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.

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 ion’s 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: α amylases
11

12. Hydrolase
3.2.1.1
12

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 enzyme’s 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

27. 27
EC
3.4.24.27
34.6 kDa
hydrolase

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 ATP’s 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
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