Course HSS 2305 A

Molecular Mechanism of Disease

Lecture-3
Proteins and Biocatalysis/Enzymes: Function and Regulation

Ajoy Basak, Ph. D.

Adjunct and Part-time Professor, Pathology and Laboratory Medicine,
Faculty of Medicine, U Ottawa,
Roger Guindon Building
451 Smyth Road
Ottawa, ON K1H 8M5
Tel 613-878-7043 (Cell)
E-mail: [email protected]
Alternate: [email protected]
Affiliate Investigator, Chronic Disease Program,
Ottawa Hospital Research Institute
Web:https://med.uottawa.ca/pathology/people/basak-ajoy
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Proteins, Enzymes, Their
Functions and Regulators

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Zwitter ion or bipolar character

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Dextro-rotatory compounds rotate the plane of polarization of
a plane polarized light in a clockwise direction (Right).

Laevo-rotatory compounds rotate the plane of polarization of

a plane polarized light in a counter-clockwise direction
(Light).

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 Proteins can be
 Soluble (secreted form)
 Insoluble
 Membrane bound

Membrane Proteins
Depending on the cell type and particular
organelle within that cell, a membrane may
contain hundreds of different proteins bound to it.

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 The Structure and Functions of
Membrane Proteins
• Membrane proteins attach to the bilayer asymmetrically, giving the membrane a distinct “sidedness”
• Membrane proteins can be grouped into three distinct classes:
1. Integral proteins - penetrate and pass through lipid bilayer; make up 20 -30% of all encoded proteins
• Are amphipathic, with hydrophilic domains anchoring them in the bilayer and hydrophilic regions
forming functional domains outside of the bilayer.
• Channel proteins have hydrophilic cores that form aqueous channels in the membrane-spanning region.

Integral proteins

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The Structure and Functions of
Membrane Proteins

Driven by van der Waals forces between amino

acids and lipids, proteins can be surrounded by a
closely applied shell of lipid molecules.

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 The Structure and Functions of
Membrane Proteins

• Membrane proteins attach to the bilayer asymmetrically, giving the membrane a

distinct “sidedness”
• Membrane proteins can be grouped into three distinct classes:
2. Peripheral proteins are attached to the membrane by weak bonds and are easily solubilized.
– Located entirely outside of bilayer on either the extracellular or cytoplasmic side; associated
with membrane surface by non-covalent bonds.

Peripheral proteins

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 The Structure and Functions of
Membrane Proteins
• Membrane proteins attach to the bilayer asymmetrically, giving the membrane a distinct
“sidedness”
• Membrane proteins can be grouped into three distinct classes:
3. Lipid-anchored membrane proteins are distinguished both by the types of lipid anchor and their
orientation.
– Glycophosphatidylinositol (GPI)-linked proteins found on the outer leaflet can be released by inositol-
specific phospholipases.
– Some inner-leaflet proteins are anchored to membrane lipids by long hydrocarbon chains.

Lipid-anchored
proteins

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 The Structure and Functions of
Membrane Proteins

Ectoplasmic vs
protoplasmic

• Distribution of Integral Proteins: Freeze-

Fracture Analysis
– Freeze-fracture technique divides the
phospholipid leaflets of the membrane.
– Integral membrane proteins appear as
bumps and pits using the electron
microscope.
– The heterogeneity of protein distribution AB: carb group for
is shown. glycophorin
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 The Structure and Functions of
Membrane Proteins

Solubilization of membrane An integral protein as it resides

proteins with detergents within the plasma membrane

• Studying the Structure and Properties of Integral Membrane Proteins

– Determining membrane sidedness: The orientation of integral proteins
can be determined using non-penetrating agents that label the proteins.
– SDS (ionic)-denatures proteins
– Triton X-100 (non-ionic)- does not alter protein tertiary structure
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 The Structure and Functions of
Membrane Proteins

Glycophorin A, an integral protein with a

single transmembrane domain with a Gly- Hydropathy plot for glycophorin
X-X-X-Gly sequence A demonstrates a single pass
domain
• Studying the Structure and Properties of Integral Membrane Proteins
– Identifying transmembrane domains: A string of 20-30 hydrophobic amino
acids from hydropathy plots identifies a membrane-spanning domain.
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 The Structure and Functions of
Membrane Proteins

Accommodating nonpolar amino acid residues within transmembrane

helices

Studying the Structure and Properties of Integral Membrane Proteins

– Spatial relationships within an integral membrane protein
• Site-directed mutagenesis—replacing specific amino acids with others—
identifies some spatial relationships.
• Electron spin resonance identifies some conformational changes that
occur when integral proteins function.
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 The Structure and Functions of Membrane Proteins

Site-directed
mutagenesis to learn
about dynamic changes
in the conformation of
a membrane protein as
it carries out its activity

• Determining spatial relationships between

amino acids within integral membrane
proteins
• Use of site-directed mutagenesis to replace
amino acids residues
• Replacing residues in neighboring helices
with cysteine residues can lead to disulfide
bond formation to reveal proximity.
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 The Structure and Functions of Membrane Proteins

Use of EPR spectroscopy to monitor changes

in conformation of a bacterial K-on channel
as it opens and closes
• Dynamic events occur as a protein functions which can be monitored:
• Introduce chemical groups whose properties are sensitive to
distance
• Introduce nitroxides at any site in protein by first mutating the
amino acid residue to cysteine via site-directed mutagenesis,
then attach nitroxide to thiol group of cysteine.
• Monitor by technique called electron paramagnetic resonance
[EPR] spectroscopy
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 Biocatalysts (Enzymes)

Proteo-Lytic Enzymes (Proteases/Proteinases)

Cleave proteins and polypeptides

Proteo-Ligase Enzymes (Protein Ligases)

Join protein and polypeptide chains to generate a

single larger protein

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 Ligases
 In Molecular Biology DNA Ligases are more common (involved in
DNA repair, DNA replication & Recombinant DNA experiments)

 DNA-Ligation occurs in the presence DNA-Ligase Enzyme

DNA-ligases

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 Proteoligase
 Less common
 Mediates in Joining or Attaching Proteins
 Example: “E3 Ubiquitin Ligase” of Ubiquitin system

Ubiquitin
Ubiquitin
Target
Protein Protein
Ubiquitination
Process

Degradation by
Proteosome of
Ubiquitinated
Protein

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 Enzymatic Reaction
 Temperature
 Light
 pH
 Oxygen *Fast
E + S E:S E + P
 Metal ions Irreversible
 Co-factors
(1 or multiples)
*RDS
Slow and
Reversible

E = Enzyme. S = Substrate, P = Reaction Products

E:S = Complex, RDS = Rate Determining Step
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