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Metalloenzymes preethi

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The document discusses proteins that require metals to function, including enzymes, transport proteins, storage proteins, and signal transduction proteins. It describes how metals are used as cofactors in metalloenzymes, helping with electron transfer and substrate binding. Specific metalloenzymes discussed include carbonic anhydrase, pyruvate kinase, alpha-amylases, nitric oxide reductase, and zinc-containing enzymes. The roles of metals like calcium, magnesium, zinc, molybdenum, and iron in these enzyme active sites and their coordination geometries are summarized.

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Proteins which require metals to carryout function Enzymes Transport proteins Storage proteins Signal transduction proteins 2
Contains metals as cofactor- Metalloenzyme and metal activated enzyme Metals help in electron transfer Amino acid groups form coordinate- covalent bonds with metal 3
By binding to substrates to orient them properly for reaction. By mediating redox reactions through reversible changes in the metal ions oxidation state. By electrostatically stabilizing or shielding negative charges. 4
Diverse Industrial importance in small molecule reactions Metals are usually light metals eg: Ca, Mg surrounded by amino acid ligands; normally these are carboxylate, S2-, or N2 ligands Multiple metal ions coordinated to S2- and S aa- forming a small cluster 5
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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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
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Tetramer 4 metal binding sites PK has an absolute requirement for a divalent metal ion and a monovalent metal ion. Mg2+ and K+ probably fill these needs in vivo Inhibitors- Ca, fluro phosphate, ATP 10
Stronger Octahedral complexes Extracellular activation- Ca2+ Intracellular- Mg2+ Invitro- Mn2+ Eg: 留 amylases 11
Hydrolase 3.2.1.1 12
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
Binds more strongly Eg: nitric oxide reductase (Mo and Fe) Zinc metalloenzymes 14
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
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
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
A class of catalytic zinc sites has in which two or more zinc atoms are in close proximity to one another 19
Phospholipase C:- 3 Zn ion sites, Zn1(catalytic Zn ion)contains a bound water that is essential for catalysis and has an His2glu 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
CO2 + H2O H2CO3 a zinc ion coordinated by three imidazole nitrogen atoms from three histidine units fourth coordination site is occupied by a water molecule 21
22
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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 enzymes 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
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
Zn2+-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
Zn responsible for catalyzing peptide hydrolysis and stabilizing intermediates Normal tetrahedral catalysis -pentacoordinate 28
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
Action : Carbonyl O2 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
Competitive inhibition- transition state analog: phosphorous UV light 31
Oxidizing agent 2 O2 + 2 H+ O2 + H2O2 Oxidation: M(n+1)+ + O2 Mn+ + O2 Reduction: Mn+ + O2 + 2H+ M(n+1)+ + H2O2 In human SOD the active metal is Cu, as Cu2+ or Cu+, coordinated tetrahedrally by four histidine residues, also contains Zn ions for stabilization 32
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
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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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 N2 split into 2 NH3 For each electrons,2 ATPs are needed 37
38
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
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
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
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
43
Terminal oxidase for respiration 2 iron sites and 2 copper sites in addition to zinc & magnesium sites 13 different polypeptides 44
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
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
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
Zn inhibits ribonuclease. So ,dietary intake is important for the production of some enzymes and the inhibition of others 48
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
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
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
http://www.cs.stedwards.edu/chem/Chemistry/CHEM 43/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
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Metalloenzymes preethi

  • 1. 1
  • 2. Proteins which require metals to carryout function Enzymes Transport proteins Storage proteins Signal transduction proteins 2
  • 3. 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. By binding to substrates to orient them properly for reaction. By mediating redox reactions through reversible changes in the metal ions oxidation state. By electrostatically stabilizing or shielding negative charges. 4
  • 5. Diverse Industrial importance in small molecule reactions Metals are usually light metals eg: Ca, Mg surrounded by amino acid ligands; normally these are carboxylate, S2-, or N2 ligands Multiple metal ions coordinated to S2- and S aa- forming a small cluster 5
  • 6. 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
  • 7. 7
  • 8. 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. 9
  • 10. Tetramer 4 metal binding sites PK has an absolute requirement for a divalent metal ion and a monovalent metal ion. Mg2+ and K+ probably fill these needs in vivo Inhibitors- Ca, fluro phosphate, ATP 10
  • 11. Stronger Octahedral complexes Extracellular activation- Ca2+ Intracellular- Mg2+ Invitro- Mn2+ Eg: 留 amylases 11
  • 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. Binds more strongly Eg: nitric oxide reductase (Mo and Fe) Zinc metalloenzymes 14
  • 15. 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. 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. 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. 18
  • 19. 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:- 3 Zn ion sites, Zn1(catalytic Zn ion)contains a bound water that is essential for catalysis and has an His2glu 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. CO2 + H2O H2CO3 a zinc ion coordinated by three imidazole nitrogen atoms from three histidine units fourth coordination site is occupied by a water molecule 21
  • 22. 22
  • 23. 23
  • 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 enzymes 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. 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. Zn2+-endopeptidase Bacillus thermoproteolyticus. first metalloproteases to be completely sequenced peptide sequencing and is used in the production of the artificial sweetener aspartame 26
  • 28. Zn responsible for catalyzing peptide hydrolysis and stabilizing intermediates Normal tetrahedral catalysis -pentacoordinate 28
  • 29. 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 O2 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. Oxidizing agent 2 O2 + 2 H+ O2 + H2O2 Oxidation: M(n+1)+ + O2 Mn+ + O2 Reduction: Mn+ + O2 + 2H+ M(n+1)+ + H2O2 In human SOD the active metal is Cu, as Cu2+ or Cu+, coordinated tetrahedrally by four histidine residues, also contains Zn ions for stabilization 32
  • 33. 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. 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
  • 35. 35
  • 36. 36
  • 37. 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 N2 split into 2 NH3 For each electrons,2 ATPs are needed 37
  • 38. 38
  • 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. 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. 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
  • 43. 43
  • 44. Terminal oxidase for respiration 2 iron sites and 2 copper sites in addition to zinc & magnesium sites 13 different polypeptides 44
  • 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. 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. 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. 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. http://www.cs.stedwards.edu/chem/Chemistry/CHEM 43/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. 53

Editor's Notes

  • #3: Str of Hb
  • #6: nitrogen to ammonia, the oxidation of methane to methanol, and the oxidation of ammonium ions to nitritemetals are surrounded by amino acid ligands; normally these are carboxylate (glutamate or aspartate), sulfide (cysteine, occasionally methionine), or nitrogen (normally histidine) ligands,..,.sulfide s2-
  • #7: The metals resemble protons (H+) in that they are electrophiles that are able to accept an electron pair to form a chemical bond. In this aspect, metals may act as general acids to react with anionic and neutral ligandsA ligand is whatever molecule the metal interacts with
  • #11: ATP inhibits the reaction by removal of Mg2+from the substrate MgADP-. ATP also appears to inhibit the reaction competitively with respect to both ADP and PEP if the Mg2+concentration is higher than that of ATP
  • #13: The active site of alpha-amylase contains a trio of acidic groups (colored white and red) that do most of the work. In the amylase shown here (PDB entry1ppi), glutamate 233, aspartate 197, and aspartate 300 work together to cleave the connection between two sugars in a starch chain. This structure contains a short chain of five sugar units (colored yellow and orange) bound in the active site. The site of cleavage is shown in pink. A calcium ion, shown as the large gray sphere, is found nearby where it stabilizes the structure of the enzyme. A chloride ion, shown as a green sphere, is bound underneath the active site in many amylases, where it may assist the reaction.
  • #16: Lewis acids accepts electrons, Filled d orbitals
  • #17: Tissue degrdtion, prtntrnovr,psttrnsltnlmodifictn,asctacin
  • #18: coordination sphere; that is, the zinc-binding polyhedron contains at least one water molecule in addition to three or four protein ligands
  • #20: These sites are termed cocatalytic because all three metals play crucial roles in catalysis despite only the zinc activating the attacking water being termed catalytic.PHOSPHOLIPASE C
  • #24: In the first step, zinc-bound hydroxide attacks the carbonyl carbon of CO2 to form zinc-bound bicarbonate;bicarbonate is subsequently displaced with water by a ligand-exchange step. In the second step, H+ is transferred from zinc-bound water to external buffer via a shuttle group (H64 in CA II) to regenerate the catalytically active species, the zinc-bound hydroxide.
  • #25: Carbonic Anhydrase contains a bound zinc ion essential for catalytic activity. Since zinc is positive, it attracts a water molecule to its active site.1. Zinc facilitates the release of a proton from a water molecule, which generates a hydroxide ion. A zinc-bound hydroxide ion is sufficiently nucleophilic to attack2. The carbon dioxide substrate binds to the enzymes active site and is positioned to react with the hydroxide ion.3. The hydroxide ion attacks the carbon dioxide, converting it into bicarbonate ion.4. The catalytic site is regenerated with the release of the bicarbonate ion and the binding of another molecule of water.12Nitrogen atoms of three histidines--numbered 94, 96 and 119 (colored in yellow)--directly coordinate the zinc. 11 Atoms from threonine 199 and glutamate 106 interact indirectly through the bound water.Carbonicanhydrase inhibitors
  • #29: original three residues (His142, His146, and Glu166), the oxygen of the nucleophilic water, and the carbonyl oxygen of the substrateRemoval of Zn2+yields an inactive enzyme. Exogenous addition of other divalent transition metals, specifically Zn2+,Co2+, Fe2+, and Mn2+, results in the regaining of 100%, 200%, 60%, and 10% enzymatic activity
  • #32: Ultraviolet-visible radiation (400 W, 了=250-750 nm) has been shown to cause uncompetitive inhibition. Exposure times of greater than 24 minutes adversely affect the structure of CPA, form protein aggregates.
  • #41: Ni ion bridged to an Fe atom via two bridging thiolates supplied by cysteine residues of the protein
  • #43: Notice how the zinc atom, shown in light blue, is cradled by three amino acids from the protein: cysteine 46 to the left, cysteine 174 to the right, and histidine 67 above. The ethanol, shown in green and magenta, binds to the zinc and is positioned next to the NAD cofactor,