bonds and a cleft containing the active site.  Substrate of lysozyme is peptidoglycan, a carbohydrate found in many bacterial cell walls. © 2008, YCMOU. All Rights Reserved.25

26. School of Science and Technology, Online Counseling Resource… Hen egg white lysozyme © 2008, YCMOU. All Rights Reserved.26 Ribbon diagram of the enzyme with the active-site residues Glu35 and Asp52 shown as blue stick structures and bound substrate shownin red (PDB ID1LZE).

27. School of Science and Technology, Online Counseling Resource… © 2008, YCMOU. All Rights Reserved.27 Lysozyme-2

28. School of Science and Technology, Online Counseling Resource… Lysozyme-3  Lysozyme cleaves the (1n4) glycosidic COO bond between the two types of sugar residue in the molecule, N-acetylmuramic acid (Mur2Ac) and N-acetylglucosamine(GlcNAc), often referred to as NAM and NAG, respectively.  There are two chemically reasonable mechanisms that could generate the observed product of lysozyme mediated cleavage of the glycosidic bond.  Phillips and colleagues proposed a dissociative (SN1-type) mechanism, in which the GlcNAc initially dissociates to leave behind a glycosyl cation a carbocation intermediate. © 2008, YCMOU. All Rights Reserved.28

29. School of Science and Technology, Online Counseling Resource… Lysozyme-4 © 2008, YCMOU. All Rights Reserved.29 A ribbon diagram of the covalent enzymesubstrate intermediate with the activesite residues (blue) and bound substrat (red) shown as stick structures (PDB ID 1H6M).

30. School of Science and Technology, Online Counseling Resource… What You Learn…  You have learnt : Bovine pancreatic chymotrypsin is a protease, an enzyme that catalyzes the hydrolytic cleavage of peptide bonds. Chymotrypsin is a good example of the use of covalent modification as a catalytic strategy. Chymotrypsin is roughly spherical and comprises three polypeptide chains, linked by disulfide bonds. Lysozyme is a natural antibacterial agent found in tears and egg whites. The structure revealed four stabilizing disulfide bonds and a cleft containing the active site. There are two chemically reasonable mechanisms that could generate the observed product of lysozyme mediated cleavage of the glycosidic bond. 30© 2008, YCMOU. All Rights Reserved.

31. School of Science and Technology, Online Counseling Resource… Critical Thinking Questions 1.Write a detail note on chymotrypsin 3d structure and mechanism of action. 2.Write a detail note on lysozyme 3d structure and mechanism of action. © 2008, YCMOU. All Rights Reserved.31

32. School of Science and Technology, Online Counseling Resource… Hints For Critical Thinking Question 1.Details of chymotrypsin, structural details like spherical and comprises three polypeptide chains, linked by disulfide bonds and mechanism featuring general acid base catalysis, covalent catalysis, and transition- state stabilization 2.Details of Lysozyme, structural details revealing four stabilizing disulfide bonds and a cleft containing the active site. There are two chemically reasonable mechanisms. © 2008, YCMOU. All Rights Reserved.32

33. School of Science and Technology, Online Counseling Resource… Study Tips:1  Book1 Title: Molecular Cell Biology Author: Harvey Lodish, David Baltimore Publisher:Publishers: W. H. Freeman and Company  Book2 Title: Principles of Biochemistry Author: AlbertL Lehninger Publisher:CBS Publishers & Distributors 33© 2008, YCMOU. All Rights Reserved.

34. School of Science and Technology, Online Counseling Resource… Study Tips:2  Book3 Title: Biochemistry Author: Lubert stryer Publishers: Freeman International  Book4 Title: Biochemistry Author: Keshav Trehan Publishers: Wiley Eastern 34© 2008, YCMOU. All Rights Reserved.

35. School of Science and Technology, Online Counseling Resource… Study Tips www.en.wikipedia.org Microsoft Encarta Encyclopedia http://en.wikipedia.org/wiki/ Wikipedia the free encyclopedia (www.chem.qmul.ac.uk/iubmb/enzyme). 35© 2008, YCMOU. All Rights Reserved.

36. School of Science and Technology, Online Counseling Resource… End of the Presentation Thank You ! 36© 2008, YCMOU. All Rights Reserved.