Transcript Biochemistry for the Radiation Biologist

Illinois Institute of Technology
Physics 561
Radiation Biophysics, Summer 2014
Lecture 11: Biochemistry Blitz
Andrew Howard
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Why biochemistry matters
u
u
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Biochemists point out how
chemical processes occur in
biological systems, inside and
between cells.
We need to know that because
ionizing radiation will exert its
effects on these chemical
systems.
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Lecture 17 Plans
Unity and diversity
u Thermodynamics
u Organic chemistry
u Small biomolecules
u Polymers
u Proteins
u Enzymes
u Carbohydrates
u Lipids & Membranes
u
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Metabolism (general)
u Carbohydrate metabolism
u Lipid metabolism
u Amino acid metabolism
u Nucleic acid metabolism
u Replication
u Transcription
u Translation
u
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Biochemical unity
There is a remarkable unity
to biochemical processes all across eubacteria,
archaea, and eukaryota
The genetic code is almost uniform throughout
all three kingdoms (two extra amino acids in a
few organisms, but that’s it!)
Specific proteins look the same across wide
gaps of evolutionary history
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PDB 1emy
Elephant myoglobin
Biochemical diversity
The morphological and
functional differences
between you and an
elephant, or between you
and a flowering plant, or
between you and a bacterium, arise from
differences in biochemical phenomena
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Some classes of proteins are present only in
certain kinds of organisms
Structural components in vertebrates differ
significantly from those in arthropods
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Thermodynamics
and Kinetics
Systems of molecules tend toward
an equilibrium state in which the
minimum free energy is obtained
In order to go from an initial state R
to a final, lower-energy state P, a
higher-energy transition state T may
have to be overcome
Free energy has both enthalpic
(heat) and entropic (organizational)
components (Boltzmann):
G = H - TS
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T
G
G‡
R
G
P
Reaction
Coordinate
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Bioorganic chemistry
Most biological reactions
involve carbon-containing
compounds
Therefore organic chemistry
(the chemistry of compounds
that contain C-C or C-H
bonds) explains a large
fraction of what’s happening
below the surface in
biochemical systems
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NADPH
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We can ignore a lot of organic reaction
mechanisms!
Only a subset of all possible organic reactions
occur in biochemistry:
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They’re almost all aqueous
Generally occur at temperatures between 3ºC
and 50ºC
Most occur between pH 5 and pH 9 (exception
in humans: the stomach, which is highly acidic)
Entire classes of organic reactions are
unrepresented
Claisen
condensation
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What reactions do occur?
Mostly nucleophilic substitutions (SN1 and
SN2)
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These are two-electron transformations, as
we’ve discussed in previous lectures
These take place under enzymatic control
Therefore they can proceed at measurable
rates even though the nucleophiles aren’t
very powerful by organic lab standards
Some free radical reactions, as we’ve said
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There are enzymatic free-radical reactions
But there are non-enzymatic ones too
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Chirality
Many biomolecules are chiral:
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At least one carbon atom has 4 distinct substituents
Therefore the mirror image of the molecule is
different from the original molecule
Specific classes of biomolecules are chiral
Standard amino acids are L- and so are their
polymers
Most sugars are D- and so are their polymers
Lipids are usually not chiral
Nucleic acid bases: achiral; but ribose is chiral
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Small biomolecules
Most common small molecule in
biological systems is water
There are other important small
molecules and ions:
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CO2, O2, glyceraldehyde, glucose
HCO3-, PO43-, Na+, Cl-, K+, Ca2+,
Fe2+,Mg2+
Palmitate, oleate, stearate, choline,
sphingomyelin
Amino acids (20 ribosomal +
others)
Nucleic acids
(A,C,G,U,dA,dC,dG,dT, …)
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Biopolymers
Most complex activities in biology involve polymers
Biology achieves catalysis, reproduction, specificity,
and many other things by making polymers rather than
by building complex monomeric molecules
Biological polymers are built up by formal (and actual)
elimination of water (M1 + M2  M1-2 + H2O)
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Protein: polymer of amino acids
RNA: polymer of ribonucleotides
DNA: polymer of deoxyribonucleotides
Polysaccharides: polymers of sugars
Except for polysaccharides, they’re unbranched
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Proteins
Proteins are polymers of L-amino acids
There are 20 amino acids in proteins from 99%
of all organisms; a few use 2 others
Variety of functions:
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Enzymes (catalysts)
Structural proteins
Storage and transport proteins
Hormones
Receptors
Nucleic-acid binding proteins
Molecular machines
Specialized functions (e.g. sweetness)
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How do proteins perform so
many functions?
Amino acid side chains have a wide variety of
reactivities
Scaffolding surrounding active or binding site can
significantly change reactivity of specific moieties
within the protein
Side chains that would normally be hard to ionize
become easy to ionize because the ion is
stabilized by interactions with neighboring groups
Regions that would be hydrophilic if exposed
become hydrophobic when buried within protein
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Is that all?
No; Some proteins have non-amino-acid
components bound, loosely or tightly, to the amino
acid chain(s); thse entities are cofactors
Cofactors can be inorganic (e.g. Mg2+ ions) or
organic (coenzymes)
These provide functionalities that would otherwise
be unavailable to the polypeptide
Many coenzymes are derived from vitamins,
particularly the B-complex vitamins
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Why are proteins so big?
Typical enzyme active site or
carrier-protein binding site involves
< 5% of the amino acids in the protein
So what are all those other amino acids doing?
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Creating environment perfectly suited to the electrostatics and
hydrophobicity or hydrophilicity required for proper function
Guarantees that the active site or binding site keeps its shape
and electrostatic properties over time
Enables binding of cofactors and other effectors
Allows for appropriate interactions with other macromolecules
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Does every protein have
exactly one function?
No.
Some proteins that have evolved
for one purpose become pressed
into service in a different guise
Example: some of the proteins
that make up the eye lens are
actually enzymes, reused as
structural proteins
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Enzymes
Enzymes are defined as biological catalysts
Biochemists figured out in the 1920’s that
all enzymes were proteins, although it took
until the 1930’s for that notion to be
definitively accepted
James
In the 1970’s this assertion had to be modified:
Sumner
some enzymes (biocatalysts) are actually RNA
molecules
1990’s: Recognition that the catalytic step in protein
synthesis is performed by a specific adenine base
in ribosomal RNA
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What properties must an
enzyme have?
Catalysis:
must lower the activation energy of a reaction
Specificity:
must act preferentially on certain substrates
Regulation:
must be subject to some sort of regulation
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How is catalysis accomplished?
Multiple mechanisms, often acting in concert:
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Acid-base catalysis (specific amino acid side-chains
participate temporarily in reactions by acting as
nucleophiles or electrophiles)
Proximity effect (enzyme contains channels with
appropriate shape and electrostatic properties to cause
substrates to travel down them toward one another)
Induced fit (enzyme reshapes itself to bind substrate,
but substrate gets reshaped as well)
Transition state stabilization
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How effective are enzymes, and how
are they regulated?
The best can speed up a reaction by a
factor of 1012 or more
They don’t change equilibrium: they just
make the reaction go faster.
Regulation by:
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Control of transcription
Control of translation
Interference with activity via inhibitors
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Enzyme kinetics
v0
Michaelis-Menten kinetics:
v0 = Vmax [S] / (Km + [S])
v0 is the reaction velocity
under enzymatic influence
[S] is substrate concentration
Vmax and Km are constants
characteristic of the system
1/v0 = (Km/Vmax)(1/[S]) + 1/Vmax
Not all reactions obey M-M
kinetics,but many do.
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Vmax
[S]
1/v0
1/Vmax
-1/Km
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1/[S]
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Inherent properties of enzymes
Km is an inherent property of an enzyme
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Can be measured for a specific substrate, independent
of enzyme and substrate concentration
Measures binding affinity of enzyme for substrate
Vmax clearly depends on how much enzyme we put in:
the more we put in, the faster the reaction
But kcat = Vmax/[E]tot is an inherent property
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Measures turnover rate
kcat = number of substrate molecules converted to
product per unit time by a single enzyme molecule
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Enzyme inhibition
Various small molecules can bind to an
enzyme and slow it down
Competitive inhibitors compete with the
substrate for the active site and raise Km
Noncompetitive inhibitors interfere with
catalysis and decrease Vmax
Uncompetitive inhibitors decrease both Vmax
and Km,
but do so in a way that slows the reaction
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Carbohydrates
Monomers are three-carbon to sevencarbon polyalcohols with one carbonyl
group at either C1 or C2
Simplest: glyceraldehyde &
dihydroxyacetone - C3
Most important other ones:
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Ribose, xylose (C5)
Glucose, fructose, galactose (C6)
Monomers are fuel, signals, intermediaries
(especially when phosphorylated)
Monomers are very soluble
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Sugar derivatives
Sugar monomers that have been modified
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Deoxyribose
Phosphorylated sugars (especially at C1 and C5 or C6)
Aminated sugars (mostly C6 sugars)
N-acetyl sugars
Sugar acids
Sugar alcohols (carbonyl converted to alcohol)
These derivatives are often the active agents in
intermediary metabolism
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Disaccharides
Compounds made up of
two sugar monomers
Glucose + glucose  Maltose + H2O
Glucose + fructose  Sucrose + H2O
Glucose + galactose  Lactose + H2O
These are instances of oligomers
(few rather than many building blocks)
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Storage polysaccharides
Starch: storage form of glucose in plants
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Amylose: 1-4 linkages only
Amylopectin: mostly 1-4 linkages, a few 1-6
Glycogen: storage form of glucose in animals,
some bacteria
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mostly 1-4 linkages, some 1-6
Glycogen is primary short-term fuel in humans;
long-term fuel is usually fats (more efficient)
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Glycogen
structure
Single glucose
units broken off
and
phosphorylated to
use as fuel
Amylases cleave
the 1-4 linkages;
Other enzymes
deal with the 1-6
linkages
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Sugar-peptide
complexes
Sugars are often complexed
either to peptides
(oligomers of amino acids)
or proteins
(polymers of amino acids)
Smaller-peptide complexes make up cell walls
Full-size proteins that have been decorated with a
few sugar molecules are ubiquitous in eukaryotes
and often alter the properties of the protein or
enable it to be involved in cell-cell communication
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Lipids
Hydrophobic molecules
Many contain fatty acids (hydrocarbons with
carboxylate groups at one end) esterified to glycerol
Others, including steroids like cholesterol,
are built from a five-carbon nucleus called isoprene
Roles:
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Long-term energy storage
Primary components of lipid bilayers, out of which cell
membranes and organellar membranes are made
Signaling (steroid hormones, others)
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Membranes
Phospholipid bilayer self-assembles
Typically contains cholesterol and integral
membrane proteins as well
Keeps the outside out and the inside in
Enables selective passage of certain
molecules
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Passive transport: permits passage down a
concentration gradient
Active transport: permits passage against a
concentration gradient
Highly dynamic system
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Metabolism: General Principles
Metabolism: the collection of chemical reactions
that take place in a biological system
Two broad categories:
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Catabolism: breakdown of complex molecules
into simpler ones, generally yielding energy
Anabolism: buildup of complex molecules from
simpler ones, generally requiring energy
Some reactions are amphibolic, i.e. have both
catabolic and anabolic characteristics
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Energy
currency
Adenosine triphosphate is a readily
available compound in almost all cells
The terminal phosphoanhydride linkage
(and the previous one) can be hydrolyzed,
yielding energy:
ATP + H2O  ADP + Pi,
Go ~ -30 kJ mol-1
Energy drives other reactions, performs
mechanical work in molecular machines
ATP itself produced through oxidative
phosphorylation: organicCO2
Thus ATP becomes an energy currency in
the cell
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Metabolic distinctions
Certain tissues or cell types perform some
metabolic functions and others perform
other functions
This is controlled primarily by which
proteins get expressed (transcribed &
translated) in a given cell
It’s also controlled by availability of
substrates
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Organismal distinctions
Organisms can be distinguished by:
Whence do they get their carbon?
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By converting CO2 to organic molecules
(autotrophs)
By intake of organic molecules
(heterotrophs)
Whence do they get their energy?
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From sunlight (phototrophs)
From oxidation of organic molecules
(chemotrophs)
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Combining these distinctions: I
Chemoheterotrophs: get energy and
carbon by intake of organic molecules:
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Almost all animals
Many bacteria, some archaea
Chemoautotrophs: get carbon from CO2
but get energy from inorganic compounds
or heat vents in ocean
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Specialized bacteria—methanogens,
sulfur oxidizers …
Certain archaea
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Combining these distinctions: II
Photoheterotrophs: use light for energy but can’t
obtain all their carbon from CO2
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Need an organic carbon source
Purple non-sulfur bacteria, a few other bacteria
Photoautotrophs: use light for energy and can
obtain all needed carbon from CO2 in air
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Green plants, algae
Blue-greens and certain other eubacteria
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Metabolism: kinetics
Remember our free-energy diagram:
The purpose of an enzyme is to reduce the activation
energy G‡
Can be entropic or enthalpic
Can be measured via temperature dependence of
reaction rate k = Qexp(-G‡/RT)
For typical biochemical reactions, G‡ ~ 54 kJ mol-1 in
the absence of the catalyst, ~ 24 kJ mol-1 with catalyst
Therefore reaction rate increases twofold every 10ºC
without catalyst, only 1.35-fold with catalyst
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Carbohydrate
catabolism
Typically starts with glucose
Broken down to two three-carbon
molecules called pyruvate with net release of modest
amounts of energy in the form of ATP
In aerobic organisms (like us, but unlike some of our
gut bacteria) pyruvate is converted to acetyl CoA and
then subjected to a cycle of reactions called the
tricarboxylic acid cycle, producing considerable ATP
and converting much of the carbon to CO2
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Where did the glucose come from?
We mentioned storage polysaccharides
earlier
Glucose is derived from those
One glucose unit at a time is split off from
starch or glycogen, phosphorylated, and
then inserted into the glycolysis pathway
and the TCA cycle
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Carbohydrate anabolism
In photoautotrophs, CO2 is captured out of the air
and condensed with a C5 compound called
ribulose bisphosphate to make two C3
compounds, netting one extra carbon that
eventually enables production of more glucose or
other C6 and C5 sugars
Chemoheterotrophs get sugars or building blocks
from food
These are built up to glucose 6-phosphate and
then used to make glycogen or starch
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Regulation of these pathways
All of these systems are carefully regulated
Enzymes are set up so that futile cycles (build up 
break down  build up again, losing energy each
time due to inefficiencies) are rare
Hormonal regulation assists in turning on catabolism
when energy is needed, anabolism when storage
molecules are needed
Spatial compartmentation helps too:
catabolic reactions mostly occur in mitochondria and
peroxisomes, anabolic reactions mostly in cytoplasm
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Lipid catabolism
Fatty acids are broken off of triacylglycerol (common
name: triglycerides)
Resulting fatty acids are oxidized, 2C at a time
Final products: acetyl CoA and considerable ATP
Lipids are more than twice as efficient energy sources
than saccharides or proteins
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The raw energy calculations show twofold preference
Lipids are stored almost without water, whereas sugars
and proteins are surrounded by water
Other catabolic pathways for lipids yield energy too
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Lipid anabolism
Fatty acids are built up by adding two carbons at a
time to acetyl ACP using malonyl ACP as the 2-carbon
donor; one CO2 molecule gets lost for each cycle
Requires a lot of ATP equivalents for energy
The cycle involves five enzymes, each of which must
contribute to each 2-carbon growth
Builds up to C16 or C18; then enzymes release products
But until then the reactions are very tightly coupled
because the enzymes are grouped together in large
(~3 megadalton) complex called fatty acid synthase:
it’s bigger than the ribosome!
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Steroid
anabolism
Pathway starts by converting acetyl CoA through a
couple of steps to hydroxymethylglutaryl CoA
This HMGCoA is starting material both for steroids and
for energy-yielding metabolites called ketone bodies
Steroid pathway converts to C5, then 2 C5 -> C10, then
add another C5 to get to C15.
Two C15 molecules come together to make a C30, which
then gets extensively remodeled to make cholesterol
Almost all other steroids are derived from cholesterol
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Amino acid
anabolism
Many amino acids are built
up by adding nitrogen to
TCA cycle intermediates
Nitrogen comes from
NH3 or from amino transfers:
aa1 + -ketoacid2  aa2 + -ketoacid1
A few bacteria can grab N2 from the air and
convert it to NH3: energetically expensive
process!
Glutamate (a medium-sized ribosomal amino
acid) is a critical intermediate in making many
of the other amino acids
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Essential & nonessential
amino acids
Higher organisms have lost the pathways
required to make ~ half of the 20 amino acids
They must get the missing half from breaking
down proteins in their diet
Amino acids that we can’t synthesize are called
essential amino acids
Healthy diet requires a minimum amount of
each of the essential amino acids
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Why can’t we make all 20?
It takes energy to synthesize amino acids
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Most of them are present in a mixed diet
High correlation with the amount of energy
required:
Essential amino acids require > 35 ATP
hydrolyses
Complex pathways require complex
regulation
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Amino acid catabolism
Intact proteins are broken down into oligomeric
fragments and then down to individual amino
acids through the action of peptidases or
proteases (enzymes that cleave peptide bonds)
Amino acids are either recycled or deaminated
and converted in the TCA cycle intermediates
Nitrogenous component (~ ammonia) is typically
excreted; see nucleic acid catabolism, below
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