06/23/22 Biochem in Rad Bio p. 1 of 54 Illinois Institute of Technology Physics 561 Radiation Biophysics, Summer 2014 Lecture 11: Biochemistry Blitz Andrew Howard
Dec 30, 2015
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Illinois Institute of Technology
Physics 561 Radiation Biophysics, Summer 2014
Lecture 11: Biochemistry BlitzAndrew Howard
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Why biochemistry matters
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 Thermodynamics Organic chemistry Small biomolecules Polymers Proteins Enzymes Carbohydrates Lipids & Membranes
Metabolism (general) Carbohydrate metabolism Lipid metabolism Amino acid metabolism Nucleic acid metabolism Replication Transcription Translation
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Biochemical unity
There is a remarkable unityto 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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Biochemical diversity
The morphological andfunctional differencesbetween you and anelephant, or between youand a flowering plant, orbetween you and a bacterium, arise from differences in biochemical phenomena
– Some classes of proteins are present only in certain kinds of organisms
– Structural components in vertebrates differ significantly from those in arthropods
PDB 1emyElephant myoglobin
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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 - TS
G
ReactionCoordinate
G
T
RP
G‡
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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
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:
– 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
unrepresentedClaisen condensation
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What reactions do occur?
Mostly nucleophilic substitutions (SN1 and SN2)
– 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– There are enzymatic free-radical reactions– But there are non-enzymatic ones too
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Chirality
Many biomolecules are chiral:– 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:– 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)
– 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:
– 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 orcarrier-protein binding site involves< 5% of the amino acids in the protein
So what are all those other amino acids doing?– 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 tookuntil the 1930’s for that notion to bedefinitively accepted
In the 1970’s this assertion had to be modified: 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
James Sumner
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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:– 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:– Control of transcription– Control of translation– Interference with activity via inhibitors
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Enzyme kinetics
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.
v0
[S]
Vmax
1/[S]
1/v0
1/Vmax
-1/Km
Inherent properties of enzymes
Km is an inherent property of an enzyme– 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– 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 seven-
carbon polyalcohols with one carbonyl group at either C1 or C2
Simplest: glyceraldehyde & dihydroxyacetone - C3
Most important other ones:– 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– 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 oftwo 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)
sucrose
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Storage polysaccharides
Starch: storage form of glucose in plants– Amylose: 1-4 linkages only– Amylopectin: mostly 1-4 linkages, a few 1-6
Glycogen: storage form of glucose in animals, some bacteria
– 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 complexedeither 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:
– 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– 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:– 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: organicCO2
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?
– By converting CO2 to organic molecules (autotrophs)
– By intake of organic molecules (heterotrophs)
Whence do they get their energy?– 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:– Almost all animals– Many bacteria, some archaea
Chemoautotrophs: get carbon from CO2 but get energy from inorganic compounds or heat vents in ocean
– 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
– 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
– 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– 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 builtup by adding nitrogen toTCA cycle intermediates
Nitrogen comes fromNH3 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– 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