Bio-Inorganic Chemistry - WordPress.com€¦ · B12 DEPENDANT ENZYMES: COENZYME B12. COPPER -ZINC...
Transcript of Bio-Inorganic Chemistry - WordPress.com€¦ · B12 DEPENDANT ENZYMES: COENZYME B12. COPPER -ZINC...
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D R . R . P . J O H N
Bio-Inorganic Chemistry
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Bio-inorganic Chemistry -by R. P. John
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Syllabus
Metal Storage Transport and Biomineralization: Ferritin, transferrin, and siderophores
Calcium in Biology: Calcium in living cells, transport and regulation, molecular aspects of intramolecular processes, extracellular binding proteins
Metalloenzymes: Zinc enzymes-carboxypeptidase and carbonic anhydrase. Iron enzymes-catalase, peroxidase and cytochrome P-450. Copper enzymes – superoxide dismutase. Molybdenum oxatransferase enzymes- xanthine oxidase. Coenzyme vitamin B12.
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I R O N E N Z Y M E S : P U R P L E A C I D P H O S P H A T A S E , A C O N I T A S E , C Y T O C H R O M E C O X I D A S E , C Y T O C H R O M E P 4 5 0 , X A N T H I N E O X I D A S E
M g D E P E N D A N T E N Z Y M E S : R u b i s c oB 1 2 D E P E N D A N T E N Z Y M E S : C O E N Z Y M E B 1 2
C O P P E R - Z I N C S U P E R O X I D E D I S M U T A S EM n C O N T A I N I N G E N Z Y M E S : A R G I N A S E , M n - S O D
Bio-inorganic Chemistry: Part 3-2Enzymes
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ENZYMES: Introduction
The shape and chemical environment inside the active site facilitates specific catalysis.
Cofactors: They are additional non-protein molecules or groups that are required to catalyse the reaction
Prosthetic groups: They are tightly bound cofactors.
Coenzymes: A reversibly bound group that combines with an enzyme for a particular reaction and release when the reaction is over
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Enzymes: Classification
Class Function
Oxidoreductases Catalyses oxidation and reduction
Transferases Catalyses Transfer of groups of atoms
Hydrolases Catalyses hydrolysis
Lyases Catalyses addition and removal of atoms to/from a double bond
Isomerases Catalyses rearrangement of atoms
Ligases Combines molecules using ATP
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Iron Ezyme: Purple Acid Phosphatase
Function: Hydrolysis of phosphorylated proteins Mammalian origin PAPs -bridged FeIII-FeIII/FeIII-
FeII centers Mammalian PAPs: metal centers 5 coordinate in
distorted TBP geometry Bridging groups: OH- & Asp/Glu (µ:η1- η1O) Plant origin PAPs –Bridged FeIII-ZnII or FeIII-MnII
centers Plant PAPs: metal centers 6 coordinate Bridging groups: Asp (µO) & OH-
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Source: BioMedCentral-Structural Biology 2008: http://www.biomedcentral.com/1472-6807/8/6
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PAPs: Mechanism-steps involved
♣ A precatalytic complex is formed where, the phosphate bound substrate is H-bondedin the second coordination sphere
♣ The bridging OH plays a key role in orienting the substrate♣ The phosphate attack M2 and binds, Then M1 binds via the other O forming a µ-1,3
phosphate bridge♣ Bridged OH makes a nucleophilic attack at the phosphate, esterolysis occur, and the
phosphate bridges M1 &M2 in a tripodal fashion. ♣ The triply bridged phosphate switch back to µ-1,3 bridging♣ An H2O attack M2 which prompt Phosphate to leave M2♣ The phosphate remains H-bonded to the newly bound H2O♣ Another H2O enters M2’s coordination sphere, Phosphate takes up a proton from H-
bonded bound water.♣ HPO42- leaves M1, OH- bound to M1 attacks M2 and form µ-(OH) bridge♣ ROPO32- enters site, M2 expels bound water and pre-catalytic complex is formed
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Iron containing Enzymes: Aconitase
Aconitase belongs to class of isomerases
Reversibly catalyses conversion of Citrate to isocitrate
Process involve dehydration and rehydration
Proceeds through an intermediate Aconitate
The active site contain a ferredoxin type, 4Fe-4S cluster
The Fe-S cluster is bound to Domain3 through Cys-358, Cys-421, Cys-424
The labile Fe, lost during catalysis is bound to 1 H2O instead of a Cystein S
Source: S. J. Lloyd et al, Protein Science 1999, (8) 2655
PresenterPresentation NotesM-aconitase: single polypeptide 82.8kDa, 754 amino acidsDomian 1: 1-200; Domain 2: 201-319; Domain 3: 320-512; Domain 4: 537-754; Hinge btn 3 n 4: 512-536PDB no 7ACN
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Aconitase: Mechanism
Iso-citrate bind to Fea site via Cαhydroxyl group and Cα carboxyl O
Ser-642 alkoxide attacks H at Cβleaving a carbanion transition state with excess –ve charge on the carboxyl O
This TS is stabilized by a low barrier H-bond to a coordinated H2O
Cβ-Ohydroxyl bond cleaves and the bond pair abstrat a proton from His-101
The bound Cis-aconitate flip 180°about Cα-Cβ double bond
The Fea bound H2O acts as a nucleophile and adds a OH to Cβ
This leave a carbanion TS that is stabilised by a low barrier H-bondto the 2nd Bound water
The Carbanion abstract a proton from Ser642 forming citrate
PresenterPresentation NotesThe Fea is 6 coordinate upon binding citrate. 5 coordinate when isocitrate is bound. Once the cis-aconitate is formed it flip 180 deg. About Ca-Cb bond. When Cis-aconitate is bound by Cb carboxyl O it is in citrate mode and will be converted to citrate. When Cis-aconitate is bound via C-a Carboxyl O, it is in iso-citrate mode and will be converted to isocitrate
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PresenterPresentation NotesIsocitrate binds to Fea via Ca carboxyl oxygen. The Cb hydroxyl is H-bonded to Asp 165 and His101. A Cb carboxyl O is H-bonded to water. Ser642 alkoxide acts as a base, taking away Cb proton annd forming a carbanion intermediate. This intermediate collapse when Cb-O is transferred to a proton in His101, forming cis-aconitate and water. Cis-aconitate leaves, Ser642 become unprotonated and His101 takes up proton
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Mg Dependant Enzymes: Rubisco
Ribulose-1,5-bisphosphate(RuBP)
OH
H2C
CH
C
C
OHH
H2C OPO32-
OPO32-
O
3-Phosphoglycerate(3PG)
OH
H2C
CH
COO
OPO32-
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Rubisco convert RuBP to 2 molecules of 3-phosphoglycerate by combination with water and CO2
It exist as L8S8, where L is the large subunit and S is the small subunit
O
H
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2
C
C
H
C
C
O
H
H
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C
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P
O
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P
O
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O
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C
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C
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H
H
H
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C
O
P
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O
P
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Ribulose-1,5-bisphosphate
(RuBP)
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O
H
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C
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O
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3-Phosphoglycerate
(3PG)
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Rubisco: Mechanism
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Rubisco: Mechanism
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Cytochromes
Electron and H+ transfer proteins having one or more Haem groups Classified according to the type of Fe coordination, and groups bordering haem Plays a major role in ET mechanism involving oxygen and its subsequent reduction
to H2O, releasing energy and phosphorylating ADP
Cytochrome a Cytochrome b Cytochrome c
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Mechanism of electron transfer
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Cytochrome c Oxidase
4 Fe2+-cytochrome c + 8 H+in + O2 → 4 Fe3+-cytochrome c + 2 H2O + 4 H+out
PresenterPresentation NotesIt converts dioxygen to H2O or H2O2 without incorporation of O in oxidisable substrate. Besides for every molecule of O2 converted it pumps 4 protons out of the cell against a potential gradient- Electrogenic ion pump
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Cytochrome c Oxidase
The simplest CcO contains two subunits and contains 3 Cu, 2 Fe-bound-haem, 1 Mg & 1Zn
Subunit I: the active site has a Myoglobin like Haem a3 centre situated close to a semi-hemocyanin like CuBbound by 3 Histidine residues
A nearby Haem aprovides electrons to the active site
Subunit II: Has a binuclear CuA centre that receive electronfrom Cytochrome c
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Cytochrome c oxidase
Fe is hexa-coordinate with two His residues at the axial positions.
The Haem involved is hem a; In hemoglobin it is haem b
The enzyme has two proton channels one for supply of H+ for conversion of O2 to H2O while the other for pumping out H+ out of the cell
Haema3 Fe is 5 coordinate with a His at theproximal axial site
Haem a Fe is six coordinate with 2 Histidines at the axial sites
Cytochrome c in complex III transfer e- to CuAcentre, then to Haema followed by binuclear Haema3-CuB centre
The electron transfer re-dox process is assisted by the flow of electron from the t2g orbitals of Low spin Fe2+ to π* orbitals of porphyrin ring
Fe and CuB are 4.5Å apart, in the oxidised state a OH- bridges both
C6 of Tyr244 and εN of His240 are covalently linked Cytochrome c oxidase
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Mechanism of CcO
The active site in oxidised form (O) is reduced by a 2 e- reduction from Cytochrome c (R)
The reduced form has FeII haem and CuIcentres
O2 is bound by the binuclear centre forming a transient ferrous-oxy intermediate (A)
A immediately transforms into a bridging peroxide (P), absorb a 607nm
Alternately the P form is argued to be a Ferryl (FeIV=O, absorb @ 580nm) and a cation radical on His-Tyr pair
The oxygen is reduced with 2 e- from FeIIhaema3, while the other O is reduced by an e- from CuI, a proton and an e- from Tyr-His pair forming OH-
Another e- from Cyt-c and 2H+ convert Cu2+ bound OH- to H2O & Tyrosine
Source: Metallo.scripps.edu
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COX: Proton pathway
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Courtesy: PNAS, 2007, 104, 2685
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Cytochrome c Peroxidase Peroxidases converts harmful peroxides to
water The core unit consists of Haemb with FeIII at
the porphyrin pocket The proximal axial site is bound by His 175,
while distal site is manned by His 52 at 5.6Åo Close to the distal site also lies Arg48 and
Trp51 residues H2O2 binds to Fe His 52 abstract an H+ from bound O The Arginine in proximity to remote O
polarises O-O bond Remote O leaves as H2O The other remains bound to Fe to create a
Ferryl (FeIV=O) intermediate, while a cation radial remains in porphyring ring (HRP) or in proximal Trp-191(CcP)
1 e- reduction & 2H+ converts bound O to H2O and regenerate the active site
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CCP + H2O2 + 2 ferro-cyt c + 2H+ → CCP + 2H2O + 2 ferri-cyt c
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Cytochrome-P450
Unlike other cytochromes, Cytochrome P450 is not an electron transfer protein
It’s a mono-oxygenase enzyme. Plays important role in intra cellular and drug metabolism P450 based enzymes found in liver, kidney, lungs & brainDerives its name from the Soret band at 450nm for the CO bound form Reactions they catalyze include
Aliphatic compounds to alcoholsAromatic compounds to phenolsSulfides to sulfoxideAmines to amine oxidesOlefin to epoxideOxidative dealkylation of hetero atoms
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Cytochrome P450: structure
The protein is a single polypeptide chain
The heme b group is sandwitched between two α- helices.
No covalent attachment between protein and Heme
14 α –helices, 5 antiparallel β-sheets Helix rich right side β-sheet rich left side Cys-357 bound at the axial position 2nd axial site is bound by water Resting state Fe3+ low spin, changes
to high spin upon substrate binding Its then reduced to HS Fe2+
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Mechanism of Cytochrome P450
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Key features of mechanism
The proposed mechanism involve the following key steps1. Initially substrate binds near the site of heme ligand, when Fe3+ (LS) gets
converted to Fe3+ (HS)2. One-electron reduction by Flav-protein NADPH and Cytochrome p450
reductase3. Reaction with dioxygen to give a dioxygen adduct4. Addition of a 2nd electron from NADPH or cytochrome b55. Heterolytic scission of the FeO-O(H) bond to generate a formal (FeO)3+
6. Oxidation of the substrate.1. Formal abstraction of hydrogen atom or electron2. Radical recombination
7. Release of the product.
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Methane mono oxygenase
Structure has 3 parts Hydroxylase(α2,β2,γ2), b-unit,
reductase The catalytic active site resides in a
four-helix bundle of the a subunit The Di-iron center is coordinated by
two Histidine and four Glutamateresidues
Rest of pseudo octahedral coordination is satisfied by solvent water
In the resting state (MMOHox) the iron centers are bridged by Hydroxide ions
Fe- Fe distance at rest is ∼3.1Å In the reduced state (MMOHred)
E243 displaces a OH- ion forming the bridge
CH4 + O2 + NAD(P)H + H+ → CH3OH + NAD(P)+ + H2OCourtesy: Acc. Chem. Res. 2011, 44(4), 280-288
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sMMO: Mechanism
The mechanism involve the following steps Dioxygen insert into the di-iron (FeII-
FeII) core of MMOHred to form P (FeIII-FeIII core)
P contains a peroxide ring bound symmetrically
P changes into the reactive intermediate Q with a diamond core structure
Q then goes over a H-abstraction- rate determining step- to form MMOHox
The bridged OH weakly interact with methyl radical
The intermediate MMOHox rearranges to eliminate the alcohol and regenerate the enzyme in the reduced state MMOHred.
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Xanthine Oxidase family
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XO hydroxylases have Cofactor dithiolene ligand coordinated to Mo in facMoOS(H2O) unit SO have single thiolene moiety coordinated to cisMoO2 unit DMSO reductase have bis-dithiolene group coordinated to Mo=X group where X can be
O, S, Se etc. & remaining position is taken up by serine, Cisteine or selenocisteine
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Hydroxylase: Xanthine Oxidase
XO belongs to the class of oxido reductasesXO serves to convert Xanthine to Uric acidMol wt. 270kDaXO is a homodimerEach monomer consists of 3 domainsDomain1 contains FAD, Domain 2 Fe2S2-I & Fe2S2-II & Domain 3has the active site Mo-ptInter subunit Mo-pt distance is 50ÅThe electron transfer takes place in the order FAD→Fe2S2-I →Fe2S2-II → Mo-pt→SubstrateAllopurinol -a drug for goutserves to inhibit XOMo-pt is near the interface of domain 1 & domain 2
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Xanthine Oxidase: Active site
First domain contain Fe2S2-I & Fe2S2-II (residue 1-165) Second domain contains Moco is the active site (residue 226-531) Third Domain (residue 590-1331) contain the Mo-pt cofactor In Eukaryotes R=H in Moco/Mo-p; In Prokaryotes R= AMP, CMP, GMP
Flavin Adenine Dinucleotide [FAD]
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Xanthine Oxidase: Mechanism
The XO mechanism involve the following steps
A base assisted nucleophilic attack by Mo-OHeq on the C8 carbon atom with simultaneous Hydride transfer to Mo=S, while MoVI gets reduced to MoIV
Re-oxidation of the Molybdenum centre occurs with electron transfer to the other ET centers followed by H+transfer
Displacement of the product by OH- from Mo site
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Courtesy: Cao H et al. J. Biol. Chem. 2010;285:28044-28053
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Catalyze reversible 2 e- oxidation of formate to CO2
Belongs to DMSO reductase family Has two subunits 977 and 214 residues W is bound to Molybdopterin cofactors, a
selenocystein and a hydroxyl/S2-group Domain 1 also contain an Fe4S4 unit Substrate is accessible via +vely charged
tunnel, H+ is eliminated via buried H2O & protonable amino acid side chain channel, CO2 is excluded via a hydrophobicchannel
The smaller subunit contains 3 Fe4S4 units MGD 1002 is involved in extensive H-
bonding interactions with Domain III, IVand with K56 in Domain I.
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Formate Dehydrogenase
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Bio-inorganic Chemistry -by R. P. John
37o The H-bonding between K56 and pterin Cofactor provides
contact with 1st Fe4S4 unit oAn R407 lines and points towards the entry channel near the
active siteo The R407 stabilized & orients the substrate
o The funnel shaped substrate entry channel is progressively lined with several 4H, 3K, and 3Rresidues
oHis 159 provide π-interaction with Se-Cys
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Formate Dehydrogenase: Mechanism
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o H+ channel is located perpendicular to Substrate channelo Acidic residues from D and E lines H+ channelo H, R, V, W etc line the Hydrophobic channel along with several H2O
moleculeso The electrons are eliminated via the series of Fe4S4 cluster located in
small subunit
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Cu-Zn Superoxide Dismutase
Dismutation is an antioxidant defense mechanism of cells against superoxide(O2-)
Dismutation involves conversion of superoxide into O2 and H2O2
Three types of SOD found in Humans SOD1, a dimer (found in cytoplasm)
and SOD 3, a tetramer (found in extracellular fluids) contain Cu-Znactive site
SOD 2 is a tetramer with Mn at the active site
Cu-Zn SOD is a homo-dimer of 32.5 kDa
It is a b-barrel of 8 anti-parallelstrands
Each subunit is connected by hydrophobic/electrostatic interactions
[Superoxide is a byproduct of mitochondrial respiration & fatty acid oxidation]
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Cu-Zn SOD
The enzyme is so designed that only superoxide or water can enter the site.
The size of the cavity towards CuII in active site progressively decreases
Arg 141 lines the cavity preceding active site pit. This interacts with superoxideduring reaction
At the mouth of the reaction center the cavity diameter is 4Å, which is lined with lysine residues
The active site of Cu-Zn SOD contain 4Histidines and one water bound to Cu; 3Histidines and one Aspartate coordinated to Zn centre
Out of which one His (His 63) is bridgingCu & Zn
While Cu acts as the catalytic center Znprovide only structural role.
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Cu-Zn SOD: Mechanism
Bio-inorganic Chemistry -by R. P. John
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processM(n+1)+-SOD + O2− → Mn+-SOD + O2Mn+-SOD + O2− + 2H+ → M(n+1)+-
SOD + H2O2. In Cu-Zn SOD, Cu shuttles between I &
II oxidation sate The incoming HO2- displaces the bound
water and bind with the CuII centre The bridging His 63 abstract the H+
from bound HO2-. This is followed by a one electron
reduction of CuII and release of O2 Another HO2- enter the site and binds
to CuI. The bound HO2- radical abstract an H+
from His 63, and released as H2O2.
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Mn Superoxide Dismutase
Fe/Mn-SOD is tetramer found in mitochondria
Each subunit consists of two domains –an α-N terminal domain and a mixed α/β C-terminal domain
The active site metal ions sits at the interface of the two domains
Two Histidines from domain 1 (His 26 & His 81) & Asp 167 and His 171 from domain 2
The Mn2+ in each subunit is coordinated by 3 Histidines, one Aspartate and OneH2O (MnII) or One OH- (MnIII)
Glu 170 & Tyr 174 form the inter subunit contacts through H-bonds
A pair of gateway residues His 30 Tyr 34lie in front of equatorial His (81, 171)
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Mn-SOD: Mechanism
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Coenzyme B12
Coenzymes are reversibly bound groups or molecules to assist a particular enzyme catalysis
Coenzyme B12 is a group transferenzyme
Co3+ (LS) at the center, six-coordinate Two axial positions- α and β α- occupied by α-D-ribofuranose-3-
phosphate linked with 5,6-dimethyl benzimidazole
β-can be occupied by 5’-deoxyadenosyl, Me, OH or CN
When CN- at β it is Vitamin-B12
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Coenzyme B12 mediated Mechanism
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Ethanolamine Ammonia Lyase
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Bio-Inorganic ChemistrySyllabusBio-inorganic Chemistry: Part 3-2�EnzymesENZYMES: Introduction Enzymes: ClassificationIron Ezyme: Purple Acid PhosphataseSlide Number 7PAPs: Mechanism-steps involvedIron containing Enzymes: AconitaseAconitase: MechanismSlide Number 11Slide Number 12Mg Dependant Enzymes: RubiscoSlide Number 14Rubisco: MechanismRubisco: MechanismSlide Number 17CytochromesMechanism of electron transferCytochrome c OxidaseCytochrome c OxidaseCytochrome c oxidaseMechanism of CcOCOX: Proton pathwayCytochrome c PeroxidaseCytochrome-P450Cytochrome P450: structureMechanism of Cytochrome P450Key features of mechanismMethane mono oxygenasesMMO: MechanismXanthine Oxidase familyHydroxylase: Xanthine OxidaseXanthine Oxidase: Active siteXanthine Oxidase: MechanismFormate DehydrogenaseSlide Number 37Formate Dehydrogenase: MechanismCu-Zn Superoxide DismutaseCu-Zn SODCu-Zn SOD: MechanismMn Superoxide DismutaseMn-SOD: MechanismSlide Number 44Coenzyme B12Coenzyme B12 mediated MechanismEthanolamine Ammonia Lyase