Metalloenzymes preethi

1. METALLOENZYMES SUBMITTED BY PREETHI G U II SEM MSc BIOTECHNOLOGY 1

2. METALLOPROTEINS Proteins which require metals to carryout function Enzymes Transport proteins Storage proteins Signal transduction proteins 2

3. METALLOENZYMES Contains metals as cofactor- Metalloenzyme and metal activated enzyme Metals help in electron transfer Amino acid groups form coordinate- covalent bonds with metal 3

4. FUNCTIONS By binding to substrates to orient them properly for reaction. By mediating redox reactions through reversible changes in the metal ion’s oxidation state. By electrostatically stabilizing or shielding negative charges. 4

5. CHEMISTRY Diverse Industrial importance in small molecule reactions Metals are usually light metals eg : Ca, Mg surrounded by amino acid ligands ; normally these are carboxylate , S 2- , or N 2 ligands Multiple metal ions coordinated to S 2- and S aa - forming a small cluster 5

6. STRUCTURE Metals found in active site Metals resembles proton or electrophiles 2 ligands - linear 4 ligands - planar or tetrahedral 6 ligands - octahedron Aid in tertiary structure 6

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8. ACTIVATION BY ALKALI METALS Weak binding K + bind to negatively charged gps of inactive to active confirmation aid in substrate binding Catalyse phosphoryl transfer and elimination Eg : pyruvate kinase 8

9. PYRUVATE KINASE 9

10. PK Tetramer 4 metal binding sites PK has an absolute requirement for a divalent metal ion and a monovalent metal ion. Mg 2+  and K + probably fill these needs in vivo Inhibitors- Ca, fluro phosphate, ATP 10

11. ACTIVATION BY ALKALINE EARTH METALS Stronger Octahedral complexes Extracellular activation- Ca 2+ Intracellular- Mg 2+ Invitro - Mn 2+ Eg : α amylases 11

12. α AMYLASE Hydrolase 3.2.1.1 12

13. Active site is trio of acidic gps Calcium ion stabilizes the structure A chloride ion assist the reaction Breaks starch into smaller pieces with 2 or 3 glucose units 13

14. ACTIVATION BY TRANSITION METAL CATIONS Binds more strongly Eg : nitric oxide reductase (Mo and Fe) Zinc metalloenzymes 14

15. ZINC METALLOENZYMES Zinc is required for the activity of > 300 enzymes Binding sites- distorted tetrahedral or trigonal bipyramidal Functions as Lewis acids Stable- no redox activity 15

16. CLASSES Six Metzincins : mononuclear zinc proteins Contains three histidine residue which are zinc ligands Contains zinc proteins with combination of H and C ligands Contains mononuclear zinc proteins coordinated by two histidines Contains predominantly acidic ligands Contain other ligand composition 16

17. CATALYTIC SITES Active site Open coordination sphere The Zinc-bound water is a critical component for a catalytic zinc site, because :- it can be either ionized to zinc-bound hydroxide (as in CA) polarized by a general base (as in carboxypeptidase A) to generate a nucleophile for catalysis displacement of substrate(as in alkaline phosphatase ) 17

18. ZINC BOUND WATER 18

19. CO CATALYTIC SITES A class of catalytic zinc sites has in which two or more zinc atoms are in close proximity to one another 19

20. PHOSPHOLIPASE C Phospholipase C:- 3 Zn ion sites, Zn1(catalytic Zn ion)contains a bound water that is essential for catalysis and has an His 2 glu metal polyhedron. Zn2 and Zn3/Mg ion sites may have unusual ligands such as the oxygen of serine/ threonine or the nitrogen of the N-terminal group . 20

21. Carbonic anhydrase CO 2 + H 2 O H 2 CO 3 a zinc ion coordinated by three imidazole nitrogen atoms from three histidine units fourth coordination site is occupied by a water molecule 21

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24. Carbonic Anhydrase contains a bound zinc ion 1. Zn facilitates the release of a proton from a water molecule, which generates a OH-. A Zn-bound OH is sufficiently nucleophilic to attack  2. The CO2 substrate binds to the enzyme’s active site and is positioned to react with the OH-.  3. The OH- attacks the CO2 converting it into HCO3 4. The catalytic site is regenerated with the release of the HCO3 and the binding of another molecule of H2O. 24

25. METALLOPROTEASES proteases that contain a metal ion at their active site which acts as a catalyst in the hydrolysis peptide binds Commonly Zn or Co/ Mn Metalloendopeptidases Metalloexopeptidase 25

26. METALLOENDOPEPTIDASE THERMOLYSIN Zn 2+ - endopeptidase Bacillus thermoproteolyticus . first metalloproteases to be completely sequenced peptide sequencing and is used in the production of the artificial sweetener aspartame 26

27. EC 3.4.24.27 34.6 kDa hydrolase 27

28. Zn responsible for catalyzing peptide hydrolysis and stabilizing intermediates Normal tetrahedral catalysis - pentacoordinate 28

29. METALLOEXOPEPTIDASE CARBOXYPEPTIDASE 3.4.17.1 Zinc hydrolase hydrolysis of C-terminal esters and peptides with large hydrophobic side chains commercial applications- hydrolysis of cheese whey protein & the production of phenylalanine-free protein hydrolysates for use by individuals with phenylketonuria 29

30. Action : Carbonyl O 2 of the peptide bond being hydrolysed replaces the water molecule bound to Zn. metal ion facilitates cleavage of the peptide bond by withdrawing electron from this carbonyl group. 30

31. Competitive inhibition- transition state analog: phosphorous UV light 31

32. SUPEROXIDE DISMUTASE Oxidizing agent 2 O 2 − + 2 H + → O 2 + H 2 O 2 Oxidation: M (n+1)+ + O 2 − → M n + + O 2 Reduction: M n + + O 2 − + 2H + → M (n+1)+ + H 2 O 2 In human SOD the active metal is Cu, as Cu 2+ or Cu + , coordinated tetrahedrally by four histidine residues, also contains Zn ions for stabilization 32

33. DISMUTATION Two equal but opposite reactions occur on two separate molecules. SOD takes two molecules of superoxide, take the extra electron from one, and places it on the other. so,one is electron less-form normal oxygen other-pick H and form peroxide 33

34. CLINICAL APPLICATION Amyotrophic lateral sclerosis, more commonly known as Lou Gehrig's disease. This disease is a degenerative disorder that leads to selective death of neurons in the brain and spinal chord, leading to gradual increasing paralysis over a few years. Due to mutation in SOD coding gene. 34

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36. OTHER EXAMPLES 36

37. NITROGENASE Nitrogen fixation Components a molybdenum atom at the active site, Iron-sulfur clusters which are involved in transporting the electrons needed to reduce the nitrogen and an abundant energy source. MoFe protein to perform the reaction and Fe break ATP to pump electrons. Require 6 electrons for each N 2 split into 2 NH 3 For each electrons,2 ATP’s are needed 37

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39. The Fe protein- uses the breakage of ATP to pump these electrons into the MoFe protein. The metal clusters are the centerpiece of nitrogenase . it contains both the MoFe protein and two copies of the Fe protein dimer bound on either end. iron-sulfur cluster, the P-cluster, and the FeMo -cluster arranged in a row. The ATP binding site is revealed in this structure by using an unusual analogue of ATP: an ADP molecule with an aluminum fluoride ion. Two of these molecules bind at each end, forming a stable but inactive complex with the Fe protein, essentially gluing the Fe protein to the FeMo protein so its structure can be solved.   39

40. HYDROGENASE Reversible H2 oxidation exist in either NiFe or Ni-independent, or Fe-only, forms. Active site heterobimetallic The active sites are all different, but they have compelling structural similarities. All are centered around an iron atom with several unusual ligands , such as cyanide ions and carbon monoxide. Each has another metal ion or cofactor to assist the iron atom with the reduction/oxidation reaction. And they all use cys amino acids to hold everything in place. 40

41. The active site complexes are an unusual combination of metal ions and strange molecules such as cyanide and carbon monoxide, held in place by cysteine amino acids. These complicated active sites are constructed by a dedicated set of maturation enzymes. For instance, the nickel-iron hydrogenases require at least seven enzymes, powered by GTP and ATP, to build their active sites. One of these enzymes acts as a chaperone, bonding to a key cysteine in the active site and wrenching the protein open to make it accessible to the other enzymes. They load in metal ions and add the cyanide and carbon monoxide ligands . Finally, the chaperone protein releases the cysteine and the mature hydrogenase snaps shut around its new active site. 41

42. ALCOHOL DEHYDROGENASE Defense against alcohol two molecular "tools" to perform its reaction on ethanol. The first is a zinc atom, which is used to hold and position the alcoholic group on ethanol. The second is a large NAD cofactor 42

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44. CYTOCHROME C OXIDASE 44 Terminal oxidase for respiration 2 iron sites and 2 copper sites in addition to zinc & magnesium sites 13 different polypeptides

45. Evolution Endosymbiotic theory. Mammals Cyt.C oxidase has 13 chains. 3 large at core. 10 smaller. Bacteria 4 chains similar to core. So in our cells,3 chains made in mitochondria 10 in cytoplasm 45

46. The oxygen molecule itself binds lower, in the middle of the enzyme. The oxygen is pinioned between a heme iron atom and another copper atom, denoted as site "B." A second heme group, off to the left in this picture, assists in the transfer of electrons 46

47. REGULATION pH- disrupts e- flow Diet- source of metals Zinc metalloenzymes Exclusively through diet. Deficiency will inhibit many enzymes. Cause stunted growth, Enlarged liver and spleen , underdevelopment of genitals and secondary sexual characteristics. 47

48. Zn inhibits ribonuclease . So ,dietary intake is important for the production of some enzymes and the inhibition of others 48

49. INHIBITION Transition state analogs -competitive inhibition they mimic the structure of the substrates transition state in the reaction of enzyme and substrate. Substitution of foreign metals for the metals in metalloenzymes is an important mode of toxic action by metals. Cd toxicity is the substitution of this metal for Zn, a metal that is present in many metalloenzymes . This substitution occurs readily because of the chemical similarities between the two metals , however, Cd does not fulfill the biochemical function of Zn and a toxic effect results. Eg : alcohol dehydrogenase , and carbonic anhydrase 49

50. ARTIFICIAL METALLOENZYMES Inorganic catalyst incorporated in an inactive protein structure. Each constituent plays its part : The inorganic catalyst determines the nature of the reaction by acting as the active site. protein structure controls the production of the molecular form of interest and the efficiency of the reaction. In green chemistry 50

51. An understanding of naturally occurring zinc-binding sites will aid in creating de novo zinc-binding proteins and in designing new metal sites in existing proteins for novel purposes such as to serve as metal ion biosensors 51

52. REFERENCES http://www.cs.stedwards.edu/chem/Chemistry/CHEM43/CHEM43/Metallo/Metallo.HTML www. Sciencedirect.com Surprising cofactors in metalloenzymes Catherine L Drennan and John W Peters Trevor Palmer (2004), enzymes biochemistry, biotechnology, clinical chemistry, Horwood publishing ltd, pp:202- 206 The journal of nutrition.nutrition.org PDB database Meenakshi Meena , Deepak Chauhan (2009) fundamentals of enzymology , Aavishkar publishers, pp: 371-403 52

53. THANK YOU 53