Biochemistry Exam 2 Flashcards
exergonic reaction
releases energy as a part of the reaction
endergonic reaction
needs energy for the reaction to take place
Enzymes are proteins that catalyze chemical runs by
Binding Site Ligand Specificity Temperature sensitivity pH Sensitivity
Binding Site
active sires are binding sites that contain residues with high affinity for substrate and are the site of catalysis
allosteric sites are ligand binding sites that influence enzyme conformation and thus catalytic activity
Ligand
ligand is the reactant and the substrate, or just the molecule that binds to active site and undergoes chemical change
allosteric regulator is a molecule that binds an enzyme outside the active site and influences catalytic activity
Specificity
Specificity varies with enzyme, some enzymes have a single active site that is highly specific for one form of ligand, while other enzyme active sites can accommodate multiple substrates
Examples of specificity
Hexokinase- enzyme that has 4 isozymes
Hexokinase I/A has a wide tissue expression, and catalyzes hydrolysis of ATP followed by phosphorylation of a hexose in the reaction
Hexokinase IV/D or glucokinase - selectively expressed in liver, pancreas, smal intestine, and has a lower affinity for glucose
it only catalyzes rxn with glucose
Isosyme
catalyzing the same chemical reactions but all have different amino acid sequences, tissue expression patterns, affinity for substrate
Temperature Sensitivity
each enzyme has an optimal temp. humans is at 35-40 degrees C. Higher temp results in faster molecule motion and catalysis where as cooler temp results in slower motion and catalysis
too much heat will denature a protein
pH Sensitivity
active sites contain amino acid residues with distinct ionization stats
altering the hydrogen ion concentration in solution will alter the ionization and therefore denature the protein
Apoenzyme
the protein portion of the enzyme
Prosthetic Group
chemical component, either a metal ion or molecule is called the prosthetic group.
it is usually covalently bonded to the protein
How enzymes work
enzymes catalyze a reaction by increasing the rate at which a reactant is converted into product
the key to catalysis is the formation of ES, enzyme plus substrate
Proof of ES existing before P
electron microscopy and x-ray crystallography
physical properties of an enzyme change upon binding substrate
high specificity for substrate
ES can be isolate in pure form
when enzyme is saturated with substrate, the rate of product formation is constant
Enzyme Thermodynamics
catalyze the conversion of energy forms
there must be a energy source to do the work of conversion
-dG is spontaneous, +dG is non-spontaneus
Ways to drive run are by energy coupling and equilibrium influences
Energy Coupling
combine a chemical reaction with excess free energy, or -dG with a +dG rxn
dG
the magnitude of G depends on how far from equilibrium you system is initially
Keq
equilibrium constant reflecting molar concetration of product/reactant at equilibrium
if Keq is large then the reaction favours the products and is more likely to go to completion
if Keq is small then the reaction favours the reactants and is less likely to go to completion
S–>P
goes thru a transition state, in which the molecule is no longer S or P
the energy create the transition molecule is not calculated in the final dG b/c this energy is release upon making product
we get a value of energy that represents the amount needed to activate the rxn known as energy of activation
E + S going to ES
substrate binding to the active site lowers the activation
it is down by multiple weak non-covalent bonds that release energy upon substrate:active site binding, satisfying the energy debt by enzyme
Structure of the Active Site
binds substrate, but not in a perfect complementary fit
with non-covalent bonds, conformation of both substrate and enzyme change forming induced fit
as substrate is encountered, binding energy is low and reaches a maximal binding energy to satisfy activation energy debt
enzyme thus most complementary to the transition state
Active Structure dictates function
non-covalent binding energies are driving force of catalysis
microenvironment of active site is non-polar, unless water is used as a substrate
-in a non-polar envirnment, polar residue have special catalytic or binding properties
active site residues are oriented due to 3D folding; this gives more range of motion in the active site compared to steric hinderance seen in primary structure
1st order reaction
when the rate of reaction is directly related to concentration of substrate
Zero order reaction
when rate is independent of substrate
usually when substrate is much greater than enzyme
Second order reaction
when rate is dependant on 2 substrates
Steady State Assumptions
- consider only the forward reaction
- measure rate from initial introduction of enzyme
- enzyme concentration is constant
- substrate is typically greater than enzyme concentration
- concentration of ES stays the same
- Vmax can’t be obtained b/c of diffusion, temp, pressure
E + S ES
is a fast reversible reaction
rate of formation is k1
rate of dissociation is k-1
ES E +P
is a slower reversible reaction
rate of formation is k2
rate of dissociation is k-2
What is the overall reaction rate proportional to
The concentration of ES, the product formation is the rate limiting step due to slower reaction of ES to P
due to the steady state assumption we assume negligible product reversion to substrate, so k-2 approaches 0
How do we know ES proceeds to P
initial reaction rate is dependant on rate productiomn formation of ES
ES can be formed and broken down, and in steady state k values for ES are equal
k1 [E][S] = (k-1+k2)[ES]
M and M developed a rate constant to represent the proportion of ES breakdown/formation, km
we cant measure [ES], but in vitro we have a known [E]t that is equal to the amount of [E] and [ES] together
Michaelis and Menton equation vs. Lineweaver Burk
from the M and M algebraic equation gives a hyperbolic curve. To predict km and Vmax using MM equation you need several points to make an accurate line
Lineweaver Burk is a double reciprocal plot of MM equation that creates the equation y=mx+b, and for this graph is requires fewer data points and you also know the x and y intercepts
Using km and kcat
km reflects the affinity for an enzyme for substrate ONLY when k2 is much smaller than k-1
if the dissociation of enzyme and substrate together is high, then affinity of enzyme for substrate is low
if k2 is greater than k-1, then km is not a measure of affinity
As enzyme kinetics grow in substrate and transistion states, the number of rate constants increase
Not all enzymes have the same RLS and can be dependant on each other, kcat measure the overall rate of catalysis
kcat/km is a measure or catalytic effeciency
by determining the ratio of kcat/km we know the rate of catalysis and affinity, most use is comparing an enzyme preference for substrate
The most efficient enzymes have limitations to Vmax due to diffusion in water
Allosteric Enzymes
do not obey MM kinetics
have multiple binding sites on a single enzyme or holoenzyme that influences the velocity of enzyme activity in the presence of substrate
The two type of allosteric enzymes are homotropic and heterotropic
Homotropic Enzyme/Allosterism
Binding of one substrate alters the conformation of the enzyme such that the second substrate binds more cooperatively
Converting from T to R state with each bound substrate increases the velocity, creating a sigmoid curve
Cooperativity allows for more regulation
Heterotropic Enzyme/Allosterism
non-substrate molecules binding outside the active site to influence velocity
they can be positive or negative
in stimulation the R state is favoured, Vmax/2 is achieved with less substrate
in inhibition the T state is favoured, Vmax/2 is achieved with more substrate
Inhibition
enzymes can be inhitbited by specific molecules, altering catalytic rates measured by classic MM kinetics
molecules can be reversible when they are joined by non-covalent interactions, the types of inhibitors have a rapid dissociation from the [EI] complex
molecules can be irreversible when they are joined with covalent or strong non-covalent bonds, key is the inhibitor dissociates at a very slow rate
eg. Suicide inhibitor such as penecillin form covalent bonds at the catalytic site and prevent further use of enzyme
Inhibitors such as asparin make a covalent modificattion to cyclooxygenase
Competitive Inhibition
A competitor [I] competes with substrate for the active site
- inhibitor resembles the substrate
- no product is formed
- does not change the Vmax b/c sufficiently high [S] can overcome inhibition
- creates apparent increase km making rxn slower b/c it appears the affinity for substrate is reduced when reality says enzyme is bound by inhibitor
on the LIneweaver Burk graph the Vmax does not change but the km increases and moves towards the y axis (x increases)
Naturally Occuring competitive examples
Digitalis from foxglove bind to NaK ATPase
Tetradotoxin from puffer fish binds Na channels
Cytochalasin B from fungus binds glucose transporter
Atropine from nightshade binds acetycholine receptor
Synthetic Competitive Inhibitors
Sulfanilimide is an antibacterial that inhibits folate synthesis
Neostigmine inhibits acetylcholinesterase, prolongs neuromuscular transmission in diseases like myasthenia gravis
Indinavir inhibits HIV protease II to treat HIV infection
Uncompetitive Inhibition
Inhibitor binds the ES complex, at site distinct from the active site
- inhibitor only binds ES complex, so if [E] > [I] some enzyme activity will occur
- Vmax decreases b/c functional [E] decreases
- Apparent km decreases b/c ES is functionally inactive. As less active enzyme is available, less substrate is required to load enzyme and thus apparent km decreases
In the Lineweaver Burk the km decreases (moves from y axis, x decreases) and the Vmax decreases (moves up y axis)
Uncompetitive Inhibition Example
Glyceraldehyde 3-phosphotase Dehydrogenase (GAPDH) is one of 10 enzymes in the process of glycolysis (6th step)
GAPDH binds 3 substrates in the precise following order ebfore making product, glycerate 1,3 biphosphate
-NAD+
-Glyceraldehyde 3-phosphate
-PO4-
AsO4- is an uncompetitive inhibitor b/c it has similar shape as PO4- and b/c PO4- is the last in the order above the ASo4- binds the ES complex
Non-Competitive Inihibtion
Inhibitor and substrate bind simultaneously at distinct binding sites, thus inhibitor binds either the E or ES complex
-Vmax decreases b/c functional enzyme is depleted from solution
km is unchanged b/c active site is not altered
-common theme is feedback inhibition
On the Lineweaver Burk the km does not change bu the Vmax is decreasing (moves up the y axis)
Non-competitive example
Heavy metal poisoning causes wide spread denaturation of proteins (Lead, Mercury and sliver). React with sulfhydryl groups on cysteines to cause misfiling of tertiary structures of proteins
Naturally occuring- Caffeic acid found in tomatoes inhibit lypooxygenase
Caffeine found in tea and coffee inhibits cAMP phosphodiesterase Synthetic
Synthetic - trichostatin A inhibits histone acetyltransferase and is anti-cancer
Mycophenolic acid inhibits inosine monophosphate transferase and used to treat flavivirus infections like west nile virus and dengue virus
Suicide Inhibition
Compounds that bind to the active side of an enzyme and undergo chemical steps of the reaction, but form a covalent or stable non- covalent bond with the enzyme
-compounds would be substrate analogs, and thus you would not anticipate competitive inhibition graphs
-since suicide inhibitors inactivate the enzyme, Vmax decreases
-suicide inhibitors do not affect the km
Therefore, suicide inhibitors would have a graph similar to non-competitive b/c inhibition cannot be relieved at high substrate concentrations
Suicide Inhibtion of Aspirin
Aspirin as a substrate analogs for cyclooxygenase-1 (best inhibition) and cyclooxygenase-2 (induced specifically by cytokines and other immune stimulation).
COX enzymes normally bind archaadonic acid to make prostaglandins (PGG) which are pro-inflammatory precursors
Aspirin acetylates a serine residue in the upper channel of the active site preventing arachodonic acid binding
Suicide Inhibition of Monoamine Oxidase
MAO is important for breaking down dopamine and seratonin. In disorders such as Parkinson’s and depression, which less neurotransmitter is made, inhibiting MAO is therapeutic leaving the dopamine and seratonin in the system longer
N,N dimethylpropargylamine makes a covalent bond with the flavin prosthetic group in MAO
Catalytic Strategies
Enzyme catalyzed reactions begin with substrate binding
Binding Energy not only ensure optimal structural associations, but also facilitates catalysis
Induced fit dictates that the ES complex is oriented such that the nucleophiles are able to attack electrophiles, and the electrophile is in approximation with the nuc.
4 Types of catalytic Strategies
Covalent catalyis - the active site contains a powerful nuc that leads to a covalently bound transition intermediate
General Acid-base catalysis - a molecule in the active site other than water is the proton donor/acceptor.
Metal Ion catalysis - a metal ion may facilitate the formation of nuc, or the metal ion itself may be the elec. or nuc. A metal ion may also participate in increasing the binding energy by orienting the substrate for catalysis.
Catalysis by approximation - ensures that 2 distinct substrates are localized together along 2 active sites of an enzyme
The Protease
Protease are enzymes involved in digestion, but also in AA scavenging at protein turnover
All proteases us the process of hydrolysis
-the covalent peptide bond and the covalent H-OH bond is broken. The unpaired electrons resulting from the broken bonds are covalently joined, such that the H bonds with the amino group and the OH bonds with the carbonyl carbon
Proteins are more stable than other molecules containing carbonyl groups b/c the bond resonance about the peptide bond stabilize the bond as well as decrease the reactivity of the carbonyl carbon
Chymotrypsin as a Serine Protease and the 3 components
Chymotrypsin uses 2 catalytic strategies: acid/base and covalent catalysis 3 components of the active site: -hydrophobic pocket -oxyanion hole -catalytic triad
Hydrophobic pocket
to attract Trp, Tyr, Phe or Met.
the hydrophobic pocket aligns the protein such that the peptide bond between the hydrophobic residue and the next residue is oriented next to the catalytic site of serene.
Oxyanion Hole
near the catalytic site has protruding amino group that form stabilizing H-bonds with oxygen of the substrate carbonyl group, for chymotrypsin, the amino group are the from the catalytic serene and an adjacent amino group from glycine.
Catalytic Triad
the catalyze both the acid/base and covalent catalysis reactions
- His in a serine prtease is unprotonated and forms an H-bond with the hydroxyl of Serine
- -If the pH is less than 8, His is protonated and no longer supports base catalysis
- The NH of the imidazole ring of His forms a stabilizing H-bond (salt bridge) with Asp. This makes the His a better proton acceptor for base catalysis to extract the H from an alkoxide ion on Serine.
Mechanism of Chymotrypsin
The goal is to hydrolyze peptide bond in 2 steps
- covalent acylation of enzyme (trade H from serene hydroxyl for the carbonyl group of the peptide)
- Water dependant deacylation (restore H to serine and form carboxylic acid of the peptide)
Protease Homologs
Trypsin and elastase also use the catalytic triad to hydrolyze protein but their residue binding pockets differ
Cysteine, aspartyl, and metalloproteases also provide nucleophilic attack on the carbonyl group of a peptide
Serine hydrolase enzymes attack the carbonyl carbon
Trypsin
binds positively charged lysine and arginine
Elastase
binds short residues
Cysteine proteases
papain in papaya, are important as cathepsins in immune system and caspases in apoptosis pathways
Aspartyl proteases
are important in blood pressure (renin) and digestion (pepsin)
Metalloproteases
are important in tissue remodelling and degradation
Serine Hydrolase
Acetylcholinesterase is a serine enzyme that uses the same mechanism as trypsin and chymotrypsin to hydrolize acetylcholine
acytelcholinesterase is an enzyme at neuromuscular junction and cholinergic synapses that hydrolyze acetylcholine, a neurotransmitter
Carbonic Anhydrase
uses metal ion catalysis, incorporating Zinc into the active site to make bicarbonate
-CO2 and H2O rapidly make bicarbonate without catalyst, but carbonic anhydrase makes this reaction proceed at the limit of diffusion
Zinc is bound by 3 histidines in covalent coordination, and has a 4th coordination bond with water
Catalytic Strategy of Carbonic Anhydrase
- an adjacent histidine serves as a proton shuttle to abstract the proton from water to create a reactive nucleophile that will attack the carbon of CO2
- the hydroxide ion attacks carbon of CO2 and forms HCO3-, still bond to zinc
- incoming water displaces the HCO3- from zinc
- to restore the protonated histidine proton shuffle, which can regenerate the hydroxide nucleophile, a buffer removes the H+
Chemical Modifications of Enzymes
Acetylation, Myristoylation, ADP ribosylation, Ubiquitination, Adenylation, Methylation, Phosphorylation
Acetylation
chromatin remodelling regulation
metabolic enzyme regulation
Myristoylation
targets the enzyme to the membrane to localize a cell signalling event
ADP ribosylation
chromatin remodelling
cholera toxin in an ADP riboslyase that modifies G proteins to cause dairrhea
Ubiquitination
targets proteins for degradation -or- involved in complex cell signalling events
Adenylation
nitrogen sensor regulating glutamine amidotransferase
Methylation
histone/chromatin modification
Phosphorylation
1/3 of all eukaryotic proteins are phosphorylated. Transfer of phosphoric groups occur via prtein kinase, acquiring the gamma-phosphate from ATP or another nucleotide. The phosphate is transferred to a Ser, The, Tyr or occasionally a His.
Phosphorylation can be stimulatory or inhibitory, and cumulative phosphorylations can lead to subtle, yet important modifications of enzyme activity, especially in cell signalling events
Phosphorylation can be reversed by phosphotase, and enzyme that removes the phospate
Glycogen Phosphorylase
is an enzyme in muscle and liver that release glucose-1-phosphate monomers from glycogen polymers
It is regulated by phosphorylase kinase and phosphotase
the more active the enzyme has a phosphorylated serene and causes greater enzyme activity
Zymogen
is an active enzyme precursor that must be made active by chemical modification
- most commonly activation of zymogens is proteolysis cleavage of a regulatory portion of the amino acid sequence that exposes the active site
- proteolytic cleavage of symogens is irreversible, so further regulation involves inhibition strategies
- fibrinogen is a zymogen in blood converted to fibrin to form blood clots. Proteolysis is initiated by the serene protease thrombin. Thrombin is also a zymogen in blood, activated by serene proteases upstream, also zymogens, which are triggered to activate by vascular insult
nucleic acids
are polymers of nucleotides linked by phosphodiesterase bonds
nucleotides
are phosphate esters of nucleoside
the phosphate is bonded to one of the pentose hydroxyl groups
nucleosides
are N-glycosides of either D-ribose or 2-deoxy-D-ribose
N-glycosides
are a purine or pyrimidine nitrogenous base attached to the anomeric carbon via a glycosidic bond
Pyrimidine
a nitrogenous heterocyclic base
- cytosine
- thymine
- uracil
Purine
a nitrogenous heteocyclic base that contains fused pyrimidine and imidazole rings
- adenine
- guanine
Making a Nucleoside
Nucleosides are a product of a dehydration reaction b/w nitrogenous base and cyclized ribose
-the NH group forms a glycosidic bond with the C-1 of the pentose. Water and a C-N bond forms
Making of a Nucleotide
Nucleotides are a product of a dehydration reaction between a nucleoside and a phosphate
-creates the nucleotide and water
Unusual Nucleosides
- 5-methylcytidine is an epigenetic marker that protects against restriction enzymes in bacteria and is part of regulation of gene expression in plants and animals
- N6 methyladenosine is also an epigenetic marker, but recently, this modification has been found in mRNA as well as DNA
- N2 methylguanosine stabilizes RNA
- 5-hydroxylmethylcytidine is also an epigenetic marker, recently discovered, and plays a role in pluripotency
- Inosine is a precursor in purine synthesis but also found in tRNA to support wobble base pairing in translation
- Pseudourine helps initiate translation
- 7 methylguanosine is the 5 cap of mature mRNA exiting the nucleus and is protected from cytoplasmic nucleases
- 4 thiouridine is a photo-protective base found in tRNAs
Making a Nucleic Acid
are a product of a dehydration reaction between 2 nucleotides
-the products are the nucleic acid and water
DNA vs RNA
- Deoxyribonucleic acids lack the hydroxyl at the 2’ carbine of the ribose, while the ribonucleic acid retains the hydroxyl
- DNA is more stable than RNA, in alkaline solution, the 2’ OH of an RNA polymer can cause intramolecular displacement of the phosphate
Nucleic Acid Properties
-nucleic acids containing less than 50 nucleotides are called oglionucleotides
-purines and pyramidines are weakly basic, thus called bases
-all nucleotides absorb UV light and can be characterized by peak absorptions near 260nm
-though purines and pyrimidines have nitrogens, carbonyl groups, and exocylic amino groups, they are hydrophobic at physiological pH, Hbonds occur via the functional group and non-covalent interactions among bases in a nucleic acid cause stacking like coins along the polymer
-H bonding patterns among the functional groups of purine and pyrrimidines were established by watson and crick, these distinct H bonding patterns only occur with another nucleic acid
Chargaff established the A=T and G=C among all known species, A only bonds to T using 2 H bonds and C only bonds to G using 3 H bonds
Deoxyribonucleic Acid Structure
- hydrophobic bonding, hydrophillic sugar-phosphate makes contact with water while hydrophobic bases are stacked nearly planar, bases are stacked tightly at 3.4 A per base pair supported by Van De Waal
- One purine one Pyrimidine, to satisfy X ray diffraction studies width can only be this combo
- Complementary Base Pairs A=T, C=G
- DNA Polymers are antiparallel, for best fit H bonds
- Watson and Crick double helix, B form DNA
- Periodicity, helix forms a major and minor groove, minor represents one set of base paris in the same plane, major represents one full turn of helix
- Forces holding helix together, major contribution from hydrophobic non-specific base stacking interaction and then by H bonds b/w base pairs
Other DNA Structures
- Structural rotation in the sugar phosphate backbone, and free rotation at the glycosidic bond allows alternate helix structures, purines can be anti, or sun positions; In B-form they take on anti.
- A form DNA does not exist in nature but is favoured in solutions lacking water, so this structure occurs in labs working with crystals
- Z form DNA is left handed, and does occur in nature based on DNA sequence motifs, such as CG repeats or 5-methylCG repeats
DNA Packaging
-A chromosone is a linear DNA helix in complex with protein
the helix would be about 3.2 cm long but the average diameter of a nucleus is 6um
-chromatin is filamentous DNA in complex with histone proteins and non-histone proteins
-DNA undergoes supercoiling, or coiled coils. Histones support the supercoiling
the basic unit of DNA packaging is the nucleosome, containing 200 bp DNA,
histone modifications which help tighten or relax DNA occur at the amino terminal tails
RNA Structures
-RNA forms a single stranded polymer, and takes on a right handed curve
supported by base stacking interactions, purine-purine stronger than pyrimidine interactions
-palindromic and inverted repeat sequences are complimentary
single strand RNA will acquire various structures based on complement pairing
Nucleic Acid Denaturation
- DNA is highly stable at pH 7 and 25 C
altering pH disrupts ionic and H bond interactions, increasing temp increases kinetic energy causing denaturation, no covalent bonds are broken during denaturation
-After temp and pH manipulation is removed the DNA will spontaneously rewind, or anneal
the RLS in annealing is complementary base pairing by random collision
-™ is the mid point, or melting point at which half of the DNA has been denatured due to temperature, there is more energy in GC base pairs due to the 3 H bonds
Nuceic Acid Chemistry Mutations
- Deamination
- Depurination
- UV Irradiation
- Alkylating agents
- Reactive Oxygen Species (ROS)
Deamination
spontaneous loos of the exocyclic amino group on purines or pyrimidines
DNA containing the thymine is an adaptive advantage b/c deamination of cytosine to uracil occurs about one in every 10’7 residues every 24 hours, deamination of 5-methylcytosine make thymine, chemical deamination also occurs upon reaction with nitrate nitrite and nitrosamine
Depurination
creates lesion in DNA in which phosphodiester bond remains, but hydrolysis reaction removes the nitrogenous base. If DNA repair mechanisms don’t address deprivation upon replication the daughter cell strand will be 1 bp short
UV Irradiation
can induce 2 ethylene groups to form cyclobutane. Thymine dimers create kink in the DNA and upon replication, only one thymine is used for replication and daughter cell is again 1 bp short. The light energy excites a cohort of electrons in a particular molecular formation which move the e from is ground state to a higher orbital making it chemically reactive
Alkylating Agents
modify bases and prevent base pairing. DMSO makes 06-methylguanine which does to pair with cytosine
Reactive Oxygen Species
(ROS) are the best source for DNA damage, the hydroxyl radicals cause the most damage
catalase, superoxide dismutase, antioxidants and other similar enzymes the neutralize ROS
-oxidation of base pairs (attacking C=C bonds to make OH-C-C)
-phosphodiester bond breaks
-oxidation of deoxyribose, in general the oxidation generate a base that can’t form the correct complement and upon replication leads to a base substitution, oxidation of a base can also create a product that in turn attacks the deoxyribose causing a strand break
Nucleotides as Chemical Energy
Hydrolysis of anhydride bonds yield more than double the energy of ester bonds
Nucleotides as Enzyme Co-factors
- mant structurally unrelated coenzymes contain adenosine
- adenosine does not play a role in catalysis, nor is any part of adenosine transferred to a substrate
- adenosine appears to play a role in binding energy
- adenosine is seen above other nucleosides due to cellular economy; similar motif already used in cellular energy that can be easily obtained in coenzyme formation
Nucleotides as Regulatory Factors
- second messengers are small soluble molecules that can easily diffuse in the cytoplasm, they are release upon extracellular protein:ligand binding to amplify the extracellular signal
- most common, adenyl cyclase catalyzes the conversion of ATP to 3’ and 5’ cAMP, adenyl cyclase is associated with the plasma membrane and regulated by intercellular components of a transmembrane receptor
- cGMP is used similarly to cAMP less commonly
- ppGpp is a bacterial regulator of RNA synthesis
DNA Replication
Requires the following:
- template DNA
- DNA polymerases, not more than one
- dNTPs, must be triptosphates to provide an energy source
- support enzymes such as helicases, topiosomerases, primes, ssDNA binding proteins, nuclease, and ligase
- maintenance enzymes (DNA repair)
Semiconservative Replication
DNA replication follows a semiconservative model established by Meselson-Stahl
-the 2 DNA strands separate
-each strand is uses as a pattern to produce a complementary strand, using specific base pairing
1 generation after cells growing in heavy nitrogen were transferred into media containing N14, 100% was the hybrid
-each new DNA helix has one old strand and one new strand
still growing in N14 the second generation has 50% hybrid and 50% pure
DNA Replication
-DNA replication begins at the origins of replication, specific sequences of DNA to which proteins attach for replication
-DNA helicase separates the DNA double helix, but this creates stress on the helical structure
-Topoisomerase binds upstream and unwinds DNA to relieve helical stress from helicase
-ssDNA binding proteins stabilize and protect ssDNA from nucleases
-DNA replication occurs in the 5’->3’ direction
replication is continuous on the 3’->5’ template, replication is discontinuous on the 5’->3’ template, forming short segments called okazaki fragments
DNA polymerase III problem
- is the primary enzyme required to replicate DNA. The restrictions of replication are due to the catalytic activity of the enzyme
- Problem is that DNA polymerase III cannot add new nucleotides without an existing 3’ end of the nucleotide
- Solution is that Primase is an enzyme that adds 5-10 RNA molecules complementary to parent DNA during replication, thereby providing a 3’ end for DNA Polymerase III
- DNA polymerase enzymes require a primer because in the catalytic site, the 3’ OH becomes a nucleophile for the a-phosphate on the incoming dNTP
RNA:DNA hybrid problem
RNA:DNA hybrids cannot be part of the fully synthesized DNA molecule
-Solution, nuclease will degrade RNA. Upstream of the RNA, DNA pol I will add new dNTP’s. DNA pol I uses a 3’ OH from DNA polymerized by DNA pol III fro the replication fork
Problem making the final covalent bond
DNA polymerase cannot make the final covalent bond of the nucleotide polymer when it approaches an existing 5’ end
-solution, DNA ligase makes the final covalent bond
Telomere Problem
At the telomere for the lag gin strand (3’ to 5’)
-primase generates a primer complementary to the last 5-10 nucleotides of the parent DNA, but DNA pol III does not have any template to add new dNTPs
-RNA:DNA hybrids are not supported, nuclease degrades both primer and parent DNA
-with several rounds of replication, chromosomes become shorter, and genes at telomeres are not properly expressed
-aging or cellular death occur
Solution; is an enzyme that adds random nucleotides to both telomeres of each chromosome using an RNA template. These nucleotides do not code for protein, and are degraded by nuclease with each round of replication. The lost nucleotides are replaced once again by telomerase after replication
-stem cells and some cancer cells express telomerase
DNA Mutations
Silent-Substitution-No effect on the protein coding region
Sense-Substitution-Mutation that changes a termination codon and produces a longer protein
Nonsense-Substitution-Mutation that creates a termination codon, shortening the protein
Missense-Substitution-Mutation that substitutes one amino acid for another
Insertion-Frameshift-Inserts one or more nucleotides and alters the protein coding region
Deletion-Frameshift-Deletes one or more nucleotides and alters the protein coding region
DNA repair
repair involves nuclease-mediated excision not only of the damages nucleotide but also of flanking DNA. The undamaged DNA strand is used as a template for DNA pol I to add new dNTP and DNA ligase to make the final phosphodiester bond
-the DNA repair enzyme is specific to the damage, and is the primary nuclease, secondary nuclease are seen in cases as DNA glcosylases, which remove only the mutated base. An AP endonuclease must remove the a basic nucleotide
Transcription
DNA dependant synthesis of RNA is carried out by RNA polymerases, near identical catalytic site of DNA pol
Promotor - DNA sequence that does not code for proteins but serves as a docking site for RNA polymerase initiation
Terminator - DNA sequence that terminates RNA polymerase association with DNA
Enhancer - regulatory DNA sequence on the same chromosome or different chromosome that activators bind to increase transcription
Repressor - regulatory DNA sequence that repressors bind decrease transcription
Distinct RNA Polymerases
RNA polymerase I - ribosomal RNA synthesis
RNA polymerase II - messanger RNA, the protein coding RNA
RNA polymerase III - transfer RNA used in translation and specializes RNA’s
-all the RNA polymerase promoters differ, and for RNA pol III, regulatory regions are located within the transcript itself
-RNA polymerase transcribes in the 5’->3’, due to catalytic site specificity. RNA Polymerase can bind to either strand of the DNA helix for a given gene but each strand of DNA produces a different mRNA transcript and protein
Regulating transcription
- promotor strength wares among genes, transcriptional activity is at a low basal level. for most systems categorized, transcription initiation is a positive regulation to increase gene expression from basal levels
- heterochromatin is highly condensed chromatin that prevents transcription machinery from binding promoter
- euchromatin is relaxed chromatin that allows transcription machinery to bind
- each cell has a unique pattern of heterochromatin and euchromatin to regulate cell-specific gene expression
RNA processing
the RNA transcripts produced by RNA polymerases are immature and require chemical modifications and splicing leaving nucleus to be translated in the cytoplasm:
- ribozymes, RNA structures with catalytic activity remove introns and join axons to make continuous protein coding sequence
- to avoid degeneration by nucleases, a 5’, 5’ triphosphate linkage of 7-methylguanosine is added to the 5’ end of the transcript
- a 3’ poly-tail allows mRNA fidelity and further nuclease protection, an endonuclease cleaves pre-mRNA at the 3’ mRNA sequence AAUAAA, polyadenylate polymerase binds this sequence and adds 80-250 adenosine triptophates
snRNP
small nuclear ribonucleoprotein that composes a spliceosome. Spliceosomes initiate the catalytic spicing of pre-mRNA
Intron
sequence of pre-mRNA that does code for protein, but may have regulatory sequences guiding RNA splicing
Exon
sequence of pre-mRNA that codes for protein
Translation requires the following
- mature mRNA
- genetic code
- tRNA
- aminoacyl-tRNA synthetase
- small subunit ribosome, which loads mRNA
- large subunit ribosome, which contains translational catalytic activity
- release factor
Codon
3 nucleotides and also equals 1 amino acid
The Genetic Codon
is:
- Universal
- Degenerative, a single amino acid may be specified by more than one codon
- No “punctuation” or spaces, and ORF, open reading frame is the sequence of DNA starting with AUG and ending with one of the 3 stop codons. ORFs are divisible by 3.
tRNA
is the molecule to translate nucleotides to amino acid
- anticodons complement base pair with the codons in mRNA
- the 3’ CCA tail is loaded with an amino acid by aminoacyl transferases
- tRNA contains unusual nucleosides, sometimes occurring in the anticodon, these can form H bonds with mRNA nucleotides but are weak
- other regions of tRNA associate with rRNA for orientation support in the ribosome
tRNA Wobble
for 61 codons there are only 32 tRNAs
the 3rd base pair in a codon:anticodon complex pairs loosely
-this 3rd bp wobble allows for reduced diversity of tRNA to satisfy the 61 codons
-wobble also increases efficiency of translation, as a fully H bonded codon takes more energy to break
Rules of the wobble:
- first 2 bases of mRNA codon always form strong Watson-Crick bonds and confer most coding specificity
- when the 5; anticodon is C or A, base pairing is specific and only 1 tRNA is used
- when the 5’ anti-codon is U or G binding less specific: U= A or G and G = C or U
- when the 5’ anti-codon is Ionisine, it pairs with either A,U or C
- when an amino acid is specified by several codons, the codon that differ in the first 2 bases require different tRNA’s
Aminoacyl-tRNA Synthetase
- before initiation of translation, each of the 20 amino acids must be linked by an ester bond to their corresponding tRNA via the 3’ A
- each aminoacyl-tRNA synthetase is specific for one amino acid and one or more tRNAs
- a second genetic code is established b/w synthetase and tRNA to ensure specificity of aminoacylation
- each tRNA differs in sequence at the amino acid and anti-codon arm, and a few other places as well
- aminoacyl-tRNA synthetase is an ATPase
Eukaryotic Translation Initiation
- the small subunit ribosome loads mRNA, but 11 regulatory factors, in addition to mRNA ensure proper loading
- translation initiation involves loading the tRNA before loading mRNA
- mRNA loading is ATP-dependant
- GTP hydrolysis is required to initiate loading of the 80S large subunit
Practical Application of Translation Initiation
- mature mRNA contains a 5’ and 3’ untranslated region that form secondary structure recognized by regulatory proteins
- AUG codes for methionine and is the start codon for all known species on the planet, so it is the first nascent protein
- the first eukaryotic methionine does not differ from the subsequent methionines in the protein
- the tRNA for the start codon is different from internal tRNA
- the first prokaryotic methionine has a chemical modification, a formyl group is transferred to distinguish it from others
- humans have an immune receptor for this called fMet-Leu-phe receptor, fMLP initiates chemotacis toward the bacterial peptides, also respiratory burs by white blood cells which is an increase in oxygen consumption to generate reactive oxygen species to destroy bacteria
Eukaryote Translation Elongation requires
-ribsomal initiation complex
-aminoacyl tRNAs, forms a complex with the initiation factors and GTP and then associated with the peptidyl site of ribosome
-three elongation factors
-GTP, it becomes hydrolyzed and the elongation factors are released
Peptide bond formation is carried out by the peptidyl transferase ribozyme activity of rRNA,
Transloaction requires the 3rd elongation factor, EF2, a translocase using energy provided by a second GTP
Transloaction editing occurs in the time it takes for GTP hydrolysis. The codon:anticodon base pairing guides editing
Eukaryote Translation Termination
termination codons are recognized by release factors
-RF-1 binding UAG and UAA and RF-2 binds UGA and UAA
Upon binding termination codons, release factors induce hydrolysis of the terminal peptidyl-tRNA bond
The free tRNA is released to the cytosol
Release factor binding also causes dissociation of the 40S and 60S ribosome subunits
Post-translation modifications
Mascent polypeptides undergo folding and proteolysis and/or chemical modification before full protein function is attained
- 50% of protein lose the amino-terminal methionine and/or is N-acetylated after translation
- 15-30 amino residues comprise the signal sequence, which localizes a protein in the cell. This sequence is eventually proteolytically cleaved
- chemical modification of residues. Ser, The, Tyr Hydroxyl groups are phosporylated. Glu is carboxylated. Lys is methylated
- Carbohydrate side chains, lubricatind proteoglycans have N-linked ogliosaccharides to Asn, or O-linked ogliosaccharides to Ser or The residues
- Isoprenyl groups. cholesterol isoprene intermediates are covalently attached to Cys residues
- prosthetic groups are covalently bound (heme, biotin)
- proteolytic processing to generate multiple proteins or to activate an enzyme
- disulfide cross-links help prevent against denaturation or serve as oxidizing agents
Protein Degradation
- the lysosome contains hydrolytic enzymes that act on extracellular proteins engulfed by cells or membrane proteins within cytosol, and aging organelles
- vesicles can fuse with the lysosome
- chaparones, such as oxidative stress-induced heat shock proteins can deliver soluble proteins to the lysosome membrane for selective translocation into the lysosome
Ubiquitination
is a process that targets proteins for degradation in a proteasome
- E1 activating enzyme makes a covalent bond with the highly conserved 76 residue ubiquitin protein
- ubiquitin is then transferred to E2 conjugating enzyme
- E3 ligase is highly specific to its target and transfers the ubiquitin for E2 onto the target
- polyubiquitinated proteins are targeted for degradation by a 26S proteasome, with regulatory caps and a barrel structure that degrades the protein to short peptides
