Biochemistry
The chemistry of life
But what is “life”?...
Mammals
• “Animals” -- when
most people think of
animals, they are
really thinking about
mammals
• Mammals: warmblooded, hair, live
birth, etc.
Vertebrates
Invertebrates
The vast, vast, vast majority of animals: arthropods, molluscs,
echinoderms, annelids, etc.
“Plants”
• “Things that don’t move”
• Fungi are really more like animals than plants
• “True” plants perform photosynthesis
Bacteria
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•
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•
•
“bugs”, germs, gross stuff…
Some cause disease
Very small, single-celled (usually <10 µm)
Found everywhere
2 kinds: eubacteria & archaebacteria
New view of life
Norman Pace
A Molecular View of
Microbial Diversity and the
Biosphere
Science 276: 734-740
• Based on molecular analysis (rRNA)
• The more similar the molecules, the closer the
species are placed.
Prokaryotes
The 3 “kingdoms” of Life!
Fungi
Animal
Plants
Cells
Even a single cell represents a high order of complexity.
A cell does everything required for life.
Most organisms are unicellular.
Biology tells us how cells and cellular assemblies (organs and
organisms) behave.
Chemistry tells us how (relatively) small molecules behave.
Biochemistry provides the link between the two.
Biochemists are assisted by related disciplines:
genetics!
cell biology!
molecular biology!
biophysics
Bacteria/prokaryotes
• Extremely abundant
– There are more bacteria in your mouth than the total
number of humans that have ever existed.
(~10% of your body mass is bacteria! Microbiome)
– Estimated to be ~5x1030 on Earth
– Contain as much carbon as all plants & >10 times the
amount of nitrogen and phosphorous
– Majority of biomass on Earth is bacterial
• Contribute in essential ways to the cycling of
carbon and nitrogen
Bacteria/prokaryotes
• Use every type of metabolism known
• Inhabit every niche available
–Land, sea, air
–Geothermal vents, polar ice
–Extremes of acidity, salinity, etc.
• Single-celled, small (0.2 µm to ~100 µm),
relatively little internal structure
• Come in a wide variety of shapes
• Genomes tend to be small and compact
• 2 domains: Bacteria & Archaea
Bacteria/prokaryotes
Eubacteria ("true" bacteria) & archaebacteria
Bounded by 1 or 2 membranes. Most lack any internal membranes, but there are
several exceptions (e.g. thylakoid membranes in cyanobacteria).
Interior consists of:
nucleoid – DNA and associated proteins
cytosol – everything else (ribosomes, enzymes, metabolites, etc.)
Dimensions: on the order of a micrometer
Mycoplasma is ~0.3 µm in diameter
Escherichia coli is 2 µm long and 1 µm in diameter
Display a wide variety of lifestyles.
Eubacteria occupy almost every ecological niche and display every type of energy &
carbon utilization mode.
Archaebacteria can be found in many extreme niches (e.g. high temperature, high salt,
etc.)
Eukaryotes
• Have a nucleus (karyos = nucleus)
• Most have a more complicated internal
cellular arrangement with several
membrane-bound organelles
• While many are unicellular, contain all
known multicellular organisms:
–Plants
–Animals
–Fungi
Eukaryotes
Slightly more related to archaebacteria than to
eubacteria (proteins involved in molecular information
flow, such as transciption, translation, etc.)
Efficient energy transduction systems have all been
taken from eubacterial systems:
Respiration: mitochondria derived from proteobacterial
ancestor
Photosynthesis: chloroplasts derived from cyanobacterial
ancestor
Eukaryotic cellular architecture
Main distinguishing feature is membrane-bound
organelles:
• nucleus: houses DNA and machinery involved in DNA and
RNA manipulations
• endoplasmic reticulum: contiguous with nuclear envelope,
insertion and initial processing of proteins destined for
endosecretory system
• Golgi apparatus: further processing and sorting of proteins to
endosome/lysosome or plasma membrane
• lysosome/vacuole: contain degradative enzymes, storage of
some molecules
• plasma membrane: defines identity of cell, input/output of
small molecules
Advantages of different experimental systems:
Bacteria
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Single cell — only 1 cell type
Short generation time (grow quickly)
Inexpensive to maintain
Haploid genetics; many mutants available for certain species (E. coli)
Plasmids and bacteriophages as vectors to introduce DNA; recombination
allows gene integration and gene deletions.
Yeast (unicellular fungus)
• All of the above, but last: few viruses available
• Simple sexual system allows one to make new genetic combinations.
Viruses
• Simple nucleic acid/protein assembly — "stripped down"
only a (relatively) few genes and proteins; possible to understand
• Use cellular machinery to reproduce themselves (the cell is their
"environment")
Ways to make a living
The most important defining
characteristics are how you get
• Energy
• Carbon
Lithotrophs (Chemotrophs)
• Get their energy by oxidizing inorganic
molecules
• Most species are also autotrophic (get their carbon from CO2)
• Basic idea: pass electrons from a good
electron donor to a good acceptor, extracting energy in the process
• Several potential electron donors, but most
use O2 as the acceptor.
Examples
Bacteria
donor
acceptor
Hydrogen
H2 → H2O
O2 → H2O
Sulfide
H2S → S
O2 → H2O
Sulfur
S → SO42-
O2 → H2O
Nitrifying
NH4+ → NO2-
O2 → H2O
Nitrifying
NO2- → NO3-
O2 → H2O
Iron
Fe2+ → Fe3+
O2 → H2O
Phosphite
HPO32- → HPO42-
SO42- → H2S
Lithotrophs (cont’d)
• Most H2 bacteria are also chemoorganotrophs (not enough H2 usually)
• Sulfur is an essential element for all life forms (2 of the 20
amino acids have it), so sulfur bacteria play an important
role in planetary S cycles.
• The same is even more true for the nitrifying bacteria.
• Important anaerobic group that catalyzes anamox:
NH4+ + NO2- → N2 (gas) + 2 H2O
More fun lithotroph facts!
•
Iron oxidation does not yield much energy, so iron bacteria go through
a lot, making insoluble ferric hydroxide (rust).
Phototrophy
2 main flavors:
Anoxygenic - does not produce O2,
uses a variety of electron sources
Oxygenic - produces O2, uses H2O as
electron source
2 H2O + 2 A → 2 AH2 + O2
Photosynthesis in a nutshell
Uses molecules that can absorb visible
light (or slightly in the infrared)
Chlorophylls, carotenes, or derivatives
Absorption of light raises an electron to a
higher energy state (“excited state”)
The electron can then be transferred to
another molecule (and to another and to another…)
Charge separation
P*
Electron
Transfer
Chl
Q
Excitation
Let there be photon!
FeS
Fd
Pc
P
Anoxygenic photosynthesis
Two types of organisms, defined by the type of
reaction center that they use. Reaction center is the
protein-chlorophyll complex where the light-driven
electron transfer reactions take place.
Type 1: donate electrons to an iron-sulfur protein
strongly reducing
Type 2: donate electrons to a quinone
moderately reducing (PS II is strongly oxidizing – can oxidize water)
Oxygenic phototrophs
• Have both types of reaction center.
• The type 2 reaction center is able to oxidize water: 2 H2O → O2 + 4 H+ + 4e• The 2 reaction centers work together in series.
(This allows them to pass electrons all the way
from water up to the high-energy FeS protein.)
Oxygenic phototrophs
• Represented by prokaryotes: cyanobacteria & prochlorophytes
• eukaryotes: algae & plants (chloroplasts) Photosynthetic prokaryotes
They tend to occupy
more extreme
conditions (high/low
temperature, etc.).
Anoxygenic bacteria
use modified
chlorophyll and other
pigments to absorb
photons not absorbed
by chlorophyll.
Anaerobic chemotrophs
• Many anerobic niches out there!
• Very few eukaryotes live in this way
• They live off of respiration using electron
acceptors other than oxygen.
• Wide variety of electron donors; many are organic
molecules (acetate, lactate, ethanol, etc.)
• Major electron acceptors include
Sulfate:
Sulfur:
Carbonate:
SO42- → H2S
S → H2S
CO2 → CH4 (methane)
CO2 → CH3COOH (acetic acid)
Methanogens
• Strictly anaerobic archaebacteria
• Uses a very complicated biochemical pathway to reduce
CO2 (or acetate) to CH4
• Most use H2 in the reaction as reductant.
• Account for most/all of the natural gas we pump out of the
ground and for the methane from ruminants. (Cows belch
50 L of methane per day.)
Methanococcus jannischiiwas originally isolated
from a sample taken from a "white smoker"
chimney at an oceanic depth of 2,600 meters on the
East Pacific Rise. It can be grown in a mineral
medium containing only H2 and CO2 as sources of
energy and carbon for growth within a temperature
range of 50 to 86°C.
Organotrophs
• Use organic molecules (carbon has already been
fixed) as both a source of energy and carbon,
• Anaerobes can catalyze fermentations, where part
of the molecule is oxidized and part is reduced.
Common: carbohydrate → ethanol, lactate, butyrate, etc,
Unusual substrates: acetylene, phloroglucinol, resorcinol,
aconitate, glyoxylate, etc.
Aerobic organotrophs
• Oxidize organic compounds completely to
CO2 using oxygen as the electron acceptor
• All animals and fungi (and plants too!) and
many bacteria use this lifestyle
• Possible substrates include carbohydrates,
fats, amino acids, hydrocarbons, etc.
Making ATP
• ATP is the energetic currency of
the cell.
• Every organism on the planet
has a version of ATP synthase,
which makes ATP using energy
stored as an ion imbalance
across a membrane.
• That means that all you have to
do is couple some reaction to
making an ion imbalance, and
you can make ATP (and live).
Biomolecules
Vast majority made up of relatively few elements:
C, H, O, N, P, S
Biomolecules
Biochemistry is essentially organic chemistry in water. You should be familiar with the major types of functional
groups found in biomolecules:
methyl, ethyl, phenyl, etc.
hydroxyl
sulfhydryl
amine, amide
carboxylic acid
carbonyls (ketone, aldehyde)
ether
phosphoryl (attachment of phosphate to hydroxyl)
guanidino
imidazole, indole
(note the lack of halides!)
Linking groups
Nature makes use of some functional groups to connect
molecules, creating new functional groups.
The most common are created by condensation:
carboxyl + amine → amide
carboxyl + alcohol → ester
carboxyl + thiol → thioester
2 carboxyl → acid anhydride
carboxyl + phosphate/phosphoryl → mixed acid
anhydride
2 phosphoryl/phosphate → phosphoanhydride
2 sulfhydryl → disulfide (oxidation, not condensation)
As you examine biomolecules, pay attention to these "linking"
functional groups. They often indicate where 2 smaller
molecules were joined together.
Molecular Configuration
It is important to distinguish between:
configuration = arrangement of atoms in a molecule that
has double bonds or chiral centers; it cannot be changed
without breaking bonds
conformation = spatial arrangement of groups in a
molecule; can be interconverted by bond rotation without
breaking any bonds
Living systems discriminate between different molecular
configurations because molecular recognition is carried out by
molecules (e.g. enzymes) that are themselves chiral and spatially arranged in a specific way.
Change in conformation
X & Y cannot exchange places without breaking the C-C bond
Chiral molecules
Most biomolecules have at least 1 chiral center (C atom to which are attached 4 inequivalent
substituents).
A molecule with n chiral centers can have up to 2n
stereoisomers. These can be further classified as
enantiomers: mirror images
diastereomers: non-mirror images
Note: when a molecule has only 1 chiral center, there
are only 2 stereoisomers, and they are enantiomers.
Chiral molecules
Although enantiomers behave chemically in a
similar fashion, they display an important physical
difference – they rotate polarized light in opposite
directions.
A racemic mixture (1:1 mix of both enantiomers)
does not rotate light.
Proteins (and other biomolecules) are
stereospecific, because they also are chiral and
their binding sites can thus distinguish between
enantiomers.
Chiral molecules
Although the R/S system is the accepted system for
indicating stereoisomers, biochemists still make use of the
older D/L system, which is based upon configurations of
glyceraldehyde.
D-glyceraldehyde = (2R)-glyceraldehyde
L-glyceraldehyde = (2S)-glyceraldehyde
Amino acids and carbohydrates are commonly indicated as
D or L, based upon the analogy with glyceraldehyde.
Amino acids are found as L-amino acids in living systems,
while carbohydrates are D stereoisomers.
Biochemical reactions
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•
With very few exceptions, the reactions occurring
in living systems are catalyzed by enzymes.
However, the types of reactions are not exotic, and
generally fall into one of the following categories:
1. redox reactions
2. C-C bond formation/breakage
3. internal rearrangements
4. group transfers
5. condensation/hydrolysis reactions
Oxidation-reduction
• Some reactions involve transfer of a single
electron, and these involve radicals and/or metals.
• Most involve transfer of 2 electrons (the oxidized
molecules will typically lose 2 H+ as well). These
are commonly called dehydrogenations
(catalyzed by dehydrogenase enzymes).
• Some of these reactions will result in formation of
a new C-O bond
– the enzymes are called oxidases
– oxygenases if O2 is used
Carbon-carbon bond formation/breakage
• Homolytic cleavages are rare (in general, radicals are rare in biological
transformations).
Carbon-carbon bond formation/breakage
• Heterolytic cleavage usually involves
nucleophilic substitution:
– unimolecular (SN1)
– bimolecular (SN2) *most common
Isomerizations
(a.k.a. Internal rearrangements)
• These are reactions in which the molecular
formula does not change, but the structural
formula does.
• These can include
– internal redox reactions
– movement of double bonds
– stereoisomerization
Group transfers
• Attachment of good leaving groups can
substantially activate nucleophic
substitution reactions.
• Phosphoesters and thioesters are common
examples used in biology.
Condensation/hydrolysis
• Condensations join 2 molecules with elimination of
water. This is the major mechanism to make
macromolecules from their basic building blocks:
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–
–
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amino acids → polypeptides
nucleotides → nucleic acids (polynucleotides)
sugars → polysaccahrides
Lipids made by condensation of glycerol with fatty acids
– Biological systems typically "activate" at least
one of the functional groups in order to drive the
condensation.
• Hydrolyses are used to break down macromolecules
Review of Thermodynamics
1st Law: ΔEtotal = 0
ΔEsystem = q - w!
q = heat absorbed by system!
w = work done by system
Energy is neither created nor destroyed.
Enthalpy
ΔH = ΔE + Δ(PV)
for biochemistry, Δ(PV) ≈ 0, so ΔH ≈ ΔE
Review of Thermodynamics
2nd Law: ΔSuniverse > 0
Ssys = k ln Ω
Ω= # of ways to arrange system with same E
Entropy is always increasing in the universe.
Review of Thermodynamics
For a spontaneous process:
ΔSuniverse = ΔSsys + ΔSsurr > 0
We rarely make measurements of the surroundings, but the change in
entropy of the surroundings is directly proportional to the change in
enthalpy of the system.
ΔSsurr = ΔHsurr/T = –ΔHsys/T
ΔSuniverse = ΔSsys + (-ΔHsys/T) > 0
-TΔSuniverse = -TΔSsys + ΔHsys < 0
define free energy change (ΔG):
ΔG = ΔHsys – TΔSsys
Review of Thermodynamics
Gibbs free energy (G) or simply free energy can be used to express
spontaneity more directly.
G = H – TS
The change in free energy for the system is:
ΔG = ΔH – TΔS
understood as ΔG = ΔHsys – TΔSsys
Gibbs free energy
• Isothermal, reversible process
ΔS = ΔH/T
• Therefore, ΔH = TΔS at equilibrium
ΔH - TΔS = 0
• Gibbs free energy (G)
ΔG = ΔH - TΔS
when ΔG = 0, the system is at equilibrium
• If ΔG < 0, reaction can occur spontaneously
• If ΔG > 0, reaction does not occur spontaneously
Biochemical thermodynamics
ΔGf° = free energy of formation of compound from
elements (all in their standard states)
T = 298K (25°C)
P = 1 atm
[X] = 1 M
aA + bB → cC + dD
ΔG°rxn = Σ ΔG°f(products) - Σ ΔG°f(reactants)
ΔG = ΔG°rxn + RT ln Q
Q = Πproducts/Πreactants = reaction quotient
(in this case, Πproducts = [C]c[D]d)
Biochemical thermodynamics
• At equilibrium
ΔG = 0, Q = Keq
• Thus,
ΔG°rxn = -RT ln Keq
Keq = e-ΔG°/RT
• Free energies usually calculated at pH = 7!
(i.e. [H+] = 10-7 M, rather than 1 M)
• This is indicated with a prime (i.e. ΔG°')
ΔG => ΔG'
ΔG° => ΔG°’
Keq => Keq'
Work
1. changes in chemical bonds
2. movement of molecules & ions across
membranes
3. mechanical work
4. -ΔG = maximum amount of work
obtainable
How biological systems drive reactions
1)
Couple reactions
A+B→ C
G°1
D →E + F
G°2
A+B+D→C+E+F
G°3
G°3 = G°1 + G°2
How biological systems drive reactions
2)
Join reactions in series
A+B→ C
G°1
C→ D+E
G°2
A+B→ D+E
G°3
G°3 = G°1 + G°2
How biological systems drive reactions
3)
Modulate concentrations of reactants
and/or products
Remember:
• Thermodynamics ony concerned with
starting and ending states – ΔG does not
depend on how you get there
• ΔG determines spontaneity, not ΔG°
How biological systems drive reactions
4)
Use catalysts to choose which reactions can
proceed
ΔG determines if a reaction can proceed
spontaneously, not if it will do so or how fast
• The rate of the reaction depends upon the
activation energy.
•
Why is ATP used as !
"energetic currency"
1. ΔG' is about right (i.e. not much higher
or lower than ΔG' of typical cellular
reactions).
2. ATP is relatively stable.
3. Products of hydrolysis are useful for
other reactions.
4. The nucleoside moiety provides a
handle for enzymes to grab.