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METALLOENZYMES SUBMITTED
BY PREETHI
G U II SEM MSc BIOTECHNOLOGY
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METALLOPROTEINSProteins which require metals to carryout
function
Enzymes
Transport proteins
Storage proteins
Signal transduction proteins
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METALLOENZYMES
Contains metals as cofactor- Metalloenzyme and
metal activated enzyme
Metals help in electron transfer
Amino acid groups form coordinate- covalent
bonds with metal
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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.
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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, S2-, or N2 ligands
Multiple metal ions coordinated to S2- and S aa-
forming a small cluster
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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
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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
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PYRUVATE KINASE
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PK
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
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ACTIVATION BY ALKALINE EARTH METALS
Stronger
Octahedral complexes
Extracellular activation- Ca2+
Intracellular- Mg2+
Invitro- Mn2+
Eg: α amylases
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α AMYLASE
Hydrolase 3.2.1.1
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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
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ACTIVATION BY TRANSITION METAL CATIONS
Binds more strongly
Eg: nitric oxide reductase (Mo and
Fe)
Zinc metalloenzymes
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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
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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
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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)
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ZINC BOUND WATER
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CO CATALYTIC SITES
A class of catalytic zinc sites has in which two or more zinc atoms are in close proximity to one another
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PHOSPHOLIPASE C
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.
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Carbonic anhydrase
CO2 + H2O H2CO3
a zinc ion coordinated by three imidazole nitrogen atoms from three histidine unitsfourth coordination site is occupied by a water molecule
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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 enzyme’s active site and is positioned to react with the OH-. 3. The OH- attacks the CO2 converting it into HCO34. The catalytic site is regenerated with the release of the HCO3 and the binding of another molecule of H2O.
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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
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METALLOENDOPEPTIDASETHERMOLYSIN
Zn2+-endopeptidase
Bacillus thermoproteolyticus.
first metalloproteases to be completely
sequenced
peptide sequencing and is used in the
production of the artificial sweetener aspartame
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EC 3.4.24.2734.6 kDahydrolase
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Zn responsible for catalyzing peptide
hydrolysis and stabilizing
intermediates
Normal tetrahedral
catalysis -pentacoordinate
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METALLOEXOPEPTIDASECARBOXYPEPTIDASE
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
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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.
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Competitive inhibition- transition state analog: phosphorous
UV light
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SUPEROXIDE DISMUTASE
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
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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
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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.
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OTHER EXAMPLES
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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 N2 split into 2 NH3 For each electrons,2 ATP’s are needed
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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.
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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.
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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.
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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
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CYTOCHROME C OXIDASE
Terminal oxidase for respiration2 iron sites and 2 copper sites in addition to zinc & magnesium sites13 different polypeptides
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Evolution Endosymbiotic theory.
MammalsCyt.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 mitochondria10 in cytoplasm
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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
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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.
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Zn inhibits ribonuclease.
So ,dietary intake is important for the production of some enzymes and the inhibition of others
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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
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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
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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
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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
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