D R . R . P . J O H N

Bio-Inorganic Chemistry

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.

Z I N C E N Z Y M E S : C A R B O X Y P E P T I D A S E , C A R B O N I C A N H Y D R A S E , A L K A L I N E P H O S P H A T A S E , L A D H

Bio-inorganic Chemistry: Part 3-1Enzymes

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ENZYMES: Introduction

It is a protein with catalytic properties due to its power of specific activation

Biological systems sensitive to temperature change Increase the rate of reactions without increasing the

temperature. Increase the rate by lowering the activation energy. They create a new reaction pathway They are globular proteins with complex 3D structureThe active site is where the reaction is catalysed

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ENZYMES: Introduction

The shape & chemical environment at 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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Lock and Key Hypothesis

The fit between substrate and the enzyme is exact. Like a lock can be opened by only its own key a given substrate

can be converted to its products by a specific enzyme.The temporary structure formed between the enzyme and

substrate is called ENZYME-SUBSTRATE complexThe products are released fast, as soon as they are formed

leaving the active site free for another reaction.Adverse factors: Change in pH, Temperature, presence of

Inhibitors . Why Adverse effect? -change the enzyme structure, modify the

active site structure or bind to the active site.

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Scheme of Enzyme Catalysis

Enzyme may be used again

Enzyme-substrate complex

E

S

P

E

E

P

Reaction coordinate

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Factors affecting rate

Temperature, pH, substrate concentration and presence of inhibitors affect the rate of the reaction.

The rate of the reaction is governed by the Michaelis-Menton Equation for the reaction

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Effect of Substrate concentration

Reaction velocity

Substrate concentration

Vmax

›When all available sites are bound by the substrate the rate of reaction become constant, Vmax

›Increase in substrate concentration does not affect the rate of reaction. ›Increase in Enzyme concentration increase Vmax.

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Effect of pH

Every enzyme has a specific pH range for optimum activity Beyond the optimum pH the activity decreases.

Enzyme activity Trypsin

Pepsin

pH1 3 5 7 9 11

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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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Some Enzymes and their functions

Enzyme Metal ion function

Carboxy peptidase Zn2+ Hydrolysis of C-terminal peptides

Leucine aminopeptidase Zn2+ Hydrolysis of Leucine N-terminal peptides

Thermolysin Zn2+, Ca2+ Hydrolysis of peptides

Alkaline phosphatase Zn2+, Mg2+ Hydrolysis of phosphate esters

Carbonic anhydrase Zn2+ Hydration of CO2 and dehydration of H2CO3

Phospholipase Ca2+ Hydrolysis of phospholipids

α-amylase Ca2+/Zn2+ Hydrolysis of glucosides

Creatine kinase Mg2+ Phosphorylation of creatine

DNA polymerase Mg2+, Mn2+ Polymerisation of DNA with formation of phosphate esters

Alcohol dehydrogenase Zn2+ Hydride transfer of Alcohol to NAD+

Xanthine oxidase Mo6+/Fe2+ Oxidation of xanthine to uric acid

Coenzyme B12 Co3+ 1,2 vicinal group interchange

Cytochrome P450 Fe2+ Hydroxylation of aliphatic compounds and aromatic rings

Bio-inorganic Chemistry -by R. P. John

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Zinc Enzymes

Why Zinc?

No CFSE for d10 configuration Flexibility of coordination sphere for 4, 5 or 6 Low energy barrier for conversion of coordination sphere

between 4, 5 and 6 Bound ligands are kinetically labile The pKa value of H2O bound to a Zn2+ is lower than free water Offer same function of OH- ion at a still lower pH close to that of

neutral.

Bio-inorganic Chemistry -by R. P. John

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Carbonic Anhydrase

Its an enzyme that catalyses hydration of CO2 reversibly

It maintains the acid base equilibrium in blood

The rate of hydration is 107 times faster than the uncatalysed one.

Has a molar mass of 30kDa HCA II is the high active form kcat 106 s-1

HCA-II is shaped like a rugby ball A crevice 16Å deep runs down the

south pole Zn2+ is anchored deep in the crevice

Active site Structure of HCA II

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The active site of HCAII. The zinc ion is tetrahedrally coordinated by 3 histidines (His-94, His-96, and His-119) and catalytic water (Wat-263).

Sjöblom B et al. PNAS 2009;106:10609-10613

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Carbonic Anhydrase: Active site

Three Histidine residues (His-94, 96, 119) are bound to Zn2+.

A water molecule completes tetrahedral coordination around Zn2+

His-119 NH group is H-bonded to a glutamate residue

Coordinated water is H-Bonded to Thr-199 which in turn is H-bonded to Glu-106

Generally H2O is bound at A site but when B site is occupied H2O shuffles to C site

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Carbonic Anhydrase: mechanism of action

The proton of coordinated H2O is snapped up by neighboring His-64

This leaves a Zn-OH for CO2 activation The H-bonding of the Zinc bound O-H group

with Thr-199 orients the Zn-OH for nucleophilic attack

The Zinc bound OH group makes Nucleophilicattack on C of CO2

The polarization of CO2 is achieved by the H-bonding of CO2 O by a water molecule

Once the bicarbonate is formed the proton transfer is achieved by a H-bonding network

This is followed by 5 coordinate intermediate which eventually loses HCO3

- to leave behind H2O bound Tetrahedral Zn2+ active site

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Carboxy peptidase

CPA is produced by pancreas gland CPA is an egg shaped globular protein of a single chain of 307 amino acids

containing regions of α-helices and β-plated sheets It is involved in hydrolyzing the amide linkage of C-terminal amino acidsWeighs 34.6kDa with a maximum dimension of 50Å and a minimum of 38Å

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Crystal Structure of Carboxy Peptidase A

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The Active site: Carboxypeptidase

The Zn2+ rest in a cavity on one side of the egg shaped enzyme. 2 Histidines (His-69, 196) and 1 glutamate (Glu-72) satisfy 3 coordination

positions The 4th position of the Zinc tetrahedra is completed by an H2O A Glutamate residue (Glu-270) in the vicinity of active site provide Zn-OH by

deprotonation of the bound H2O Opposite side of the cavity has a mobile Tyrosine (Tyr-248), It’s role - to bind

the amide NH of the penultimate amino acid of the substrate by a H-bond. The cavity has a hydrophobic pocket where the incoming C-terminal amino

acid accommodate its hydrophobic part. 3 Arginine and 1 Asparagine reside at the peptide binding domain The Arg-145 and Asn-144 engage the Carboxylate residue of the C-terminal

amino acid through electrostatic interaction and by H-bond Arg-127 interact with the carbonyl oxygen of the substrate

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Carboxypeptidase: Mechanism of action

The incoming peptide interact with Arg residue through its terminal carboxylate group

Arg-71 is initially involved so that the hydrophobic part can easily slide into the pocket.

The H-bond formed by Arg-127 polarizes the carbonyl bond. At this point metal bound hydroxide O makes a nucleophilic attack on

Carbonyl carbon The next stage is the breakage of C-N bond with simultaneous abstraction of a

‘H’ by amide N of C-terminal amino acid At this stage the polarized carbonyl oxygen engage Zn2+ leading to 5

coordinate geometry To accommodate a bidentate substrate binding the Glu-72 become

monodentate Glu-270 then abstract the Zn bound OH proton which is then taken away by

cleaved C-terminal amino group

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Alkaline Phosphatase

They serve to catalyze hydrolysis of phosphate monoester

Trimetallic Active Site

Zn1 is bound by His331, 372, 412 3 exchangeable Hs near Zn1 (2His, 1 Arg) Zn2 is bound by Asp 369, His 370Mg is bound by Asp 153, Thr 155, Glu 322 Asp 51 is bridging Zn2 & Mg through Os Zn1 is 5 coordinate Zn2 & Mg 6 coordinate

Alkaline Phosphatase: Mechanism of action

Mechanism: Steps involved

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The presence of all metal centers are essential for optimum activityPresence of Serine between M1 & M2 essential for activityThe serine is phosphorylated during the reaction and [PO3OR]2- binds M2

The mechanism involve the following stepso Replacement of the bound water by a phosphate group[ROPO3]2-

o The unprotonated O of Phosphate group abstracts a H from serine O-Ho The serine O- gets bound to Zn2 and launches a SN2 type nucleophilic attack

on Phosphorouso P-OR bond cleaves, OR- takes up a proton from second Zn bound water and

hydroxylated protein leaves the siteo The Zn bound O launches an attack on the serine bound phosphateo Ser-102 O leaves Phosphorus and gets attached to Zn2o Serine takes a Phosphate bound H and regenerate O-Ho Phosphate leaves Zn1 as HPO4

2-

o Two water molecules enters and get bound to Zn1 in a two step process

LADH

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Liver Alcohol Dehydrogenase

LADH is a dimeric protein of 80kDaEach monomer contain two Znatoms at the active siteZn1 is coordinated by 4 Cistein residuesZn2 is coordinated by 2 Cistein, 1Histidine and 1 WaterZn1 has no catalytic role, Zn2 has catalytic roleEach monomer consists of two protein subunits that are free to rotate w.r.t each otherThe metal polarises the C-O bond and orient the substrate

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LADH: Mechanism

Coenzyme: NADP+

(Nicotinamide Adenine Dinucleotide Phosphate)

LADH: Mechanism in steps

At neutral pH mechanism involve the following stepsNAD+ enters the site through a channel. The pKa of bound water drops to 7.6 (from 9.2)The substrate –alcohol- enter the active site between the two domains and binds Zn2 displacing bound water. The enzyme is still in the open formA domain rotation takes place that brings the bound substrate and NAD+ closer while excluding unbound water molecules in the site. The enzyme now assumes a closed form. The combined effect Zn2+ and NAD+ cause a further drop in pKa of coordinated alcohol below 7. A near by base gobbles up the H+ bound to Zn bound O of substrateA direct hydride transfer takes place from αC of alcohol to C-4 of Nicotinamide ring resulting in a ternary NADH-aldehyde adductThe polarity of the active site drops dramaticallyThe product formed leave the site, a water molecules enter site and binds to Zn. The pKa of bound water is now 11.2More water molecules enters site favoring partial opening of the structureA loss of contact between the two parts favor complete opening. NADH leaves site.Site is ready for next cycle