Metalloenzymes preethi

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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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