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BCH 400/600

Introductory Biochemistry
Instructor: David Shintani
Office: 311C Fleischmann Ag.
Lab: 308 Fleischmann Ag.
E-mail: shintani@unr.edu
Phone: (775) 784-4631

Before BCH 400

BCH 400 is
heavy on
content!!!
After BCH 400

Come to class!!!!!!

Try not to fall behind!!

Review lecture notes.


http://www.ag.unr.edu/shintani/bch400-600/index.html

Read book chapters.

Principles of
Biochemistry
4th Edition

Principles of
Biochemistry
3rd Edition

Use tools to memorize

Histidine?

Study in groups

Participate in the clicker


system!!!

20 extra credit points!!

Feel Free
to see me!

My
office me
hours
Or e-mail
for
are
Tuedays
from
a private
audience
2:00 to 3:00 PM

What is Biochemistry?
Biochemistry = chemistry of life.
Biochemists use physical and
chemical principles to explain
biology at the molecular level.
Basic principles of biochemistry are
common to all living organism

How does biochemistry


impact you?
Medicine
Agriculture
Industrial applications
Environmental applications

Principle Areas of
Biochemistry
Structure and function of biological
macromolecules
Metabolism anabolic and catabolic
processes.
Molecular Genetics How life is
replicated. Regulation of protein
synthesis

Life Before Biochemistry

Once upon a time, a long long time ago..


Vitalism: idea that substances and processes
associated with living organisms did not
behave according to the known laws of
physics and chemistry
Evidence:
1) Only living things have a high degree of
complexity
2) Only living things extract, transform and
utilize energy from their environment
3) Only living things are capable of self
assembly and self replication

Origins of Biochemistry:
A challenge to Vitalism.

Famous Dead Biochemist!

Fallacy #1: Biochemicals can only be


produced by living organisms
Dead Biochemist #1
1828 Friedrich Wohler

Fallacy #2: Complex bioconversion of


chemical substances require living
matter
Dead Biochemists #2
1897 Eduard Buchner

Glucose + Dead Yeast = Alcohol

Fallacy #2: Complex


bioconversion of chemical
substances require living matter
Dead Biochemists #3
Emil Fischer

Fallacy #2: Complex


bioconversion of chemical
substances require living matter
Dead Biochemists #4
1926 J.B. Sumner

Findings of other famous dead biochemist


1944 Avery, MacLeod and McCarty identified
DNA as information molecules
1953 Watson (still alive) and Crick proposed the
structure of DNA
1958 Crick proposed the central dogma of
biology

Organization of Life

elements
simple organic compounds (monomers)
macromolecules (polymers)
supramolecular structures
organelles
cells
tissues
organisms

Range of the
sizes of objects
studies by
Biochemist and
Biologist

1 angstrom = 0.1 nm

Elements of Life

Most abundant, essential for all organisms: C, N, O, P, S, H


Less abundant, essential for all organisms : Na, Mg, K, Ca, Cl
Trace levels, essential for all organism: Mn, Fe, Co, Cu, Zn
Trace levels, essential for some organisms: V, Cr, Mo, B, Al, Ga, Sn, Si,
As, Se, I,

Important compounds, functional groups

Many Important Biomolecules are Polymers

monomer

lipids

proteins

carbo

nucleic acids

fatty acid

amino acid

glucose

nucleotide

protein subunit

cellulose

DNA

protein complex

cell wall

chromosome

polymer phospholipid
supramolecular
structure

membrane

Lipids
monomer

fatty acid

polymer phospholipid
supramolecular
structure

membrane

Proteins

monomer

amino acid

polymer protein subunit


supramolecular
structure Enzyme complex

Carbohydrates

monomer glucose
polymer cellulose
supramolecular
structure cell wall

Nucleic Acids
monomer

nucleotide

polymer

DNA

supramolecular
structure

chromatin

Common theme:
Monomers form
polymers through
condensations
Polymers are broken
down through
hydrolysis.

Prokaryote Cell

Cellular Organization
of an E. coli Cell

200 300 mg protein / mL cytoplasm

Eukaryote Cell

Chapter 2: Water

WHY DO WE NEED TO
DO THIS AGAIN!!!

Properties of water
Very polar

Oxygen is highly electronegative


H-bond donor and acceptor
High b.p., m.p., heat of vaporization,
surface tension

Water dissolves polar


compounds

solvation shell
or
hydration shell

Non-polar substances are insoluble in water


Many lipids are amphipathic

How detergents work?

Hydrogen Bonding of Water


One H2O molecule can
associate with 4
other H20 molecules

Ice: 4 H-bonds per water


molecule
Water: 2.3 H-bonds per water
molecule
Crystal lattice of ice

Biological
Hydrogen
Bonds

non-covalent interactions

Relative Bond Strengths


Bond type
H3C-CH3
H-H
Ionic
H-bond
Hydrophobic interaction
van der Waals

kJ/mole
88
104
40 to 200
2 - 20
3 -10
0.4 - 4

Ionization of Water

Ionization of Water
H20 + H20
H20
Keq=

[H+]

[OH-]

[H2O]

H3O+ + OHH+ + OHKeq=1.8 X 10-16M


[H2O] = 55.5 M

[H2O] Keq = [H+] [OH-]


(1.8 X 10-16M)(55.5 M ) = [H+] [OH-]
1.0 X 10-14 M2 = [H+] [OH-] = Kw
If [H+]=[OH-] then [H+] = 1.0 X 10-7

pH Scale
 Devised by Sorenson (1902)
 [H+] can range from 1M and
1 X 10-14M
 using a log scale simplifies
notation
 pH = -log [H+]
Neutral pH = 7.0

Weak Acids and Bases Equilibria


Strong acids / bases disassociate completely
Weak acids / bases disassociate only partially
Enzyme activity sensitive to pH
weak acid/bases play important role in
protein structure/function

Acid/conjugate base pairs


HA + H2O
HA

A- + H3O+
A- + H+

HA = acid ( donates H+)(Bronstad Acid)


A- = Conjugate base (accepts H+)(Bronstad Base)

Ka =

[H+][A-]
[HA]

Ka & pKa value describe tendency to


loose H+
large Ka = stronger acid
small Ka = weaker acid

pKa = - log Ka

pKa values determined by titration

Phosphate has three


ionizable H+ and three pKas

Buffers
Buffers are aqueous systems that resist
changes in pH when small amounts of a strong
acid or base are added.
A buffered system consist of a weak acid and
its conjugate base.
The most effective buffering occurs at the
region of minimum slope on a titration curve
(i.e. around the pKa).
Buffers are effective at pHs that are within
+/-1 pH unit of the pKa

Henderson-Hasselbach Equation
1) Ka =

[H+][A-]
[HA]

HA = weak acid
A- = Conjugate base

2) [H+] = Ka [HA]
[A-]
3) -log[H+] = -log Ka -log [HA]
[A-]
4) -log[H+] = -log Ka +log [A-]
[HA]
5) pH = pKa +log [A-]
[HA]

* H-H equation describes


the relationship between
pH, pKa and buffer
concentration

Case where 10% acetate ion 90%


acetic acid

pH = pKa + log10 [0.1 ]

[0.9]

pH = 4.76 + (-0.95)
pH = 3.81

Case where 50% acetate ion 50% acetic acid

pH = pKa + log10 [0.5 ]

[0.5]

pH = 4.76 + 0
pH = 4.76 = pKa

Case where 90% acetate ion 10% acetic acid

pH = pKa + log10 [0.9 ]

[0.1]

pH = 4.76 + 0.95
pH = 5.71

Cases when buffering fails


pH = pKa + log10 [0.99 ]

[0.01]

pH = 4.76 + 2.00
pH = 6.76
pH = pKa + log10 [0.01 ]

[0.99]

pH = 4.76 - 2.00
pH = 2.76

Chapter 3
Amino acids, peptides, and
proteins

Properties of Amino Acids


capacity to polymerize
novel acid-base properties
varied structure and chemical
functionality
chirality

Basic Amino Acid Structure


carboxyl group

 -carbon is
chiral (except for
glycine)
 at pH 7.0
uncharged amino
acids are
zwitterions
 amino acids have
a tetrahedral
structure

amino group

-carbon
side chain

Amino Acid Enantiomers

Steroisomers / enantiomers
Biological system only
synthesize and use L-aminoacids

Amino Acid Classification

Aliphatic
Aromatic
Sulfur containing
Polar/uncharged
basic/acidic

Hydrophobic

Hydrophillic

Aliphatic (alkane) Amino Acids

Hydrophobicity

Proline (pro, P)- cyclic imino acid

Glycine(gly, G)-only non-chiral amino acid, not hydrophobic

Alanine (ala, A) R-group = methyl-group

Valine (Val, V) Think V!

Leucine (Leu, L)

Isoleucine (Ile, I) -2 chiral carbons

Aromatic Amino Acids

All very hydrophobic


All contain aromatic group
Absorb UV at 280 nm
Phenylalanine (Phe, F)
Tyrosine (Tyr, Y) -OH ionizable (pKa = 10.5), H-Bonding
Tryptophan (Trp, W) bicyclic indole ring, H-Bonding

Sulfur Containing Amino


Acids
Methionine (Met, M) start amino
acid, very hydrophobic
Cysteine (Cys, C) sulfur in form of
sulfhydroyl, important in disulfide
linkages, weak acid, can form
hydrogen bonds.

Acidic Amino Acids


Contain carboxyl groups (weaker acids than a-carboxylgroup)
Negatively charged at physiological pH, present as conjugate
bases (therefore ate not ic acids)
Carboxyl groups function as nucleophiles in some enzymatic
reactions
Aspartate

Glutamate

Basic Amino Acids


Hydrophillic nitrogenous bases
Positively charged at physiological pH
Histidine imidazole ring protonated/ionized, only amino
acid that functions as buffer in physiol range.
Lysine - diamino acid, protonated at pH 7.0
Arginine - guianidinium ion always protonated, most basic
amino acid

Polar Uncharged Amino Acids


Polar side groups, hydrophillic in nature, can form hydrogen
bonds
Hydroxyls of Ser and Thr weakly ionizable
Serine (Ser, S) looks like Ala w/ -OH

Threonine (Thr, T) 2 chiral carbons

Asparagine (Asn, N) amide of aspartic acid

Glutamine (Gln, Q) amide of glutamic acid

Essential/Non-Essential Amino Acids


Essential histidine, isoleucine,
leucine, lysine, methionine,
phenylalanine, threonine, tryptophan,
valine
Non-essential alanine, arginine*,
aspartate, asparagine, cysteine*,
glutamate, glutamine, glycine*,
proline*, serine, tyrosine*

Titration Curve for Alanine


pK1 carboxylic acid = 2
pK2 amino group = 10
pI = (pK1+ pK2)/2

pI (isoelectric point) = the pH at which the number of positive and


negative charges on a population of molecules is equal (i.e. no net charge).

Titration Curve for


Glutamic Acid
pK1 carboxylic acid = 2.2
pK2 R group = 4.3
pK3 amino group = 9.7
pI = (pK1+ pK2)/2
pI = (2.2+4.3)/2
pI = 3.25

Titration Curve
for Lysine
pK1 carboxylic acid = 2.2
pK2 amino group = 9.0
pK3 R group = 10.5
pI = (pK2+ pK3)/2
pI = (9+10.5)/2
pI = 9.75

pKas of charged amino acids R-groups

Aspartate/Glutamate = 4.0
Histidine = 6.0
Cysteine = 8.4
Tyrosine = 10.5
Lysine = 10.5
Arginine = 12.5

Protein Nomenclature
Peptides 2 50 amino acids
Proteins >50 amino acids
Amino acid with free -amino group is the
amino-terminal or N-terminal residue
Amino acid with free -carboxyl group is the
carboxyl-terminal or C-terminal residue
Three letter code Met-Gly-Glu-Thr-Arg-His
Single letter code M-G-E-T-R-H

Peptide Bond Formation

Partial double bond nature of peptide bond

Stability and Formation of the Peptide Bond


Hydrolysis of peptide bond favored energetically,
but uncatalyzed reaction very slow.
Strong mineral acid, such as 6 M HCl, good
catalyst for hydrolysis
Amino acids must be "activated" by ATP-driven
reaction to be incorporated into proteins

Enzymatic and Chemical


Cleavage of Peptide Linkage

Titration Curve of a Tetrapeptide


+H3N-Glu-Gly-Ala-Lys-COO-

Proteins have pIs

Assigment
Ala-Cys-Glu-Tyr-Trp-Lys-Arg-His-Pro-Gly

Draw the decapeptide at pH 1, 7, and


12. (pay attention to the form the Nand C- terminal and each R-group
takes on at each pH)
Calculate the overall charge at each
pH.
Write out the one letter code for the
decapeptide

Chapter 3 (part 2)
Protein purification and Analysis

Why purify proteins?


Pure proteins are required to study enzyme
function
Pure proteins are required for structural
analysis (x-ray crystallography, NMR
spectroscopy)
Pure proteins are required to obtain amino
acid sequence

Steps in protein purification


Develop assay
Choose source of protein
Prepare tissue extract
cell disruption
subcellular fractionation
Protein fractionation (several steps)
Determination of purity

Differential Centrifugation
transfer
supernatant

1000 g

tissue
homogenate

10,000 g

Pellet
unbroken cells
nuclei
chloroplast

transfer
supernatant

transfer
supernatant

100,000 g

Pellet
mitochondria

Pellet
microsomal
Fraction
(ER, golgi,
lysosomes,
peroxisomes)

Super.
Cytosol,
Soluble
enzymes

Chromatography

Gel Permeation
Chromatography

Ion-exchange
Chromatography
+++

---

+++

----+ + +++
+ ++ + +
++
+ ++
+ ++ + +
+ ++
++
+ ++ + +
++
+ ++
+ ++ + +
+ ++

low salt buffer

high salt buffer

++
-+ -+ -+
+
+
+ ++
---

+ -+
Cl
Cl+- + +- + Cl- +
+ +
++
+ Cl
+
+
Cl++- + +Cl
++
++
+ ++ + +
++
+ ++
+ ++ + +
+ ++

++
+ ++ + +
+ ++

+++

+++

++
- -+ +++ +
+ ++
++
+ ++ + +
+ ++
+++

---

---

---

Affinity Chromatography
Add excess ligand

SDS poly acrylamide electrophoresis (PAGE)


SDS = H3C-(CH2)10-CH2-OSO3SDS denatures protein
coats w/ negative charge
--- -- - - - -- ---

Used to determine protein MW


And purity of protein prep

Isoelectric Focusing

pH 3

Decreasing pH

Decreasing pH

pH 9

2-D Electrophoresis

Decreasing MW

large

Decreasing pH

Decreasing MW

SDS-PAGE

small

+
Decreasing pH

Amino Acid Analysis


1) Acid hydrolyze protein
2) Treat with phenylisothiocyanate (PICT)
N

H 3N

S
C

O-

HN
C

N
C
O

3) Separate derivatized AAs by HPLC

Protein Sequencing
(Edman Degradation)
N

H3N

NH

2)

C HN

NH

Trifluoroacetic acid
S
C HN

3)

N
C

2HN

Can sequence in 30 to 60 AAs from N-terminus

Repeat

1)

Generate Proteolytic
Fragments
Endopeptidases
Typsin
Chymotrypsin

cleaves at COOH end of Lys and Arg


cleaves at COOH end of Phe, Tyr, Trp

Chemical Cleavages
Cyanogen Bromide

cleaves at COOH end of Met

Generate overlapping fragments


Sequence individual fragments and piece together sequence

Peptide mapping exercise


Met-Ala-Arg- Gly-Glu-Tyr-Met-Cys-Lys-Phe-Ala-Glu-Gln-Asp
Trypsin
Met-Ala-Arg
Phe-Ala-Glu-Gln-Asp
Gly-Glu-Tyr-Met-Cys-Lys

Chymotrysin
Met-Ala-Arg- Gly-Glu-Tyr
Met-Cys-Lys Phe
Ala-Glu-Gln-Asp

CNBr
Met
Ala-Arg-Gly-Glu-Tyr-Met
Cys-Lys-Phe-Ala-Glu-Gln-Asp

Proteomic Analysis

Matrix Assisted Laser Desorption Ionization Time of Flight


(MALDI-TOF)

Chapter 4
Protein 3-Dimensional Structure and
Function

Terminology
Conformation spatial arrangement
of atoms in a protein
Native conformation conformation
of functional protein

Protein Classification
Simple composed only of amino acid residues
Conjugated contain prosthetic groups
(metal ions, co-factors, lipids, carbohydrates)
Example: Hemoglobin Heme

Protein Classification

One polypeptide chain - monomeric protein


More than one - multimeric protein
Homomultimer - one kind of chain
Heteromultimer - two or more different
chains

(e.g. Hemoglobin is a heterotetramer. It has


two alpha chains and two beta chains.)

Protein Classification
Fibrous
1)
2)
3)
4)

polypeptides arranged in long strands or


sheets
water insoluble (lots of hydrophobic AAs)
strong but flexible
Structural (keratin, collagen)

Globular
1)
2)
3)
4)

polypeptide chains folded into spherical or


globular form
water soluble
contain several types of secondary structure
diverse functions (enzymes, regulatory
proteins)

catalase

keratin

collagen

Protein Function

Catalysis enzymes
Structural keratin
Transport hemoglobin
Trans-membrane transport Na+/K+ ATPases
Toxins rattle snake venom, ricin
Contractile function actin, myosin
Hormones insulin
Storage Proteins seeds and eggs
Defensive proteins antibodies

4 Levels of Protein Structure

Non-covalent forces
important in determining
protein structure

van der Waals: 0.4 - 4 kJ/mol


hydrogen bonds: 12-30 kJ/mol
ionic bonds: 20 kJ/mol
hydrophobic interactions: <40 kJ/mol

1o Structure Determines 2o, 3o, 4o


Structure
Sickle Cell Anemia single amino
acid change in hemoglobin related to
disease
Osteoarthritis single amino acid
change in collagen protein causes
joint damage

Classes of
Alpha helix
B-sheet
Loops and turns

o
2

Structure

2o Structure Related to Peptide Backbone


Double bond nature of peptide
bond cause planar geometry
Free rotation at N - C and Ccarbonyl C bonds
Angle about the C(alpha)-N bond
is denoted phi ()
Angle about the C(alpha)-C bond is
denoted psi ()
The entire path of the peptide
backbone is known if all phi and psi
angles are specified

Not all / angles are possible

Ramachandran Plots

Describes acceptable / angles for individual


AAs in a polypeptide chain.
Helps determine what types of 2o structure
are present

Alpha-Helix
First proposed by Linus Pauling and
Robert Corey in 1951
Identified in keratin by Max Perutz
A ubiquitous component of proteins
Stabilized by H-bonds

Alpha-Helix
Residues per
Right handed turn: 3.6
helix
Rise per residue:
1.5 Angstroms
Rise per turn
(pitch): 3.6 x 1.5A
= 5.4 Angstroms
amino hydrogen
H-bonds with
carbonyl oxygen
located 4 AAs
away forms 13
atom loop

Alpha-Helix
All H-bonds in the
alpha-helix are
oriented in the
same direction
giving the helix a
dipole with the Nterminus being
positive and the
C-terminus being
negative

Alpha-Helix
Side chain groups
point outwards from
the helix
AAs with bulky side
chains less common in
alpha-helix
Glycine and proline
destabilizes alphahelix

Amphipathic Alpha-Helices

+
One side of the helix (dark) has mostly hydrophobic
AAs
Two amphipathic helices can associate through
hydrophobic interactions

Beta-Strands and Beta-Sheets


Also first postulated by Pauling and
Corey, 1951
Strands may be parallel or antiparallel
Rise per residue:

3.47 Angstroms for antiparallel


strands
3.25 Angstroms for parallel strands
Each strand of a beta sheet may be
pictured as a helix with two residues
per turn

Beta-Sheets
Beta-sheets
formed from
multiple side-byside beta-strands.
Can be in parallel
or anti-parallel
configuration
Anti-parallel betasheets more stable

Beta-Sheets
Side chains point alternately above and below the
plane of the beta-sheet
2- to 15 beta-strands/beta-sheet
Each strand made of ~ 6 amino acids

Loops and turns


Loops
Loops usually contain hydrophillic
residues.
Found on surfaces of proteins
Connect alpha-helices and beta-sheets
Turns
Loops with < 5 AAs are called turns
Beta-turns are common

Beta-turns
allows the peptide chain to reverse direction
carbonyl C of one residue is H-bonded to the
amide proton of a residue three residues away
proline and glycine are prevalent in beta turns

Chapter 4: Part 2
Protein 3-D structure: 3o and 4o
structure and protein folding.

o
3

Structure

third level of protein organization


folding of polypeptide chain causes 2o
structures to interact
formation of motifs and domains

Proteins with similar 1o structure also


have similar 3o structure
tuna
yeast
rice

tuna
yeast
rice

1 GDVAKGKKTFVQKCAQCHTVENGGKHKVGPNLWGLFGRKTGQAEGYSYTDANKSKGIVWN
1 GSAKKGATLFKTRCLQCHTVEKGGPHKVGPNLHGIFGRHSGQAEGYSYTDANIKKNVWDE
1 GNPKAGEKIFKTKCAQCHTVDKGAGHKQGPNLNGLFGRQSGTTPGYSYSTANKMAVIWEE

61 ETLMEYLENPKKYIPGTKMIFAGIKKKGERQDLVAYLKSATS
61 NNMSEYLTNPKKYIPGTKMAFGGLKKEKDRNDLITYLKKACE
61 NTLYDYLLNPKKYIPGTKMVFPGLKKPQERADLISYLKEATS

Common
Motifs

Motifs Combine to form Domains


Domains are independent
folding units in a 3o
structure of a protein
Individual domains have
specific function
Parallel twisted sheet

Hydrophobic
interactions are the
major driving force in
folding domains
Alpha/beta barrel

Protein family members share common


domain structures
lactate dehydrogenase

malate dehydrogenase

COO-

COOO

C + NADH
CH3

COOHO

CH + NAD+
CH 3

COOHO

CH

+ NADH
H2C
COO-

+ NAD+

CH2
COO-

o
4

Structure

Quaternary structure describes the organization of


subunits in a protein with multiple subunits
(oligomeric protein)
Can have homo-multimers or hetero-multimers

22
2

4o Structure
Determine molecular weight of native protein by
gel permeation chromatography
Determine molecular weight of individual
subunits by SDS-PAGE
Can use the information to determine subunit
composition
If.
Native protein 160,000 daltons and
-Subunit 50,000 daltons -Subunit 30,000 daltons
Then
Protein can have 22 structure

4o Structure
Subunits held together by non-covalent
interactions
Oligomeric protein is more stable than
disassociated subunits
Active site often made up of AA residues from
different subunits
4o and 3o structure is often affected by ligand
(substrate or inhibitor) binding. Important in
enzyme regulation

Protein denaturation
Denaturation disruption of native
conformation
Heat commonly used to denature
proteins
Tm = temperature where 50%
folded/50% unfolded.
Typical Tm = 40-60oC
Tm for thermophiles >100oC
(Taq DNA polymerase)
Chemical denaturants Chaotrophic
agents = Urea, KCN detergents = SDS

Tm

Protein Folding
Ribonuclease A (RNase A) will refold to
native structure spontaneously (1 minute)
>1050 possible conformations
If 10-13 sec per conformation would take
1030 years to sample enough to determine
structure
How do proteins fold so quickly?

Factors driving protein folding


Conformational entropy
A+B  C decreases
entropy (unfavorable)
Non-covalent interactions
give favorable enthalpy
value
Hydrophobic effect
increases entropy by
freeing water (favorable)

G = H - TS

Protein Folding
Structures of globular proteins are not static
Proteins breathing between different
conformations
Proteins fold towards lowest energy conformation
Multiple paths to lowest energy form
All folding paths funnel towards lowest energy form
Local low energy minimum can slow progress towards
lowest energy form

Pathway of
Protein Folding
1) Nucleation of folding - Rapid
and reversible formation of
local 2o structures form

2) Formation of domains
(Molten Globular intermediates)
through aggregation of local 2o
structures
3) Domain conformations adjust
to form native protein

Chaperonins

Protein complexes that promote protein folding


Chaperonins dont determine native structure
Prevent misfolding and aggregation of protein
Sequesters unfolded protein from other proteins
Require ATP for protein binding, after ATP hydrolysis
native protein released
Thought to bind unfolded regions of protein

Disulfides Bonds
Stabilize native structure
Formed after native conformation achieved
Abundant in secreted proteins but not in intracellular
proteins
Protein disulfide isomerase catalyzes reduction of
incorrect disulfide linkages

Chapter 4 (part 3)
3-D Structure / Function

Animal
Scrapie: sheep
TME (transmissible mink encephalopathy): mink
CWD (chronic wasting disease): muledeer, elk
BSE (bovine spongiform encephalopathy): cows
Human
CJD: Creutzfeld-Jacob Disease
FFI: Fatal familial Insomnia
Kuru

PrPC

PrPSc

solubility

soluble

non soluble

structure

predominantly alphahelical

predominantly betasheeted

multimerisation
state

monomeric

multimeric
(aggregates)

infectivity

non infectious

infectious

Susan W. Liebman, and James A. Mastrianni (2005) Trends in Molecular


Medicine 11: 439-441

Myoglobin/
Hemoglobin
First protein
structures determined
Oxygen carriers
Hemoglobin transport
O2 from lungs to
tissues
Myoglobin O2 storage
protein

Mb and Hb subunits structurally


similar
8 alpha-helices
Contain heme group
Mb monomeric protein
Hb heterotetramer (
22)
myoglobin

hemoglobin

Heme group
Heme = Fe++ bound to
tertapyrrole ring (protoporphyrin
IX complex)
Heme non-covalently bound to
globin proteins through His
residue
O2 binds non-covalently to heme
Fe++, stabilized through Hbonding with another His residue
Heme group in hydrophobic
crevice of globin protein

Oxygen Binding Curves


Mb has hyberbolic
O2 binding curve
Mb binds O2 tightly.
Releases at very low
pO2
Hb has sigmoidal O2
binding curve
Hb high affinity for
O2 at high pO2 (lungs)
Hb low affinity for
O2 at low pO2
(tissues)

Oxygen Binding Curve

Oxygen Binding Curve

O2 Binding to Hb shows positive


cooperativity

Hb binds four O2 molecules


O2 affinity increases as each O2 molecule binds
Increased affinity due to conformation change
Deoxygenated form = T (tense) form = low affinity
Oxygenated form = R (relaxed) form = high affinity

O2 Binding to Hb shows positive


cooperativity

O2 Binding
induces
conformation
change

T-conformation

R-conformation

Show Movie
Heme moves 0.34 nm
Exposing crystal of
deoxy-form to air
cause crystal to crack

Allosteric Interactions
Allosteric interaction occur when specific
molecules bind a protein and modulates activity
Allosteric modulators or allosteric effectors
Bind reversibly to site separate from functional
binding or active site
Modulation of activity occurs through change in
protein conformation
2,3 bisphosphoglycerate (BPG), CO2 and protons
are allosteric effectors of Hb binding of O2

Bohr Effect
Increased CO2 leads to decreased
pH
CO2 + H2O <-> HCO3- + H+
At decreased pH several key AAs
protonated, causes Hb to take on
T-conformation (low affinty)
In R-form same AAs deprotonated,
form charge charge interactions
with positive groups, stabilize Rconformation (High affinity)
HCO3- combines with N-terminal
alpha-amino group to form
carbamate group.
--N3H+ + HCO3-  --NHCOO Carbamation stabilizes Tconformation

Bisphosphoglycerate (BPG)
BPG involved acclimation
to high altitude
Binding of BPG to Hb
causes low O2 affinity
BPG binds in the cavity
between beta-Hb
subunits
Stabilizes T-conformation
Feta Hb (
22) has low
affinity for BPG, allows
fetus to compete for O2
with mothers Hb (
22) in
placenta.

Mutations in - or -globin genes


can cause disease state
Sickle cell anemia E6 to
V6
Causes V6 to bind to
hydrophobic pocket in
deoxy-Hb
Polymerizes to form long
filaments
Cause sickling of cells
Sickle cell trait offers
advantage against malaria
Fragile sickle cells can not
support parasite

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Chapter 5 (part 2)
Enzyme Kinetics

Rate constants and reaction order


Rate constant (k) measures how rapidly a rxn occurs
k1
A

k-1

B + C

Rate (v, velocity) = (rate constant) (concentration of reactants)


v= k1 [A]
1st order rxn (rate dependent on concentration of 1 reactant)
v= k-1[B][C]
2nd order rxn (rate dependent on concentration of 2 reactants)
Zero order rxn (rate is independent of reactant concentration)

E S

+ S

E+S

k1
k-1

ES

k2
k-2

E+P

Initial Velocities
Hold [E] constant
[E]<<<<<[S]

[P]
D[P]/DT = Vo1 mM

[S] = 1 mM

time

Initial Velocities

[P]/T = Vo10 mM

[P]

D[P]/DT = Vo5 mM

[S] = 10 mM

[S] = 5 mM

D[P]/DT = Vo1 mM

[S] = 1 mM

time

Plot Vo vs. [S]

Vo 10 mM
Vo 5 mM
Vo 1 mM

Michaelis-Menton Equation
Describes rectangular hyperbolic plot
Vo = Vmax [S]
Km + [S]

Vmax = velocity where all of the


enzyme is bound to substrate
(enzyme is saturated with S)

Km = [S] @ Vmax
(units moles/L=M)
(1/2 of enzyme bound to S)

Initial Velocity Assumption


1) Measurements made to measure initial velocity (vo).
At vo very little product formed. Therefore, the
rate at which E + P react to form ES is negligible
and k-2 is 0. Therefore

E S

+ S

E+S

k1
k-1

ES

k2
k-2

E+P

Data from a single experiment


performed with at a single [S].
(single point on Vo vs. [S] plot)

Initial Velocities

[P]/T = Vo10 mM

[P]

D[P]/DT = Vo5 mM

[S] = 10 mM

[S] = 5 mM

D[P]/DT = Vo1 mM

[S] = 1 mM

time

Steady State Assumption


Steady state Assumption = [ES] is constant. The rate
of ES formation equals the rate of ES breakdown

E S

+ S

E+S

k1
k-1

ES

k2

E+P

Rate of ES formation
E

E S

+ S

E+S

k1

ES

Rate = k1 [E] [S]

Rate of ES breakdown
E S
ES

E
k2

ES

k-1

E+P

E S

+ S

E+S

Rate = (k2 [ES]) + (k-1[ES])


Rate = [ES](k2 + k-1)

Thereforeif the rate of ES formation


equals the rate of ES breakdown

1) k1[E][S] = [ES](k-1+ k2)


2) (k-1+ k2) / k1 = [E][S] / [ES]
3) (k-1+ k2) / k1 = Km (Michaelis constant)

What does Km mean?


1. Km = [S] at Vmax
2. Km is a combination of rate constants
describing the formation and breakdown of
the ES complex
3. Km is usually a little higher than the
physiological [S]

What does Km mean?


4. Km represents the amount of substrate
required to bind of the available enzyme
(binding constant of the enzyme for
substrate)
5. Km can be used to evaluate the specificity of
an enzyme for a substrate (if obeys M-M)
6. Small Km means tight binding; high Km means
weak binding
Hexose Kinase
Glucose + ATP <-> Glucose-6-P + ADP

Glucose
Allose
Mannose

Km = 8 X 10-6
Km = 8 X 10-3
Km = 5 X 10-6

What does kcat mean?


1. kcat is the 1st order rate constant describing
ES  E+P
2. Also known as the turnover # because it describes
the number of rxns a molecule of enzyme can
catalyze per second under optimal condition.
3. Most enzyme have kcat values between 102 and 103
s-1
4. For simple reactions k2 = kcat , for multistep rxns
kcat = rate limiting step
E+S

k1
k-1

ES

kcat

E+P

What does kcat/Km mean?


It measures how the enzyme
performs when S is low
kcat/Km describes an enzymes
preference for different
substrates = specificity constant
The upper limit for kcat/Km is the
diffusion limit - the rate at
which E and S diffuse together
(108 to 109 m-1 s-1)
Catalytic perfection when kcat/Km
= diffusion rate
More physiological than kcat

Limitations of M-M
1.

Some enzyme catalyzed rxns show more complex behavior


E + S<->ES<->EZ<->EP<-> E + P
With M-M can look only at rate limiting step

2.

Often more than one substrate


E+S1<->ES1+S2<->ES1S2<->EP1P2<-> EP2+P1<-> E+P2
Must optimize one substrate then calculate kinetic
parameters for the other

3.

Assumes k-2 = 0

4.

Assume steady state conditions

Chapter 5 (part 1)

Enzymes: Introduction

Catalyst

substance that increase rates of a


chemical reaction
does not effect equilibrium
remain unchanged in overall
process
reactants bind to catalyst,
products are released

Catalysts increase product formation by


(1) lowering the energy barrier (activation energy)
for the product to form
(2) increases the favorable orientation of
colliding reactant molecules for product
formation to be successful (stabilize transition
state intermediate)

Catalytic Power
Enzymes can accelerate reactions
as much as 1016 over uncatalyzed
rates!
Urease is a good example:
Catalyzed rate: 3x104/sec
Uncatalyzed rate: 3x10 -10/sec
Ratio is 1x1014 !

Specificity
Enzymes selectively recognize
proper substrates over other
molecules
Enzymes produce products in very
high yields - often much greater
than 95%
Specificity is controlled by
structure - the unique fit of
substrate with enzyme controls the
selectivity for substrate and the
product yield

Classes of enzymes
1. Oxidoreductases = catalyze oxidationreduction reactions (NADH)
2. Transferases = catalyze transfer of functional
groups from one molecule to another.
3. Hydrolases = catalyze hydrolytic cleavage
4. Lyases = catalyze removal of a group from or
addition of a group to a double bond, or other
cleavages involving electron rearrangement.
5. Isomerases = catalyze intramolecular
rearrangement.
6. Ligases = catalyze reactions in which two
molecules are joined.
Enzymes named for the substrates and type of
reaction

Co-enzymes
Non-protein molecules that help enzymes
function
Associate with active site of enzyme
Enzyme + Co-enzyme = holoenzyme
Enzyme alone = apoenzyme
Organic co-enzymes thiamin, riboflavin,
niacin, biotin
Inorganic co-enzymes Mg ++, Fe++,
Zn++, Mn++

Kinetics

study of reaction rate


determines number of steps involved
determines mechanism of reaction
identifies rate-limiting step

Chapter 5 (part 3)
Enzyme Kinetics (cont.)

Michaelis-Menton
E+S

k1

ES

kcat

E+P

k-1

Vo = Vmax [S]
Km + [S]

Vmax
Km

Vmax
Km
Kcat
Kcat/Km

kcat
kcat/Km

How do you get values for Vmax, Km and kcat?


Can determine Km and Vmax experimentally
Km can be determined without an absolutely
pure enzyme
Kcat values can be determined if Vmax is known
and the absolute concentration of enzyme is
known (Vmax = kcat[Etotal]

Lineweaver-Burke Plots
(double reciprocal plots)
Plot 1/[S] vs 1/Vo
L-B equation for straight
line
X-intercept = -1/Km
Y-intercept = 1/Vmax
Easier to extrapolate
values w/ straight line vs
hyperbolic curve

V max
0.25

B
0.2

[S]
0.5
0.75
2
4
6
8
10

B
B
B

Vo

0.15

0.1

B
B

0.05

Km

Km ~ 1.3 mM

0
0

Vo
0.075
0.09
0.152
0.196
0.21
0.214
0.23

5 6
[S]

9 10

Vmax ~ 0.25

14

12

[S]
2.000
1.333
0.500
0.250
0.167
0.125
0.100

10

Vo

BB
B
B

4
2
0

B
-1

-0.5

0.5
[S]

1.5

Vo
13.333
11.111
6.579
5.102
4.762
4.673
4.348

-1/Km = -0.8
Km = 1.23 mM
1/Vmax = 4.0
Vmax = 0.25

Enzyme Inhibition
Inhibitor substance that binds to an enzyme and interferes
with its activity
Can prevent formation of ES complex or prevent ES
breakdown to E + P.
Irreversible and Reversible Inhibitors
Irreversible inhibitor binds to enzyme through covalent
bonds (binds irreversibly)
Reversible Inhibitors bind through non-covalent interactions
(disassociates from enzyme)
Why important?

Reversible Inhibitors
E + S <-> ES -> E + P
E + I <-> EI
Ki = [E][I]/[EI]
Competitive
Uncompetitive
Non-competitive

Types of Reversible Enzyme


Inhibitors

Competitive Inhibitor (CI)

CI binds free enzyme


Competes with substrate for enzyme binding.
Raises Km without effecting Vmax
Can relieve inhibition with more S

Competitive Inhibitors look like substrate


O

HO

NH2

H2N

NH2

PABA

Sulfanilamide

PABA precursor to folic acid in bacteria

O2C-CH2-CH2-CO2 -------> O2C-CH=CH-CO2


succinate

fumarate

Succinate dehydrogenase

O2C-CH2-CO2
Malonate

Uncompetitive Inhibitor (UI)

UI binds ES complex
Prevents ES from proceeding to E + P or back to E + S.
Lowers Km & Vmax, but ratio of Km/Vmax remains the
same
Occurs with multisubstrate enzymes

Non-competitive Inhibitor (NI)

NI can bind free E or ES complex


Lowers Vmax, but Km remains the same
NIs dont bind to S binding site therefore dont effect
Km
Alters conformation of enzyme to effect catalysis but not
substrate binding

Irreversible Inhibitors
CH3
H

F
O

CH3

CH3

CH3

H3C

Diisopropyl fluorophosphate
(nerve gas)

C
S

CH2

CH3

O
H

CH2CH3

malathion
O

CH2CH3

O
S
H3C

NO2

O
CH3

parathion

Organophosphates
Inhibit serine hydrolases
Acetylcholinesterase inhibitors

Viagra

Kinetics of Multisubstrate
Reactions
E + A + B <-> E + P + Q

Sequential Reactions
a) ordered
b) random
Ping-pong Reactions
Cleland Notation

Sequential Reactions
Ordered

EA
A

(EAB)

(EPQ)

EQ
P

E
Q

Random
EA

EQ
(EAB)(EPQ)

EB
B

EP
A

Ping-Pong Reactions
A

(EA)(FP)

(F)

(FB)(EQ)

In Ping-Pong rxns first product released before


second substrate binds
When E binds A, E changes to F
When F binds B, F changes back to E

Lineweaver-Burke Plot of
Multisubstrate Reactions
Sequential

Increasing
[B]

Ping-Pong

Increasing
[B]

1/Vo

1/Vo

1/[S]
Vmax doesnt change
Km changes

1/[S]
Both Vmax & Km change

Chapter 5 (part 4)
Enzyme Regulation

Regulation of Enzyme Activity


Enzyme quantity regulation of gene expression (Response time =
minutes to hours)
a)

Transcription

b)

Translation

c)

Enzyme turnover

Enzyme activity (rapid response time = fraction of seconds)


a)

Allosteric regulation

b)

Covalent modification

c)

Association-disassociation

d)

Proteolytic cleavage of proenzyme

Allosteric Regulation
End products are often inhibitors
Allosteric modulators bind to site other
than the active site
Allosteric enzymes usually have 4o
structure
Vo vs [S] plots give sigmoidal curve for
at least one substrate
Can remove allosteric site without
effecting enzymatic action

Regulation of Enzyme Activity


(biochemical regulation)
1st committed step of a biosynthetic pathway or
enzymes at pathway branch points often regulated
by feedback inhibition.

1 A 2 C
B X

H 4 I
3
3X
E 4 F

Efficient use of biosynthetic precursors and


energy

Phosphofructokinase( PFK)
Fructose-6-P + ATP -----> Fructose
Fructose--1,61,6-bisphosphate + ADP
PFK catalyzes 1st committed step in glycolysis (10 steps
total)
(Glucose + 2ADP + 2 NAD+ + 2Pi  2pyruvate + 2ATP + 2NADH)

Phosphoenolpyruvate is an allosteric inhibitor of PFK


ADP is an allosteric activator of PFK

Allosteric modulators bind to site other


than the active site and allosteric enzymes
have 4o structure
Fructose-6-P + ATP -----> Fructose
Fructose--1,61,6-bisphosphate + ADP

ADP
Allosteric Activator (ADP)
binds distal to active site

Vo vs [S] plots give sigmoidal


curve for at least one substrate

Binding of this allosteric inhibitor or this activator does


not effect Vmax, but does alter Km
Allosteric enzyme do not follow M-M kinetics

Allosteric T to R transition
I
ET-I

S
ET

Concerted
model

ER

ER-S
S

Sequential
model

Covalent modification
Regulation by covalent modification is slower
than allosteric regulation
Reversible
Require one enzyme for activation and one
enzyme for inactivation
Covalent modification freezes enzyme T or R
conformation

Phosphorylation /dephosphorylation
most common covalent
modification
involve protein
kinases/phosphatase
PDK inactivated by
phosphorylation
Amino acids with OH groups are
targets for phosphorylation
Phosphates are bulky (-) charged
groups which effect conformation

Enzyme Regulation by
Association/Disassociation
Acetyl-CoA Carboxylase
acetyl-CoA + CO2 + ATP  malonyl-CoA + ADP + Pi
1St committed step in fatty acid biosynthesis
In presence of citrate activated
In presence of fatty acyl-CoA inactivated
citrate
polymerized
unpolymerized

Fatty acyl-CoA

Proteolytic cleavage of
proenzyme(zymogen)

Proinsulin to Insulin

Blood Clotting
Clotting involves
series of zymogen
activations
Seven clotting
factors are serine
proteases involved in
clotting cascade rxns

X
X
X

X
X
X

Chapter 6
Mechanisms of Enzyme Action

Enzymatic Catalysis
Activation Energy
(AE) The energy
require to reach
transition state from
ground state.
AE barrier must be
exceeded for rxn to
proceed.
Lower AE barrier,
the more stable the
transition state (TS)
The higher [TS], the
move likely the rxn
will proceed.

Enzymatic Catalysis

S  Ts  P

Transition (TS) State Intermediate


Transition state = unstable high-energy
intermediate
Rate of rxn depends on the frequency at which
reactants collide and form the TS
Reactants must be in the correct orientation
and collide with sufficient energy to form TS
Bonds are in the process of being formed and
broken in TS
Short lived (1014 to 10-13 secs)

Intermediates
Intermediates are
stable.
In rxns w/
intermediates, 2
TSs are involved.
The slowest step
(rate determining)
has the highest AE
barrier.

Formation of
intermediate is the
slowest step.

Enzyme binding of substrates decrease activation energy


by increasing the initial ground state (brings reactants
into correct orientation, decrease entropy)
Need to stabilize TS to lower activation energy barrier.

ES complex must not be too stable


Raising the energy of
ES will increase the
catalyzed rate
This is accomplished
by loss of entropy due
to formation of ES and
destabilization of ES by
strain
distortion
desolvation

Transition State Stabilization


Equilibrium between ES <-> TS, enzyme drives
equilibrium towards TS
Enzyme binds more tightly to TS than substrate

Transition
state analog

Mechanistic Strategies

Polar AA Residues in Active Sites

Common types of enzymatic


mechanisms
Substitutions rxns
Bond cleavage rxns
Redox rxns
Acid base catalysis
Covalent catalysis

Substitution Rxns
Nucleophillic Substitution
O

O
R

O
X

+ X

Nucleophillic = e- rich
Electrophillic = e- poor

Direct Substitution
R2

R1
C
X

R3

R2
R1
X C Y
R3

transition state

R2

R1
C
X

+ Y
R3

Oxidation reduction (Redox) Rxns


Loose e- = oxidation (LEO)
Gain e- = reduction (GER)
Central to energy production
If something oxidized something must be
reduced (reducing agent donates e- to
oxidizing agent)
Oxidations = removal of hydrogen or
addition of oxygen or removal of e In biological systems reducing agent is
usually a co-factor (NADH of NADPH)

Cleavage Rxns
Heterolytic vs homolytic cleavage
Carbanion formation (retains both e-)
R3-C-H  R3-C:- + H+
Carbocation formation (lose both e-)
R3-C-H  R3-C+ + H:Hydride ion

Free radical formation (lose single e-)


R1-O-O-R2  R1-O* + *O-R2

Acid-Base Catalysis
X

:B

X:

Accelerates rxn by catalytic transfer of a proton


Involves AA residues that can accept a proton
Can remove proton from OH, -NH, -CH, or XH
Creates a strong nucleophillic reactant (i.e. X:-)

Acid-Base Catalysis
X

:B

X:

carbanion intermediate

OH

HN

OH
H

O
H

:B

:B

Covalent Catalysis

20% of all enzymes employ covalent catalysis


A-X + B + E <-> BX + E + A

A group from a substrate binds covalently to


enzyme
(A-X + E <-> A + X-E)

The intermediate enzyme substrate complex


(A-X) then donates the group (X) to a second
substrate (B)
(B + X-E <-> B-X + E)

Covalent Catalysis
Protein Kinases
ATP + E + Protein <-> ADP + E + Protein-P
1)

A-P-P-P(ATP) + E-OH <-> A-P-P (ADP) + E-O-PO4-

2)

E-O-PO4- + Protein-OH <-> E + Protein-O- PO4-

The Serine Proteases


Trypsin, chymotrypsin, elastase, thrombin,
subtilisin, plasmin, TPA
All involve a serine in catalysis - thus the
name
Ser is part of a "catalytic triad" of Ser,
His, Asp (show over head)
Serine proteases are homologous, but
locations of the three crucial residues
differ somewhat
Substrate specificity determined by binding
pocket

Serine Proteases are structurally


Similar

Chymotrpsin

Trypsin

Elastase

Substrate binding
specificity

Chapter 7 (part 1)
Cofactors

Cofactors
Cofactors are organic or inorganic molecules
that are required for the activity of a certain
conjugated enzymes
Apoenzyme = enzyme (-) cofactor
Holoenzyme = enzyme (+) cofactor
Inorganic cofactors essential ions
Organic cofactors coenzymes

Essential Ion Cofactors


Activator ions bind reversibly to enzyme and
often participate in substrate binding.
Metal ions of metalloenzymes cations that are
tightly bound to enzyme and participate directly
in catalysis (Fe, Zn, Cu, Co).
Metal activated enzymes require or are
stimulated by addition of metal ions (i.e. Mg2+,
is required by many ATP requiring enzymes)

Metal ions can function as


electrophiles in active site

Zinc protease (angiotensin converting enzyme)

Coenzymes
Cosubstrates- altered in rxn and regenerated to original
structure in subsequent rxn
- disassociated from active site
- shuttle chemical groups among different
enzyme rxns.
Prosthetic groups- remains bound to enzyme
- must return to original form
Both cosubstrates and prosthetic groups supply
reactive groups not present on amino acid side
chains

Coenzymes
Metabolite coenzymes synthesized from
common metabolites
Nucleoside triphosphates (ATP) can donate
phosphates, pyrophosphates, adenosyl grroups
S-adenosylmethionine (SAM) donates methyl
groups
Nucleotide sugars (uridine diphosphate glucose =
UDP-glucose) - transfer sugars in carbohydrate
metabolism

Vitamin derived coenzymes


Must be obtained from diet
Synthesized by microorganisms and
plants
Vitamin deficiencies lead to disease
state
Most vitamins must be enzymatically
transformed to function as a
coenzyme

Vitamins
Vitamin
Ascorbic acid (C)
Niacin
Riboflavin (B2)
Thiamin (B1)
Pyridoxal (B6)
Biotin
Folate
Cobalamin (B12)
Vitamin A
Vitamin K
Pantothenate (B3)

Coenzyme
not a coenzyme
NAD(P)+/NAD(P)H
FMN & FAD
Thiamin-pyrophosphate
Pyridoxal phosphate
Biotin
Tetrahydrafolate
adenosyl-and methylcobalamin
Retinal
Vitamin K
Coenzyme A

Niacin (nicotinic acid)


O

C
OH

N
NICOTINIC ACID
(NIACIN)

NH2
N
NICOTINAMIDE

Deficiencies lead to pellagra (dermatitis,


diarrhea, dementia)
Required in relatively high amounts compared to
other vitamins
Not true enzyme because can be synthesized
from tryptophan in the liver

NIC
COTINAMIDE MONOPHOSPHATE

Nicotinamide Coenzymes
H

O
O

NH2

C
OH

NH2

N
N

NICOTINIC ACID
(NIACIN)

H2
C

NICOTINAMIDE

O
PO2-

OH OH
NH2

H
ADENOSINE MONOPHOSPHATE

PO2-

C
NH2

CH2

N
O

OH OH(OPO3)

NH2

OXIDIZED

REDUCED

+
NAD

+
NADP

Serve as cofactors in oxidation/reduction


reactions
Act as co-substrates for dehydrogenases
Reduction of NAD+/NADP+ and oxidation of
NADH/NADPH occurs 2 e- at a time.
Function in hydride ion transfer
Rxns forming NADH/NADPH are catabolic
NADH is coupled with ATP production in
mitochondria
NADPH is an impt reducing agent in
biosynthetic reactions
Reduced forms (NADH/NADPH) absorb light at
340 nm, oxidized forms (NAD+/NADP+) do not

Riboflavin (B2)
Water soluble vitamin
Severe deficiencies lead to
growth retardation,
reproductive problems and
neural degeneration
Meat, dairy products and dark
green vegetables, legumes and
grains are good sources

FMN/FAD

FAD and FMN can transfer


electrons one or two at a time
Quinone
form

Hydroquinone
form

semiquinone form

Thiamin

Thiamin is the first Vitamin discovered (Vital


amine = Vitamin)
Deficiencies lead to disease called Beriberi
(neurological disorders, heart problems, anorexia)
Beriberi prevealent in undeveloped countries where
polished grains make up the majority of the diet.
Associated with alcohol related disorders
(Wernickes-Korskofff syndrome memory loss,
unstable walk)

Thiamin pyrophosphate

Serves as a cofactor in decarboxylation rxn of keto


acids
Also functions as a prosthetic group in
transketolases (catalyze the transfer of two carbon
units in carbohydrate metabolism)

Thiazolium ring is the


chemically active part of TPP

Ylid = a molecule
with opposite
charges on
adjacent atoms

Pyridoxal
OH
H2C

HC
O

HOH2C

NH3

O
H2C
O

HOH2C

CH3

HOH2C

CH3

PYRIDOXINE

PYRIDOXAL

PRYIDOXAMINE

NH3

O
HC

O
O

CH3

H2C

O
O

H2C

H2C

O
N

CH3

H
PYRIDOXAL 5' PHOSPHATE

CH3

H
PYRIDOXAMINE 5' PHOSPHATE

PYRIDOXAL-PHOSPHATE
Important in amino acid metabolism
Bound to enzyme as a Schiff base thru rxn with
lysine
O
- H2O
R

NH2

R2
+ H2O

ALDEHYDE

AMINE

C
H

R2

SCHIFF BASE

PLP functions in transamination, decarboxylation,


racemization, isomerization, side-chain elimination
rxns involving amino acids

PLP in transamination
reaction

PLP in amino acid decarboxylation reaction

Chapter 7 (part 2)
Cofactors

Biotin
Water soluble Vitamin
Produced by gut microflora
which supplies RDA
Deficiencies are rare
Consuming 6 raw eggs a day can
cause deficiencies due to the
presence avidin (biotin binding
protein).

Biotin cofactor

Involved in ATP
dependent
carboxylation
rxns
Covalently bound
to enzyme
through amide
linkage w/lysine
Impt. Biotin
enzymes =
acetyl-CoA
carboxylase,
pyruvate
carboxylase

Folate
PABA

glutamate

pterin

Water soluble vitamin


Folate impt. during pregnancy to prevent neural
tube defects in fetus (I.e. spina bifida)
Vitamin B12 deficiencies cause folate
deficiencies
Has a poly-glutamate tail formed by gammacarboxy and alpha amino groups (unusual peptide
bond).

Tetrahydrofolate (THF)
Folate is converted to THF by the addition of 4
hydrogens to the pterin ring.
Impt. in transfer of one-carbon units
Pterin ring impt. functional group

Can transfer
one carbon units
at the oxidation
level of
methanol,
formaldehyde,
or formic acid.

OH

CH

O
CH2

OH
CH3

Cobalamin (B12)
Water soluble
Vitamin
Corrin ring with
Cobalt cation
Involved in
intramolecular
rearrangements,
methyl group
transfer, reduction of
ribonucleotides to
deoxyribonucleotides.
Forms radical species

Pantothenic acid/Coenzyme A (CoA)


Pantothenic acid is
water soluble
vitamin
Co-enzyme A
involved in acyl
group transfer
Sulfhydroyl group
impt.
Hydrophobic acyl
groups (fatty acids)
are made more
water soluble w/CoA
attached

Lipoic acid/Lipoamide

Not a vitamin
Important reactive groups are the sulfur atoms
Disulfide can be reduced to form 2 sulfhydryl
groups
Involved in acyl group transfer reactions
Co-factor covalently attached to enzyme
through amide linkage with lysine residue

Lipoamide

Fat soluble Vitamins


Vitamin A (retinol) derived from bcarotene impt for vision, regulation of
gene expression during cell
differentiation, teratogenic
Vitamin D impt in Ca absorption,
regulates intestinal absorption and
deposition in bones
Vitamin E antioxidant

Vitamin K is a cofactor for the


enzyme that carboxylates
certain glutamate residues on
prothrombin to carboxyglutamate residues.
Ca+ binds -carboxyglutamate
residues causes protein to
adhere to platelet surface
reduced
form

Only fat soluble cofactor that


functions as a cofactor

Drugs inhibit reduction of


oxidized form of vitamin K

oxidized
form

Ubiquinone/Plastoquinone
Lipid soluble electron carriers.
Impt in electron transport chains
Can accept or donate electrons one
or two at a time

Protein coenzymes
Usually small proteins
Active groups are either prosthetic
groups or part of protein backbone
Participate in group transfer and
oxidation/reduction rxns
acyl carrier protein
biotin carboxyl carrier protein

cytochromes
Protein coenzyme
Heme containing proteins
Fe3+ can undergo
reversible one electron
reduction
Impt in redox rxns
Classified based on the
basis of their visible
absorbance spectra

Chapter 8 (part2)
Carbohydrates: oligo- and
polysaccharides

CH2OH

Glycosidic
Linkage

CH2OH
hemiacetal

OH

OH
OH

OH

OH
OH
alcohol

OH

Hydrolysis
H2O

H2O

Condensation
CH2OH
O

CH2OH
acetal

OH

OH

OH
O

OH
OH

OH
glycosidic linkage

OH

Disaccharides
CH2OH
H

CH2OH

O H
OH

O
OH

OH

maltose

OH
H

OH

OH

O
OH

H
H

OH
H

OH

O H

lactose

CH2OH
H

O OH

CH2OH
OH
H

OH
OH
O

OH

sucrose

OH

CH2OH
O
H
OH

O
H

H
H

CH2OH
OH

OH

H
H

OH

(-D-glucosyl-(1->4)-
-D-glucopyranose)
(

CH2OH

OH

OH

(-D-glucosyl-(1->4)-
-D-glucopyranose)
(

O OH

H
CH2OH

cellobiose

CH2OH

OH

(-D-galactosyl-(1->4)-
-D-glucopyranose)
(

(-D-glucosyl-(1->2)-
-D-fructofuranose)
(

Higher Oligosaccharides

Oligosaccharide groups are incorporated


in to many drug structures

Polysaccharides

Amylose and Amylopectin

Iodine binding to starch

glycogen

Dextrans

Cellulose

Cellulose vs Amylose
amylose

cellulose

Glucose units rotated 180o relative to next residue

Cellulose

Chitin

Chitin vs Cellulose

Glycoproteins

O-linked Glycoproteins

Blood ABO Antigens

Structure of the ABO blood group


carbohydrates,
R represents the linkage to protein
in the secreted forms, sphingolipid
in the cell-surface bound form
open square = GlcNAc, open diamond
= galactose, filled square = fucose,
filled diamond = GalNAc, filled
diamond = sialic acid (NANA)

N-linked Glycoproteins

Chapter 8 (part 1)
Carbohydrates

Carbohydrates
Most abundant class of biological
molecules on Earth
Originally produced through CO2
fixation during photosynthesis

Roles of Carbohydrates
Energy storage (glycogen,starch)
Structural components
(cellulose,chitin)
Cellular recognition
Carbohydrate derivatives include
DNA, RNA, co-factors,
glycoproteins, glycolipids

Carbohydrates
Monosaccharides (simple sugars)
cannot be broken down into simpler
sugars under mild conditions
Oligosaccharides = "a few" - usually
2 to 10
Polysaccharides are polymers of the
simple sugars

Monosaccharides
Polyhydroxy ketones (ketoses)
and aldehydes (aldoses)
Aldoses and ketoses contain
aldehyde and ketone
functions, respectively
Ketose named for equivalent
aldose + ul inserted
Triose, tetrose, etc. denotes
number of carbons
Empirical formula = (CH2O)n

H
C

CH2OH

C*

OH

HO

C*

C*

OH

CH2OH
D-ribose

HO

C*

C*

OH

CH2OH
D-ribulose

Monosaccharides are chiral


Aldoses with 3C or more and
ketoses with 4C or more are
chiral
The number of chiral carbons
present in a ketose is always
one less than the number
found in the same length
aldose
Number of possible
steroisomers = 2n (n = the
number of chiral carbons)

H
C

CH2OH

C*

OH

HO

C*

C*

C*

HO

C*

OH

C*

OH

OH

C*

OH

CH2OH

CH2OH

D-glucose

D-fructose

Stereochemistry
Enantiomers
O

Epimers

Diastereomers
O

H
C

OH

HO

C*

HO

C*

HO

C*

C*

C*

OH

C*

OH

C*

OH

C*

OH

C*

OH

HO

C*

C*

OH

HO

C*

HO

C*

HO

C*

C*

OH

C*

OH

HO

C*

OH

C*

OH

C*

OH

HO

C*

HO

C*

CH2OH

CH2OH

L-glucose

D-glucose

C*

OH

C*

C*

H
C

HO

CH2OH

CH2OH

D-mannose

D-galactose

CH2OH
D-glucose

CH2OH
D-mannose

Enantiomers = mirror images


Pairs of isomers that have opposite configurations at
one or more chiral centers but are NOT mirror images
are diastereomers
Epimers = Two sugars that differ in configuration at
only one chiral center

Cyclization of aldose and ketoses


introduces additional chiral center
Aldose sugars (glucose) can cyclize to form a
H
cyclic hemiacetal H O ALDEHYDE
NEW CHIRAL
O
CARBON

C
H

C*

R1

R1
O

ALCOHOL

R2

O
H

R2

HEMIACETAL

Ketose sugars (fructose) can cyclize to form a


H
H
cyclic hemiketal
NEW CHIRAL
KETONE
O
O
CARBON

C
R

C*

R1

R1
O

ALCOHOL

R2

O
H

R2

HEMIKETAL

Haworth Projections
O

-OH up = beta
-OH down = alpha

C1
H

C2

OH

HO

C3

C4

OH

C5

OH

6
5
4

1
2
3

CH2OH

Anomeric carbon
(most oxidized)

For all non-anomeric carbons, -OH groups


point down in Haworth projections if
pointing right in Fischer projections

Monosaccharides can cyclize to


form Pyranose / Furanose forms
= 64%
= 36%

= 21.5%
= 58.5%

= 13.5%
= 6.5%

Conformation of Monosaccharides

Pyranose sugars not planar molecules, prefer to be in


either of the two chair conformations.

Reducing Sugars
When in the uncyclized
form, monosaccharides
act as reducing agents.
Free carbonyl group from
aldoses or ketoses can
reduce Cu2+ and Ag+ ions
to insoluble products

Derivatives of
Monosaccharides

Sugar Phosphates

Deoxy Acids

Amino Sugars

Sugar alcohols

Monosaccharide structures
you need to know

Glucose
Fructose
Ribulose
Glyceraldehyde
Dihydroxyacetone

Chapter 9 (part 1)
Lipids and Membranes

Lipid Subclasses

Fatty
acids

Fatty acid nomenclature

C15

C13

C11

C9

C7

C5

C3

C1

C6

C4

C2

C18

C16

C14

C12

C10

C8

C17

HO

O
C1
HO

C6

C4

C2
C3

C5

C7

C9

C11

C13

C18

C16

C14

C12

C10

C8

C
H15

C17

Common saturated fatty


acids
common name

IUPAC name

melting point (Co)

12:0

laurate

dodeconoate

44

14:0

myristate

tetradeconoate

52

16:0

palmitate

hexadeconoate

63

18:0

stearate

octadeconoate

70

20:0

arachidate

eicosanoate

75

22:0

behenate

docosanoate

81

24:0

lignocerate

tetracosanate

84

Common unsaturated fatty


acids
common name

IUPAC name

melting
point
(Co)

16:0

palmitate

hexadeconoate

63

16:1 9

palmitoleate

cis-
9-hexadeconoate

-0.5

18:0

stearate

octadeconoate

70

18:1 9

oleate

cis-
9- octadeconoate

13

18:2 9,12

linoleate

cis-
9,12- octadeconoate

-9

18:3 9,12,15

linolenate

cis-
9,12,15- octadeconoate

-17

20:0

arachidate

eicosanoate

75

20:4 5,8,11,14

arachindonate

cis- 5,8,11,14-eicosatetraenoate

-49

18:0

18:1

18:3

Physical
Properties
of Fatty
acids

70o

13o

-17o

Unusual fatty acids can function


analogously to unsaturated fatty acids

Phospholipids

Common membrane phospholipids


CH3

H2C
O

R1

R2

Phophtidate

NH3

CH2

CH2

CH2

CH2

CH2

CH2

H2C

H
C

R1

R2

CH2

Phophatidylethanolamine

H2C

H
C

R1

R2

Phophatidylserine

P
O

CH2

CH3

CH2

COO

O
H
C

H3C

NH3

H2C

H
C

R1

R2

CH2

Phophatidylcholine

Enzymes used to Dissect


Phospholipid Structure
X
O
phospholipase D
O

O
phospholipase C

O
H2C

H
C

CH2

phospholipase A1

phospholipase A2
O

R1

R2

Chapter 9 (part 2)
Lipids and Membranes

H2 C

H
C

CH2

C1
C2

C2

C2
C3

C3

C3
C4

C4

C4
C5

Triacylglycerol
(TAG)

C5

C5
C6

C6

C6
C7

C7

C7
C8

C8

C8
C9

C9

C9
C10
C10

C10

C11

C11

C11
C12
C12

C12

C13

C13

C13
C14
C14

C14

C15

C15

C15
C16
C16
C17
C17
C18
C18

C16
C17
C18

Olestra

TAG

Head

Tail

cholesterol

Steroids

Many steroids are derived


from cholesterol

The Fluid Mosaic Model

fusion

After 40 minutes

Membranes are Asymmetric

Membrane Phase Transitions

Phase
Transitions

Chapter 9 (part 3)
Membranes

Channels
and Pores

Saturation Kinetics of transport

Na+-K+
ATPase

1o active transport of Na+

2o active transport of
glucose

Chapter 10
Introduction to Metabolism

Mutienzyme complex

Separate
enzymes

Membrane
Bound System

Organization of Pathways
Closed Loop
(intermediates recycled)

Linear
(product of rxns
are substrates for
subsequent rxns)

Spiral
(same set of
enzymes used
repeatedly)

Metabolism
Proceeds in
Discrete
Steps

Enzyme Regulation of Flux


Common mechanisms

Feedforward activation

ATP

Phosphoryl-group Transfer

Chapter 11 (Part 1)
Glycolysis

Glycolysis

Anaeorbic process
Converts hexose to two pyruvates
Generates 2 ATP and 2 NADH
For certain cells in the brain and eye, glycolysis is the
only ATP generating pathway

Glucose+2ADP+2NAD++2Pi -> 2pyruvate+2ATP+2NADH+2H++2H20

Glycolysis
Essentially all cells carry out glycolysis
Ten reactions - same in all cells - but
rates differ
Two phases:
First phase converts glucose to two G-3-P
Second phase produces two pyruvates
Products are pyruvate, ATP and NADH
Three possible fates for pyruvate

Phase I: Cleavage of 1 hexose


to 2 triose

Phase II:
Generation
of 2 ATPs,
2 NADH and
2 Pyruvates

Hexose Kinase
1st step in glycolysis; G large, negative
This is a priming reaction - ATP is
consumed here in order to get more
later
ATP makes the phosphorylation of
glucose spontaneous

Hexokinase also functions in other processes


Not 1st committed
step in glycolysis
Glucose import

Directing glucose to
other pathways

Different Hexokinase Isozymes


Two major forms hexokinase (all cells) &
glucokinase (liver)
Km for hexokinase is 10-6 to 10-4 M; cell
has 4 X 10-3 M glucose
Km for glucokinase is 10-2 M only turns
on when cell is rich in glucose
Glucokinase functions when glucose levels
are high to sequester glucose in the
liver.
Hexokinase is regulated - allosterically
inhibited by (product) glucose-6-P

Rx 2: Phosphoglucoisomerase
Uses open chain structure as substrate
Near-equilibrium rxn (reversible)
Enzyme is highly stereospecific (doesnt
work with epimers of glucose-6-phosphate

Rx 2: Phosphoglucoisomerase
Why does this reaction occur??
next step (phosphorylation at C-1)
would be tough for hemiacetal -OH,
but easy for primary -OH
isomerization activates C-3 for
cleavage in aldolase reaction

Rx 3: Phosphofructokinase
PFK is the committed step in glycolysis!
The second priming reaction of glycolysis
Committed step and large, -
G means PFK is
highly regulated
-D-fructose-6-phosphate is substrate for rxn

Phosphofructokinase is highly regulated


ATP inhibits, AMP reverses inhibition
Citrate is also an allosteric inhibitor
Fructose-2,6-bisphosphate is allosteric
activator
PFK increases activity when energy
status is low
PFK decreases activity when energy
status is high

Rx 4: Aldolase

Hexose cleaved to form two trioses


C1 thru C3 of F1,6-BP -> DHAP
C4 thru C6 -> G-3-P
Near-equilibrium rxn
Position of carbonyl group determines which
bond cleaved.
If Glucose-6 P was the substrate would end up
with 2 carbon and 4 carbon product

Rx 5: Triose Phosphate Isomerase (TPI)


Near equilibrium rxn
Conversion of DHAP to G-3-P by TPI
maintains steady state [G-3-P]
Triose phosphate isomerase is a nearperfect enzyme (Kcat/Km near diffusion
limit

Rx 5: Triose Phosphate Isomerase (TPI)


O

DHAP

H
C1
H

C1H2OPO3

C2

OH

C3

C4

OH

C4

OH

C5

OH

C5

OH

HO

HO

C2

C3

-2

HO

C3
C2

D-glucose

F 1,6-BP

Aldolase

O
H

C1H2OPO3

C6H2OPO3-2

TPI

C3

C6H2OH

G-3-P

O
C4

C5

OH

C6H2OPO3-2

G-3-P

C2

OH

-2

C1H2OPO3-2

Glycolysis - Second Phase


Metabolic energy produces 4 ATP
Net ATP yield for glycolysis is two
ATP
Second phase involves two very high
energy phosphate intermediates

1,3 BPG
Phosphoenolpyruvate

Phase II:
Generation
of 2 ATPs,
2 NADH and
2 Pyruvates

Rx 6: Glyceraldehyde-3P-Dehydrogenase

G3P is oxidized and phosphorylated to 1,3-BPG


Near equilibrium rxn
Pi is used as phosphate donor
C1 phosphoryl group has high group transfer
potential, used to phosphorylate Adp to ATP in next
step of glycolysis
Arsenate can replace phosphate in rxn (results in
lower ATP)
NADH generated in this reaction is reoxidized by
respiratory electron transport chain (generates ATP)

Rx 7: Phosphoglycerate Kinase (PGK)


ATP synthesis from a high-energy phosphate
This is referred to as "substrate-level
phosphorylation"
Although has large negative Go (-18 kJ/mole)
because PGK operates at equilibrium in vivo, the
overall G is 0.1 Kj/mole and is a near-equilibrium
rxn.
2,3-BPG (for hemoglobin) is made by circumventing
the PGK reaction

2,3-BPG (for hemoglobin) is made


by circumventing the PGK

reaction

2,3-BPG acts to maintain Hb in low oxygen


affinity form
RBC contain high levels of 2,3 BPG (4 to 5 mM)

Rx 8: Phosphoglycerate
Mutase
Phosphoryl group moves from C-3 to C-2
Mutases are isomerases that transfer
phosphates from one hydroxyl to another
Involves phosphate-histidine intermediate

Rx 9: Enolase
Near equilibrium rxn
"Energy content" of 2-PG and PEP are similar
Enolase just rearranges to a form from which
more energy can be released in hydrolysis
Requires Mg2+ for activity, one bings Carboxyl
group of substrate the other involved in
catalysis.

Rx 10: Pyruvate Kinase


Substrate level phosphorylation
generates second ATP
Large, negative G - regulation!
Allosterically activated by AMP, F-1,6bisP
Allosterically inhibited by ATP and
acetyl-CoA

Chapter 11 (part 2)
Regulation of Glycolysis

Lactate formation
NADH

NAD

OH

O
H3C

H3C

C
O
NADH

NAD

C
H

C
O

Cori
Cycle

Alcohol Fermentation

Free Energy Change in Glycolysis

Hexokinase
Phosphofructokinase-1
Pyruvate kinase

Control Points
in Glycolysis

Insulin Induced Exocytosis of


Glucose Transporter

Effect of ATP on PFK-1


Activity

Effect of ADP and AMP on PFK-1 Activity

Regulation of PFK by
Fructose-2,6-bisphosphate

Glucagon
Regulation of
PFK-1 in Liver

Pyruvate Kinase Regulation

Other
Sugars can
enter
glycolysis

Chapter 12 (part 1)
Citric Acid Cycle

Gylcolysis

Electron Transport and


Oxidative phosphorylation

TCA Cycle

Entry into the TCA Cycle

Pyruvate Dehydrogenase

Citrate Synthase
Only step in TCA cycle that involves the
formation of a C-C bond

Aconitase

Isocitrate Dehydrogenase

-Ketoglutarate Dehydrogenase

Succinyl-CoA Synthetase

Fumarase

Malate Dehydrogenase

Reduced Coenzymes Fuel ATP


Production
Acetyl-CoA + 3 NAD+ + Q + GDP + Pi +2 H20 
HS-CoA + 3NADH + QH2 + GTP + 2 CO2 + 2 H+

Isocitrate Dehydrogenase
-ketoglutarate dehydrogenase
Succinyl-CoA synthetase
Sunccinate dehydrogenase
Malate Dehydrogenase

1 NADH=2.5 ATP
1 NADH=2.5 ATP
1 GTP=1 ATP
1 QH2=1.5 ATP
1 NADH=2.5 ATP

Total of 10 ATPs gained from oxidation of 1 AcetylCoA

Regulation
of TCA
Cycle

Protein/amino acid
Catabolites feed
Into the TCA Cycle

Fats breakdown
and feed
into the TCA Cycle

TCA Cycle provides


intermediates for
many biosynthetic
processes

The Anaplerotic Reactions


The "filling up" reactions
PEP carboxylase - converts PEP to oxaloacetate
Pyruvate carboxylase - converts pyruvate to
oxaloacetate
Malic enzyme converts pyruvate into malate

Following the carbons


through the TCA cycle

Chapter 13 (part 1)
Additional Pathways in
Carbohydrate Metabolism

Hormonal Regulation of Glycogen


Metabolism

Effect of glucagon and epinephrine on glycogen


phosphorylase glycogen synthase activities

Effect of insulin on glycogen phosphorylase


glycogen synthase activities

Pyruvate Carboxylase

PEP Carboxykinase

Fructose-1,6-bisphosphatase

Glucose-6-Phosphatase

Oxidative Stage

Non-oxidative
Stage

Chapter 14 (Part 1)
Electron transport

Standard reduction potentials


of the major respiratory
electron carriers.

Complex I
NADH + H+
NAD+

FMN

Fe2+S

CoQ

FMNH2

Fe3+S

CoQH2

Complex II

Succinate
Fumarate

FAD
FADH2

Fe2+S
Fe3+S

CoQ
CoQH2

QCycle
UQ

UQ.-

UQH2

Complex III

CoQH2
CoQ

cyt b ox
cyt b red

Fe2+S
Fe3+S

cyt c1 ox
cyt c1 red

cyt c red
cyt c ox

Complex IV
cyt c red
cyt c ox

cyt a ox
cyt a red

cyt a3 red
cyt a3 ox

O2
2 H2O

Glycerol phosphate shuttle

malate/aspartate shuttle system

Electron transport is coupled to


oxidative phosphorylation

NO2

O2N

OH

NO2

O2N

Chapter 14 (part 2)
Oxidative phosphorylation

ATP Synthase

Racker & Stoeckenius


confirmed Mitchells
hypothesis using vesicles
containing the ATP
synthase and
bacteriorhodopsin

Binding Change Mechanism

ATPase is a Rotating Motor

How does proton flow cause


rotation?

Transport of ATP, ADP and Pi

NO2

O2N

OH

NO2

O2N

Chapter 15 (part1)
Photosynthesis

Conversion of Light Energy to


Chemical Energy

Chlorophyll

Accessory Pigments
Carotenoid

Phycobilin

Absorption Spectra of Major


Photosynthetic Pigments

Harvesting of Light and Transfer


of Energy to Photosystems

Resonance
Transfer

Photosynthetic ETC

Z-Scheme

P680(PSII) to PQ Pool

Excitation, Oxidation and


Re-reduction of P680

Oxygen
evolution by
PSII

Crystal structure of
Photosystem II from

Thermosynechococcu
s elongatus at 3.5
angstrom resolution
Kristina N. Ferreira, Tina M.
Iverson, Karim Maghlaoui, James
Barber, and So Iwata
Science 19 March 2004: 1831-1838.

Kristina N. Ferreira, Tina M. Iverson, Karim Maghlaoui, James Barber, and So Iwata
Science 19 March 2004: 1831-1838.

Electrons are
passed from
Pheophytin to
Plastoquinone

Transfer of efrom PQH2 to


Cytbf Complex
(another Qcycle)

Chapter 15 (part 2)
Photophosphorylation

Chloroplast
CF1CFo
ATPase

Non-cyclic photosynthetic ETC

cyclic photosynthetic ETC

Arrangement of photosystems in
thylakoid membrane

Chapter 15 (part 3)
Carbon Fixation (dark
reactions)

Carbon Dioxide Fixation

Reductive Pentose Phosphate Cycle

6CO2+9ATP+5H20  9ADP+8Pi+6NADP++(DHAP or G3P)

Ribulose-1,5-Bisphosphate
carboxylase/oxygenase (rubisco)

Activation of
Rubisco

Mg2+ plays role in binding and


activating R 1,6-BP to accept CO2

Rubisco Rxn Mechanisms


carboxylase

oxygenase

Reductive Pentose Phosphate Cycle

cytosol

Reduction
Stage
F 1,6-bisphophatase
aldolase

Glyceraldehyde
dehydrogenase

Phosphoglycerate
kinase

Reductive Pentose Phosphate Cycle

Transketolases and Aldolases are


used to make 5 carbon sugars

Formation of 5 Carbon Sugars

F-6-P + 2 G3P + DHAP + 3 ATP  3 R-1,5-BP + 3 ADP

Regulation
of RPP
Cycle

Thioredoxin

Oxygenase Activity of Rubisco

Photorespiration

C4 Photosynthesis

CAM Photosynthesis

stoma

Chapter 16 (Part 1)
Lipid Absorption and
Mobilization

Lipoproteins

LDL Receptor

Plaque Build up in Artery

Energy Reserves of a 150 lb Man


100,000 kcal of TAG, 25,000 kcal
protein, 600 kcal glycogen, 40 kcal
glucose.
24 lbs of body weight is TAG
Would need 121lbs of glycogen to
store the same amount of energy

Absorption and Mobilization of TAG

Bile Salt

Storage of FAT

Mobilization of Fat

Mobilization of Fat

Chapter 16 (Part 2)
Fatty acid Catabolism
(
-oxidation)

Import of acyl-CoA into mitochondria

-oxidation

-oxidation
B-oxidation of palmitate (C16:0) yields 106 molecules
of ATP
C 16:0-CoA + 7 FAD + 7 NAD+ + 7 H20 + 7 CoA 
8 acetyl-CoA + 7 FADH2 + 7 NADH + 7 H+
2.5 ATPs per NADH = 17.5
1.5 ATPs per FADH2 = 10.5
10 ATPs per acetyl-CoA = 80
Total = 108 ATPs
2 ATP equivalents (ATP
 AMP + PPi, PPi  2 Pi)
consumed during activation of palmitate to acyl-CoA
Net yield = 106 ATPs

Acyl-CoA Dehydrogenase

-oxidation of
odd
chain fatty acids

-oxidation of unsaturated fatty acids

-oxidation of fatty acids with even


numbered double bonds

Formation of
ketone bodies

Re-utilization
of
ketone bodies

Chapter 16 (Part 3)
Fatty acid Synthesis

ACP vs. Coenzyme A

Fatty Acid Synthesis Occurs in


the Cytosol

Citrate synthase

Citrate Lyase
Malate
dehydrogenase

Pyruvate
carboxylase

Malate Enzyme

Acetyl-CoA Carboxylase
Acetyl-CoA + HCO3- + ATP  malonyl-CoA + ADP

Acetyl-CoA
Carboxylase

Regulation of Acetyl-CoA
Carboxylase (ACCase)

Animal Fatty Acid Synthase

Further Processing
of Fatty acids:
Desaturation and
Elongation

Allosteric regulation
of fatty acid
synthesis occurs at
ACCase and the
carnitine
acyltransferase

Chapter 16 (Part 4)
Synthesis of Eicosanoids,
Glycerolipids and Isoprenoids

Eicosanoids

Eicosanoid Synthesis

cyclooxygenase

Glycerolipid
Biosynthesis

Serine
NH2

H2
C

HO

NH2

CH

R1

CH2

R2

CH
O

CMP

H2C

O
O

O-

OH

OH

CDP-DAG
O
C

CH2

R2

CH

O
R1

CH2

R2

CH
O

O
O

O
H2 C

CMP

O-

R1

Inositiol

OH

H2
C

NH2

CH

H2C

OH

OH
OH

OH

O
H

OH

O-

OH

phosphatidylserine

OH
H

phosphatidylinositol

Phosphomevalonate
kinase

Mevalonate
kinase

Formation of the isopentenyl pyrophosphate (IPP)

pyrohosphomevalonate
decarboxylase

Two Fates of HMG-CoA

Condensation of IPP into


Polyprenyl Compounds
IPP
isomerase

Dimethylallyl
pryophosphate

Cholesterol
Synthesis
from IPP

IPP Isomerase
prenyltransferase

prenyltransferase

Squalene synthase

Squalene
monooxygenase

2,3-oxidosqualene
lanosterol cyclase

20 steps

cholesterol

Chapter 17 (Part 1)
Amino Acid Metabolism:
Nitrogen Assimilation
and Amino Acid Biosynthesis

Nitrogen Cycle

Nitrogenase

Nitrate and Nitrite to Ammonia

Ammonia Assimilation

Glutamate Dehydrogenase

Glutamine Synthetase (GS)

Glutamine back to Glutamate

Allosteric
Modulation of
GS Acitivity
in Bacteria

Regulation of
Bacterial GS
by Covalent
Modification

Glutamate serves as primary N- donor for AA


synthesis through transamination reactions

Transamination rxns involve


Pyridoxol-phosphate and
formation of Schiff Base

Essential vs. Non-Essential AAs

Amino Acid Biosynthesis is


a Diverse Process

Chapter 17 (part 2)
Protein Turnover and Amino
Acid Catabolism

Dietary Protein Turnover

Protein Turnover Rates Vary

Ubiquitin Related Protein


Degradation

Protein Ubiquitination

Multiple Ubiquitins can be polymerized to each other.

What determines whether a


protein will become ubiquinated?

Ubiquitinated Proteins are


Degraded by the 26S Proteosome

26S Proteosome

Removal of nitrogen

transaminase

deaminase

Mechanisms to get rid of Ammonia


O
H
N

HN

H2N

O
C O

H2N

Urea

N
H

N
H

Uric Acid

Urea Cycle

Urea Cycle

H2N
C O
H2N

Step 1: Formation of Carbamoyl


Phosphate

Step 2: Ornithine Transcarbamyolase

Step 3: Argininosuccinate
Synthetase

Step 4: Argininosuccinase
Cytosolic enzyme

Step 5: Arginase

Urea
Cycle

Glucose-Alanine Cycle

Catabolism of Carbon
Chains From Amino Acids

Chapter 19 (part 1)
Nucleic Acids

Nucleic Acids Are Essential For


Information Transfer in Cells
Information encoded in a DNA
molecule is transcribed via synthesis
of an RNA molecule
The sequence of the RNA molecule
is "read" and is translated into the
sequence of amino acids in a
protein.

Central Dogma of Biology

Nucleic Acids
First discovered in 1869 by Miescher.
Found as a precipitate that formed when
extracts from nuclei were treated with
acid.
Compound contained C, N, O, and high
amount of P.
Was an acid compound found in nuclei
therefore named nucleic acid

Nucleic Acids
1944 Oswald, Avery, MacLeod and
McCarty demonstrated that DNA is
the molecule that carrier genetic
information.
1953 Watson and Crick proposed
the double helix model for the
structure of DNA

Nucleic Acids
Nucleic acids are long polymers of
nucleotides.
Nucleotides contain a 5 carbon sugar, a
weakly basic nitrogenous compound
(base), one or more phosphate groups.
Nucleosides are similar to nucleotides
but have no phosphate groups.

Pentoses of Nucleotides
D-ribose (in RNA)
2-deoxy-D-ribose (in
DNA)
The difference - 2'OH vs 2'-H
This difference affects
secondary structure
and stability

Nitrogenous Bases

Bases are attached by -Nglycosidic linkages to 1 carbon of


pentose sugar (Nucleoside)

Nucleosides
Base is linked via a -Nglycosidic bond
The carbon of the glycosidic
bond is anomeric
Named by adding -idine to
the root name of a pyrimidine
or -osine to the root name of
a purine
Conformation can be syn or
anti
Sugars make nucleosides more
water-soluble than free
bases

Anti- conformation
predominates in nucleic acid
polymers

Nucleotides
Phosphate ester of nucleosides

The plane of the base is oriented


perpendicular to the plane of the
pentose group

Other Functions of
Nucleotides

Nucleoside 5'-triphosphates are


carriers of energy
Bases serve as recognition units
Cyclic nucleotides are signal molecules
and regulators of cellular metabolism
and reproduction
ATP is central to energy metabolism
GTP drives protein synthesis
CTP drives lipid synthesis
UTP drives carbohydrate metabolism

Nucleotide monomers are joined by 3-5


phosphodiester linkages to form nucleic acid
(polynucleotide) polymers

Nucleic Acids
Nucleic acid backbone takes on
extended conformation.
Nucleotide residues are all oriented
in the same direction (5 to 3)
giving the polymer directionality.
The sequence of DNA molecules is
always read in the 5 to 3 direction

Bases from two adjacent DNA


strands can hydrogen bond
Guanine pairs with
cytosine
Adenine pairs with
thymine

Base pairing evident in


DNA compositions

H-bonding of adjacent antiparallel


DNA strands form double helix
structure

Properties of DNA Double Helix


Distance between the 2 sugar-phosphate backbones
is always the same, give DNA molecule a regular
shape.
Plane of bases are oriented perpendicular to
backbone
Hydrophillic sugar phosphate backbone winds around
outside of helix
Noncovalent interactions between upper and lower
surfaces of base-pairs (stacking) forms a closely
packed hydrophobic interior.
Hydrophobic environment makes H-bonding between
bases stronger (no competition with water)
Cause the sugar-phosphate backbone to twist.

View down the Double Helix


Hydrophobic
Interior with base
pair stacking

Sugar-phosphate
backbone

Structure of
DNA Double
Helix
Right handed helix
Rise = 0.33
nm/nucleotide
Pitch = 3.4 nm /
turn
10.4 nucleotides
per turn
Two groves major
and minor

Within groves,
functional groups on
the edge of base
pairs exposed to
exterior
involved in
interaction with
proteins.

Factors stabilizing DNA


double Helix
Hydrophobic interactions burying
hydrophobic purine and pyrimidine rings
in interior
Stacking interactions van der Waals
interactions between stacked bases.
Hydrogen Bonding H-bonding between
bases
Charge-Charge Interactions
Electrostatic repulsions of negatively
charged phosphate groups are minimized
by interaction with cations (e.g. Mg2+)

Chapter 19 (part 2)
Nucleic Acids

DNA
1o Structure - Linear array of
nucleotides
2o Structure double helix
3o Structure - Super-coiling, stemloop formation
4o Structure Packaging into
chromatin

Determination of the DNA 1o


Structure (DNA Sequencing)
Can determine the sequence of DNA
base pairs in any DNA molecule
Chain-termination method developed
by Sanger
Involves in vitro replication of
target DNA
Technology led to the sequencing of
the human genome

DNA Replication
DNA is a double-helical molecule
Each strand of the helix must be copied in
complementary fashion by DNA polymerase
Each strand is a template for copying
DNA polymerase requires template and
primer
Primer: an oligonucleotide that pairs with
the end of the template molecule to form
dsDNA
DNA polymerases add nucleotides in 5'-3'
direction

Chain Termination Method


Based on DNA polymerase reaction
4 separate rxns
Each reaction mixture contains dATP, dGTP,
dCTP and dTTP
Each reaction also contains a small amount of
one dideoxynucleotide (ddATP, ddGTP, ddCTP
and ddTTP).
Each of the 4 dideoxynucleotides are labeled
with a different fluorescent dye.
Dideoxynucleotides missing 3-OH group. Once
incorporated into the DNA chain, chain
elongation stops)

Chain Termination Method


Most of the time, the polymerase uses
normal nucleotides and DNA molecules
grow normally
Occasionally, the polymerase uses a
dideoxynucleotide, which adds to the
chain and then prevents further growth
in that molecule
Random insertion of dd-nucleotides
leaves (optimally) at least a few chains
terminated at every occurrence of a
given nucleotide

O
O
N
NH
N
NH
N

NH2

N
HO

NH2

O
H

NH2

HO

NH2

O
N

H
O

H
N

H
H

O-

H
H

N
O

O-

O
O
H

O
O

H
N

H
OH

NH
O
P

OH

OH
O

NH

PH

O-

O-

NH2
O
O

O
H
O

H
OH

OH

OH

NH2

O
N
NH

NH2

HO

NH2

O
H

H
N

H
O
O

H
H

O-

NO CHAIN
ELONGATION

O
O
H

H
H
N

NH
OH

OH
O

PH

O-

O
O

OH

OH

H
O

NH2

Chain Termination Method


Run each reaction mixture on electrophoresis gel
Short fragments go to bottom, long fragments
on top
Read the "sequence" from bottom of gel to top
Convert this "sequence" to the complementary
sequence
Now read from the other end and you have the
sequence you wanted - read 5' to 3'

DNA Secondary structure


DNA is double stranded with
antiparallel strands
Right hand double helix
Three different helical forms (A, B
and Z DNA.

Comparison of A, B, Z DNA
A: right-handed, short and broad, 2.3 A,
11 bp per turn
B: right-handed, longer, thinner, 3.32 A,
10 bp per turn
Z: left-handed, longest, thinnest, 3.8 A,
12 bp per turn

A-DNA

B-DNA

Z-DNA

Z-DNA
Found in G:Crich regions of
DNA
G goes to syn
conformation
C stays anti
but whole C
nucleoside
(base and
sugar) flips
180 degrees

DNA sequence Determines Melting Point


Double Strand DNA can be
denatured by heat (get strand
separation)
Can determine degree of
denturation by measuring
absorbance at 260 nm.
Conjugated double bonds in
bases absorb light at 260 nm.
Base stacking causes less
absorbance.
Increased single strandedness
causes increase in absorbance

DNA sequence Determines Melting Point


Melting
temperature
related to G:C and
A:T content.
3 H-bonds of G:C
pair require higher
temperatures to
denture than 2 Hbonds of A:T pair.

DNA

o
3

Structure

Super coiling
Cruciform structures

Supercoils
In duplex DNA, ten bp per turn of helix (relaxed
form)
DNA helix can be over-wound.
Over winding of DNA helix can be compensated by
supercoiling.
Supercoiling prevalent in circular DNA molecules
and within local regions of long linear DNA strands
Enzymes called topoisomerases or gyrases can
introduce or remove supercoils
In vivo most DNA is negatively supercoiled.
Therefore, it is easy to unwind short regions of
the molecule to allow access for enzymes

Each super coil compensates for one + or turn of


the double helix

Cruciforms occur in
palindromic regions of DNA
Can form intrachain base
pairing
Negative supercoiling may
promote cruciforms

DNA and Nanotechnology

DNA and Nanotechnology

DNA

o
4

Structure

In chromosomes, DNA is tightly


associated with proteins

Chromosome Structure
Human DNAs total length is ~2 meters!
This must be packaged into a nucleus
that is about 5 micrometers in diameter
This represents a compression of more
than 100,000!
It is made possible by wrapping the DNA
around protein spools called nucleosomes
and then packing these in helical
filaments

Nucleosome Structure
Chromatin, the nucleoprotein
complex, consists of histones and
nonhistone chromosomal proteins
% major histone proteins: H1, H2A,
H2B, H3 and H4
Histone octamers are major part of
the protein spools
Nonhistone proteins are regulators
of gene expression

4 major histone (H2A,


H2B, H3, H4) proteins
for octomer
200 base pair long
DNA strand winds
around the octomer
146 base pair DNA
spacer separates
individual nucleosomes
H1 protein involved in
higher-order chromatin
structure.
W/O H1, Chromatin
looks like beads on
string

Solenoid Structure of Chromatin

RNA
Single stranded molecule
Chemically less stable than DNA
presence of 2-OH makes RNA more susceptible
to hydrolytic attack (especially form bases)
Prone to degradation by Ribonucleases (Rnases)
Has secondary structure. Can form intrachain
base pairing (i.e.cruciform structures).
Multiple functions

Type of RNA
Ribosomal RNA (rRNA) integral part of
ribosomes (very abundant)
Transfer RNA (tRNA) carries activated
amino acids to ribosomes.
Messenger RNA (mRNA) endcodes
sequences of amino acids in proteins.
Catalytic RNA (Ribozymes) catalzye
cleavage of specific RNA species.

RNA can have extensive 2o


structure

Chapter 20
DNA Replication and Repair

Replication is bidirectional

DNA Replication is
Semidiscontinuous

Okazaki Fragments

DNA Polymerase III is a


Multisubunit Enzyme

DNA Polymerase III Subunit


Organization

DNA
Replication is
a Processive
Process.

Stages of DNA Replication


Initiation
Elongation
Termination

PCNA analogous to E. coli bsubunit of E. coli DNA


polymerase

Repair of UV
Induced Thymine
Dimers

General excisionrepair pathway

Repair of
damage
resulting from
the
deamination
of cytosine

Chapter 21 (part 1)
Transcription

Central Dogma

RNA Content of E. coli


Cells
type

Steady
State
Levels

Synthetic
Capacity

Stability

rRNA

83%

58%

High

tRNA

14%

10%

High

mRNA

3%

32%

Very Low

E. Coli RNA Polymerase

RNA polymerase core


enzyme is a multimeric
protein 2,, ,
,
The subunit is
involved in DNA
binding
The subunit contains
the polymerase active
site
The subunit acts as
scaffold on which the
other subunits
assemble.
Also requires -factor
for initiation forms
holo enzyme complex

Site of DNA
binding and RNA
polymerization

General Gene Structure


5

Promoter

Transcribed region

terminator 3

Gene Promoters

Other -Factors
Standard genes 70
Nitrogen regulated genes 54
Heat shock regulated genes 32

Transcriptional
Initiation
Closed complex
Open complex

Primer formation

Disassociation
of -factor

Pausing induces termination

3end tends to be AU
rich easily to disrupt
during pausing. Leads to
disassembly of RNA
polymerase complex

Rho Dependent Termination


rho is an ATPdependent helicase
it moves along RNA
transcript, finds the
"bubble", unwinds it
and releases RNA chain

Eukaryotic RNA
Polymerases
type

Location

Products

RNA polymerase I

Nucleolus

rRNA

RNA polymerase
Nucleoplasm
mRNA
II
RNA polymerase
rRNA, tRNA,
Nucleoplasm
III
others
Mitochondrial RNA
Mitochondrial gene
Mitochondria
polymerase
transcripts
Chloroplast RNA
Chloroplast gene
Chloroplast
polymerase
transcripts

Eukaryotic RNA
Polymerases

RNA polymerase I,
II, and III
All 3 are big,
multimeric proteins
(500-700 kD)
All have 2 large
subunits with
sequences similar to
and ' in E.coli RNA
polymerase, so
catalytic site may be
conserved

Eukaryotic Gene Promoters


Contain AT rich concensus sequence
located 19 to 27 bp from transcription
start (TATA box)
Site where RNA polymerase II binds

Transcription Factors
TFAIIA, TFAIIB
components of RNA
polymerase II holoenzyme complex
TFIID Initiation factor,
contains TATA binding
protein (TBP) subunit.
TATA box recognition.
TFIIF (RAP30/74)
decrease affinity to nonpromoter DNA

Chapter 21 (Part 2)
Transcriptional Regulation and
RNA Processing

Regulation of Gene Expression

RNA Processing

mRNA
5CAP

Active
enzyme

Post-translational
modification

AAAAAA

RNA Degradation

Protein Degradation

Activators and Repressors


co-A

RNAP

+1

RNAP

co-R

R
+1

The Helix-Turn-Helix
Motif

contain two alpha


helices separated by
a loop with a beta
turn
The C-terminal helix
fits in major groove
of DNA; N-terminal
helix stabilizes by
hydrophobic
interactions with Cterminal helix

The Zn-Finger Motif

Zn fingers form a folded beta strand and an alpha helix that


fits into the DNA major groove.

The Leucine Zipper Motif


Forms amphipathic
alpha helix and a
coiled-coil dimer
Leucine zipper proteins
dimerize, either as
homo- or heterodimers
The basic region is the
DNA-recognition site
Basic region is often
modeled as a pair of
helices that can wrap
around the major
groove

Transcription Regulation in
Prokaryotes

Binding of some trans-factors is


regulated by allosteric modification

lac operon

Glucose is E. colis
primary carbon
source.
But.. it can grow
on different carbon
sources.

Diauxic growth of E. coli on a mixture of


lactose + glucose.

The lac I protein

Operator and RNA Polymerase Bind at Overlapping Sites

Inhibition of repression of lac


operon by inducer binding to lacI

Inducer : Allolactose,
produced by side
reaction of lacZ
Lehninger: Principles of Biochemistry, 3rd Ed.

IPTG is a Gratuitous Inducer

Synthetic molecule

Repression of
the Tryptophan
operon:
A variation of
the theme

Catabolite Repression of lac Operon


(Positive regulation)

Molecular Cell Biology, 4th Edition, Lodish et. al. (2000)

Why does the Lac Operon


need an activator?

Lac promoter has lousy promoter!!!

tRNA Processing

rRNA Processing

Processing of Eukaryotic mRNA

Splicing of Pre-mRNA

Splicing of Pre-mRNA

Chapter 22 (Part 1)
Protein Synthesis

tRNAs
tRNAs are interpreters of
the genetic code
Length = 73 95 bases
Have extensive 2o
structure
Acceptor arm position
where amino acid
attached
Anticodon
complementary to mRNA
Several covalently
modified bases
Gray bases are conserved
between tRNAs

tRNAs: 2o vs 3o Structure

Aminoacyladenylate Formation
NH2

N
N

O
O

O
O

O
O

O-

O
H
H OH

O-

O-

ONH2

H
OH
O

N
N

C
CH

PPi
O

NH2

O-

O
H
H OH

O
H

OH

C
CH
NH2

Aminoacyl-tRNA Synthetase Rxn


NH2

NH2
N

N
N

5' tRNA

O
O

O
H
H OH

H
H
OH

O-

O
H

H
OH

H
C

O
O

H
CH H

NH2

NH3+

AMP

5' tRNA

O
H

H
OH

H
O

C
CH H
NH3+

N
N

Chapter 22 (Part 2)
Protein Synthesis

Ribosomes

Prokaryotic Ribosome Structure

Eukaryotic Ribosome Structure

Ribosome Assembly

Assembly is coupled w/ transcription


and pre-rRNA processing

Ribosome Structure

Identification of Initiator Codon in


Prokaryotes

Prokaryotic
Translational
Initiation

Eukaryotic
Initiation of
Translation

Recycling
of EF-Tu

Peptide Bond formation


P-Site
N
5' tRNA

H+

O
O

5' tRNA

H
OH

O
H
OH

O
O

5' tRNA

OH

H
OH

5' tRNA

N
O
H

H
OH

C
O

CH H
CH H
H

H
BASE

NH2

O
H
H

A-Site

NH2
N

CH H
NH3+

P-Site

NH2

O
H

A-Site

NH2

CH H
NH3+

N
N

Formation of
Peptide Bond

More on elongation

Regulation of Gene Expression

RNA Processing

mRNA
5CAP

Active
enzyme

Post-translational
modification

AAAAAA

RNA Degradation

Protein Degradation

Regulation of Globin
gene translation by
heme

Chapter 23 (Part 1)
Recombinant DNA Technology

Cloning Vector
Required features
1. Origin of replication
2. Selectable marker
3. Screenable marker
for recombinant
molecules
4. Cloning sites

Restriction Modification System


AAGATGCGAATTCGTACA
DNA methylase

AAGATGCGAATTCGTACA
DNA methylase

*
*
AAGATGCGAATTCGTACA

AAGATGCGAATTCGTACA

Restriction endonuclease

Restriction endonuclease

*
*
AAGATGCGAATTCGTACA

AAGATGCG

AATTCGTACA

5 ATGCGAATTCCGGTT 3
3 TACGCTTAAGGCCTT 5
EcoR1

Sticky-end cutter

5-ATGCG-3
5-AATTCCGGTT-3
3-GGCCTT-5
3-TACGCTTAA-5

5 ATGCGATATCCGGTT 3
3 TACGCTATAGGCCTT 5
Blunt-end cutter
EcoRV
5-ATGCGAT-3
3-TACGCTA-5

5-ATCCGGTT-3
3-TAGGCCTT-5

T4 DNA
Ligase

Transformation
All of the previous steps were
performed in vitro.
We have generated a very
small amount of a recombinant
plasmid
Need to amplify in bacteria to
get enough to work with.
Transformation process to
mobilize DNA into bacterial
host
Select for transformed
bacteria on specific antibiotic
that corresponds to the
antibiotic resistance gene
present on the plasmid

How to produce a
recombinant protein

0.1 to 1%
of cellular
protein

10 to 70%
of cellular
protein

cDNA

cDNA
Library

cDNA

DNA
hydridization
screening for
specific gene
Requires that you know
something about the gene
sequence
Can get sequence
information form purified
protein

Product name

Protein type

Application

Company

Adagen (Adenosine
deaminase )

An enzyme

Severe combined
immunodeficiency
disease (SCID)

Enzon

Genotropin
(Recombinant
growth hormone)

A hormone

Growth hormone
deficiency (GHD) in
children

Pharmacia & Upjohn

Humalog
(Recombinant human
insulin)

A hormone

Diabetes

Eli Lilly

Nabi-HB (AntiHepatitis B)

An antibody

Hepatitis-B

Nabi

Novo Seven
(Recombinant
coagulation factor
VIIa)

A modified factor

Hemophillia patients
with inhibitors

Novo Nordisk

Ontak (Diphtheria
toxin-interleukin-2)

A fusion protein

Cutaneous T-cell
lymphoma (CTCL)

Ligand
Pharmaceuticals

Roferon-A
(Recombinant
interferon alfa-2a)

A modifier

Hairy cell leukemia


or AIDS-related
Kaposi's sarcoma

Hoffmann-La Roche

Genetic Modification of
Higher Organisms
Can introduce gene into animals and
plants
These modified organism are powerful
research tools to study the effect of a
specific gene product on metabolism,
development etc.
Has also been used to develop improved
agricultural products

Genetically Engineered Salmon


Is Bigger Better?

http://www.agwest.sk.ca/sabic_index_tp.shtml