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CHEM F343: Inorganic Chemistry III: Pilani

The document discusses the structure and function of several metalloenzymes: 1) Hemoglobin and myoglobin have remarkably similar tertiary structures, but their primary sequences are only 18% identical. 2) Hemerythrin and hemocyanin are found in invertebrates and contain iron or copper ions respectively. They both undergo two-electron oxidation upon oxygen binding. 3) Carboxypeptidase A contains a zinc ion that assists in the hydrolysis of peptide bonds through the stabilization of reaction intermediates.

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ANANYA PUPNEJA
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31 views22 pages

CHEM F343: Inorganic Chemistry III: Pilani

The document discusses the structure and function of several metalloenzymes: 1) Hemoglobin and myoglobin have remarkably similar tertiary structures, but their primary sequences are only 18% identical. 2) Hemerythrin and hemocyanin are found in invertebrates and contain iron or copper ions respectively. They both undergo two-electron oxidation upon oxygen binding. 3) Carboxypeptidase A contains a zinc ion that assists in the hydrolysis of peptide bonds through the stabilization of reaction intermediates.

Uploaded by

ANANYA PUPNEJA
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
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Download as PDF, TXT or read online on Scribd
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BITS Pilani

Pilani Campus

CHEM F343: Inorganic Chemistry III

1/24/2019
BITSPilani, Pilani Campus
Bioinorganic Chemistry
Structure similarity of Hb and Mb

Tertiary structure of a and b are remarkably similar


both to each other and to Mb

Only 18% of the corresponding residues are identical


among these 3 polypeptides
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BITSPilani, Pilani Campus
Structure similarity of Hb and Mb

The 3-D structures of the a and b chains of Hb look like


that of Mb,
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but their primary sequences are different.
BITSPilani, Pilani Campus
Hemerythrin (Hr) and Hemocyanine (Hc)
•are found in invertebrates (Annelids, Mollusks, Arthropods)
•difficult to crystallize (single crystal) as it aggregates
• Hr isolated from the blood of invertebrates is typically in
form of octamer
• Hc exists as large aggregates
•Chemical and physical properties of Hr and Hc are available.

Terminology for the oxidation states of metal ions


Hemerythrin Hemocyanine
Fe(II).Fe(II) (deoxy) Cu(I).Cu(I) (deoxy)
Fe(III).Fe(III) (met) Cu(II).Cu(II) (met)
Fe(II).Fe(III) (Semi-met) Cu(I).Cu(II)(Semi-met)
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Hemerythrin (Hr) and Hemocyanine (Hc)

• O2 binding is essentially non cooperative for Hr


• Cooperative binding of O2 is common for the multimeric Hc
• In both cases, O2 binding is accompanied by two electron
oxidation of the binuclear center to produce bound peroxide
• Raman spectroscopy can be used to detect the nature of
O2 binding

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BITSPilani, Pilani Campus
Hemerythrin
O2  Fe(III) results in a peak at 844 cm-1 indicating
peroxide type binding

• Binding mode was


explored using
asymmetrically labeled
Fe—16O—18O and
doublet was obtained

Resonance Raman spectrum of Fe—16O—18O; central peak can


be fitted with two smooth curves for Fe—16O—18O and Fe—
18O—16O
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Hemerythrin

Possible binding mode

•III and IV: considered due to unsymmetrical mode


• IV is ultimately considered based on single crystal X-ray
diffraction along with other routine studies
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BITSPilani, Pilani Campus
O2 binding to Hemerythrin

1.High spin Fe(II) ions 2. Antiferromagnetically coupled

No proton release and hence no Bohr effect


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BITSPilani, Pilani Campus
Hemocyanin: Structure

Reversible oxygen binding derives from two features of the


binding pocket.

1. 2 Cu+ ions are each bound by 3 His residues.


Geometry: pseudo Td. With His forming base, Cu at the
Apex, favoring Cu+ not Cu2+( favors Sq. pl. geometry)

2. Cavity lined with


hydrophobic residues
disfavor charged int..
Pocket size reduces
from 0.95 to 0.72 Å,
making 2 Cu ions
closer by 0.2 - 0.3 Å.

Blue
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Enzymes: 6 types
• Oxidoreductase: Oxidation/ reduction
• Transferase: Transfers the group from one compound to
another
•Hydrolase: hydrolysis
• Isomerase: converts substrate into it’s isomeric form
• Lyase: non hydrolytic addition or removal of group
• Ligase: Synthesis of a large molecules from two smaller ones
Zn—OH2  Zn—OH- + H+ pKa = 8.8
Mg—OH2  Mg—OH- + H+ pKa = 11.4

Zinc is stronger Lewis acid


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BITSPilani, Pilani Campus
Carboxypeptidase A
• General Rule
• Zn(II) is employed where substrate bear a carbonyl
(C=O) functional group (ester, amide etc.)
• Mg(II) acts as cofactor for the enzymes responsible
for hydrolysis or formation of phosphate esters

Carboxypeptidase A
• Catalyzes the hydrolysis of C-terminal amino acid of any
polypeptide and prefers for substrate with aromatic side
chain
• Active site possesses hydrophobic pocket to recognize
such side chain
• Bovine Carboxypeptidase A has been extensively
explored
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Carboxypeptidase A

• Amide carbonyl is hydrogen bonded to Arg


• Attack of H2O is assisted by glutamate and Zn(II)
• Zn(II) does not interact directly with the C=O oxygen.
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BITSPilani, Pilani Campus
Carboxypeptidase A

Tetrahedral intermediate is stabilized and then collapse


with proton abstraction by Glu
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Carboxypeptidase A

Electrostatic interactions between the product


carboxylate and Glu 270 assists product release
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BITSPilani, Pilani Campus
From: Bertini, I.; Gray, H.B.; Lippard, S.J.; Valentine, J.S.: "Bioinorganic
Chemistry" (1994), University Science Books, pg. 83

(Chemistry Library, QP531 .B543 1994 )


Alkaline Phosphatase

• Zn(II)---Zn(II) = 4 Å
• One Mg (II) ion is
located at 5-7 Å away
from both the Zn(II)
ion
• Zn(II) ions are bound
with His and Asp
• One Zn(II) is close to
Ser-OH, which acts as
nucleophile to attack
the phosphate ester
• TBP geometry about
Zn(II)
Dimer of 94 kDa
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Alkaline Phosphatase

J. Mol. Biol. 2012, 415, p102-17


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Alkaline Phosphatase

PROTEIN SC. 2010, 19, p75—84


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Alkaline Phosphatase

• Optimum activity at pH 8
• One Zn(II) is closely
associated with Ser while
other is with water
• Water coordinated Zn(II)
stabilizes the -ve charge
developed on alkoxide group
produced
• The pKa of aquated
Zn(II) is 8.8, which yields
R-OH and displaces the
phosphoryl group from the
phosphoserin intermediate
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Alkaline Phosphatase

• K3 is the
slowest step
• ROH is rapidly
produced
• Burst kinetics:
Due to an initial
high velocity of
enzymatic
turnover when
adding enzyme
to substrate.
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BITSPilani, Pilani Campus

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