Hubi 2001 Midterm 2 Flashcards
Nucleoside
nucleobase bound to pentose (without phosphate)
why do enzymes not impact equilibria?
they impact fwd and rev rxns equally, and are not used in the process
Glycan abbreviations
Trioses
Tetroses
Pentoses
Hexoses
Heptoses
What is the codon table?
3 nucleic acid bases make up a codon
each codon encodes an amino acid (more than one codon for some amino acids)
What is the V label in IgG represent?
Variable regions
What does higher G:C content correlate with in DNA?
More stability
What Ig class(es) activates complement, i.e. destroy invaders
G, M
Helicase
separates 2 strands of DNA
What are the two faces representing orientation about the glycosidic bond?
1) Watson-Crick Face (anti, normal)
2) Hoogsteen Face (syn, after rotation)
Another term for immunoglobulins
Antibodies
True or False: RNA structure may use Watson-Crick base pairing depending on how they’re sterically positioned.
True
What is the symbol for pentoses?
Star
Is DNA parallel or antiparallel?
Antiparallel
What are the 4 types of catalysis
1) catalysis by approximation
2) general acid-base catalysis
3) covalent catalysis
4) metal ion catalysis
How do enzymes overcome the solvation shell of the substrate?
solvation shells impede rxns; desolvation replaces solvation shell with weak bonds btwn substrate and enzyme (replace by bonds w enzyme)
What factors do mixed inhibitors affect?
Usually both apparent Km and Vmax
Vmax decreases when the effective [E] decreases
Km may increase or decrease, depending on enzyme form
Does blood type depend on glycans or nucleic acids?
glycans
Activation energy
Difference btwn ground state and transition state energy levels
True or False: IgG can activate other parts of the immune response, in example the complement system that pokes holes in the pathogen.
T
Ig Classes (5): include the letter, heavy chain, nickname, and % in blood
1) G: gamma, IgG, 75%
2) M: mu, IgM, 10%
3) A, aloha, IgA, 15%
4) E, epsilon, IgE, 0.002
5) D, delta, IgD, 0.2%
What is rRNA
makes up ribsome; responsible for synthesis of proetins
Which of [ES], V0, vmax, [S], and kM cannot be measured experimentally easily?
[ES]
People with pollen allergy start out with hay fever, but then develop oral allergies to a defined group of foods. This tells us that these foods and pollen all share a common what?
Epitope
What happens in induced fit when the antigen is bound?
The antibody’s binding cavity is enlarged and several sidechains shift in position
How to do ELISA plate for the antigen (do not describe fully, just difference from the antibody)
Antigens from patient coat the surface of plate, and a specific antibody to HIV is added
What would the GalB1-4 Glc glycosidic linkage look like
gal on left, glc on right
connected through O between 1 C of Gal and 4 C of Glc
Review structures of nitrogenous bases
What is the most common DNA modification?
Methylation
What are the structural requirements for a protein with recognition and/or effector functions?
They must have domains for recognition and effectance
Guanine Quadraplex
forms at the end of chromosomes, regulates transcription
4 guanines H bond w both their Watson-Crick and Hoogsteen faces
Regulation of Chymotrypsin
Dependent on pH; optimal pH 8
Regulated by proteolytic cleavage (chymotrysinogen: inactive, stored in secretory vesicles, released following nerve stimulation)
What do catalysts do? (simple)
lower the activation energy
How does the induced fit mechanism of enzyme catalysis work?
a) the enzyme assumes a conformation identical to substrate
b) the enzyme undergoes a conformational change to maximize weak interactions to substrate
c) the substrate binds to the active site of the enzyme
d) the enzyme undergoes entropy reduction to accommodate substrate
B
Who predicted double helix structure and what technique did they use?
Rosalind Franklin and Maurice Wilkins, XRD
What is the glucose modification to make N-acetyl-glucosamine?
C2 N-acetyl
Check L20 slide 10
How to prepare monoclonal antibodies and who did this first
Cesar Milstein and Georges Kohler
Isolate a clone of B cells to produce a single, identical antibody. Issue: these cells isolated from an animal die quickly, however, there are immortal cell lines that do this derived from a type of cncer (multiple myeloma).
To get antibodies that bind a target protein, fuse short-lived antibody producing cell with a myeloma cell.
Hybridoma cells screened to find one that targets the protein of interest. A clone can be grown in a large scale culture to produce lots of monoclonal antibodies of itnerest.
Apoenzyme
protein part of a holoenzyme
What are the 7 most common modifications? (and what amino acids)
1) Phosphorylation (Y, S, T, H)
2) Adenyylylation (Y)
3) Acetylation (K, N terminus)
4) Myristoylation (N-terminus)
5) Ubiquitination (K)
6) ADP-ribosylation (R, Q, C, H)
7) methylation (E)
How does an antibody bind a macromolecular antigen?
There are different antibodies which bind to distinct areas on the surface of the macromolecule
The conversion of sucrose to CO2 and water has a very large and negative dG, but sugar left out doesn’t spontaneously turn into CO2 and water. Why?
There is a large activation energy barrier; kinetically disfavoured
True or False: RNA and DNA are polymers of nucleotides
True
Mixed inhibition (aka non-competitive)
binds at a site distinct from active site
binds to either E or ES complex
increasing [S] does not overcome the inhibition
Study diagrams in L18
What does the Fab label in IgG correspond to?
F for fragment, ab for antigen binding
True or False: enzymes bind in a “lock and key” fashion
False
Binding in the active site
major source of free E to lower activation energy through non-covalent interactions which stabilize the interaction
What does RNA do?
carries genetic info from the nucleus to the cytosol
Which part of the immune system is the one that makes vaccines effective?
Adaptive
What is an example of covalent catalysis?
Citrate synthase (review slide)
What is the name of Nucleoside and Nucleotide of guanine for RNA and DNA?
RNA: guanosine, guanylate
DNA: deoxyguanosine, deoxyguanylate
What are a couple examples of non-coding RNAs?
tRNA
rRNA
miRNA
siRNA
lncRNA
snRNA
snoRNA
Review the process of DNA replication
What does the double dagger represent?
Transition state
What can we use monoclonal antibodies to study?
Antibodies themselves. Also used for identification of other molecules
Do we prefer polyclonal or monoclonal antibodies?
Monoclonal, easier to work with
3 General Regulatory Mechanisms
1) allosteric control: rev, noncovalent binding (sec to min)
2) reversible covalent modification: min to hrs
3) proteolysis: irreversible, removal of peptide segments
How do enzymes lower free E of transition state?
It uses binding E from interactions btwn enzyme and transition state, not substrate
What is coenzyme A?
A nucleoside acting as a cofactor
What bonds link sugars?
Glycosidic bonds
What is bound to IgG?
Carbohydrates, attached to CH2 domain, stabilize structure
Where do antibodies bind antigens?
Complementary determining regions (CDRs)
Humoral Immunity
immunity driven by molecules in solution in the body (antibodies); recognize invading pathogens
Michaelis-Menten equation
rate eqn for one substrate enzyme catalyzed rxns
V0 = vmax[S] / (KM + [S])
What are the two ways to test if someone is infected with HIV?
1) do they have antibodies against HIV?
2) do they have a particular antigen known to be produced early in infection, the p24 protein?
What Ig class(es) causes histamine release
E
Equilibrium
rxn has no net change in conc of reactants or products
What Ig class(es) crosses placenta
G
Antigen
any substance capable of eliciting an immune response
What label do we give regions in IgG that are less variable?
C
How many angstroms between base pairs in DNA
3.4
tRNA
bring amino acids to ribosome for protein synthesis
Lineweaver-Burk Equation
algebraic transformation of Michaelis-Menten equation to convert hyperbolic curve into linear form
1/V0 = Km/Vmax[S] + 1/Vmax
How do we detect HIV antibodies using ELISA?
HIV antigens coated onto ELISA plate, patient serum added to plate. Non-antigen binding proteins are washed free of plate.
Second antibody added with an enzyme, that will detect the presence of the primary antibody. A substrate for the enzyme is added, and the enzyme acts on it by changing color.
Measure the intensity of the colour; the darker the color, the more antibodies in the origianl sample. No color change - no HIV.
Rank the strength of base pair stacking
Strongest: G:C - G:C
Middle: A:T - G:C
Weakest: A:T - A:T
What are the two types of the immune system?
1) innate immunity
2) adaptive immunity
Explain how influenza virus works using glycans
Infectious diseases use glycans on outside of cells to choose their host organism; influenza binds to glycans terminated with a2-6-linked sialic acid
Review L19 slide 18
What technique is best for quantifying the amount of Ag in a sample?
ELISA
What is the triangle symbol used for?
Deoxysugar
Holoenzyme
complete catalytically active enzyme with its bound coenzyme and/or metal ions
What Ig class(es) are in B lymphocyte membrane
M, D
What type of plot has the fastest v_0?
Plot with steepest tangent line at t=0
What nitrogen base is present in deoxythymidine?
Thymine
What is the specificity constant?
ratio of kcat/km
best way to compare turnover of different substrated by same enzyme
lower km = higher affinity the enzyme for S and higher rate of conversion of S to P
What does catalytic activity of enzymes depend on?
Conformation / strucutre
Activity lost if subunits dissociate or denatured (usually)
Where are proteins made in the cell?
Cytosol
Helix rise per base pair in A, B, Z forms
2.6, 3.4, 3.7
Which statement is true bout both mixed inhibitors and uncompetitive inhibitors?
a) both bind to the active site of an enzyme
b) both affect Km, but not Vmax
c) both can bind to ES complex
d) both can bind to either free enzyme or ES complex
C
How do enzymes align catalytic groups in enzyme
The enzyme undergoes conformational changes when the substrate binds, induced by multiple weak interactions, known as induced fit
What factors do competitive inhibitos affect?
Apparent Km (increased with [I])
Not Vmax
Why would the addition of an enzyme make a rxn faster?
a) active site provides area of increased pH
b) prosthetic groups act as additional substrates
c) cofactors act as additional substrates
d) amino acid residues help bring substrates together
D
Phosphodiester linkages
bonds in nucleic acid polymers; can only be added to 3’ end
Epitope
particular part of antigen bound by the antibody
Irreversible inhibitors
impact an essential functional group in enzyme
bind covalently, destroys groups, forms stable association
What are the 9 biologically relevant monosaccharides in humans? List their symbols
1) Glucose (Glc, blue circle)
2) Galactose (Gal, yellow circle)
3) Mannose (Man, green circle)
4) N-Acetylglucosamine (GlcNAc, blue square)
5) N-Acetylgalactosamine (GalNAc, yellow square)
6) L-Fucose (Fuc, red triangle)
7) Sialic Acid (Sia, purple diamond)
8) Xylose (Xyl, orange star)
9) Glucuronic acid (GlcA, half blue/half white diamond)
Innate Immunity
1st line of defense; foreign particles eliminated (within hours); no memory; detects patterns, not molecular differences
True or False: complementary DNA strands are antiparallel, so we write the DNA label backwards to get the 5’-3’ direction
True
Fractional saturation formulas (2)
= [ES] / [E_T]
= [S] / (KM + [S])
see derivation slide 26 L17
What is the name of Nucleoside and Nucleotide of adenine for RNA and DNA?
RNA: adenosine, adenylate
DNA: deoxyadenosine, deoxyadenylate
Carbohydrate abbreviations
Monosaccharide
Disaccharide
Oligosaccharide (<12 units)
Polysaccharide (>= 12 units)
Primer
sets the template for DNA replication
Where do small molecules usually bind?
In the cleft of the antigen binding region
How many angstroms per turn in DNA
36
How is DNA read vs. synthesized/replicated?
read in 3’ to 5’ direction, replicated in 5’ to 3’ direction
What are some features of polyclonal antibodies? (4)
1) they are heterogeneous
2) they have different amino acid sequences
3) they recognize different regions of the target protein
4) they bind the target protein with different strengths
Proeoglycan vs. Glycoprotein
Proteoglycan: 50-60% carb content; in connective tissues
Glycoprotein: 10-15% carb content; cell surfaces; recognition and signaling
Most common source of chemical energy (hydrolysis of phosphate bond releases energy in this form)
ATP
What form does IgA take
monomer, dimer, or trimer
IgG binds to an invading bacterium or virus, and through the Fc region, activates certain white blood cells to engulf and destroy the invader. What function of the immune system is this?
Effector
Two kinds of antigens
1) small molecules (contact with around 15 residues of antibody)
2) macromolecules (20 or more residues of the antibody)
What factors do uncompetitive inhibitors affect?
Both apparent Km and Vmax
Vmax and Km decrease with increase in [I]
What happens if you add mercaptoethanol and urea to IgG? i.e. how many chains will the protein break into?
The protein will break into 4 separate chains
Glycobiology
study of glycan functions related to their structures, recognition, biosynthesis, and molecular context
What are the differences in DNA and RNA structure?
Only difference is in the sugar, ribose (OH group on 2’ and 3’ C) vs. deoxyribose (no OH group on 2’ C)
Covalent catalysis
transient covalent bond btwn enzyme and substrate; enzyme with nucleophilic group
Glycan
polysaccharide that does not have nutrient role; carbohydrate portion of glycoconjugate
What Ig class(es) is found in secretions
A
For what conditions is the Michaelis-Menten equation valid?
[S]»_space; [E], i.e. [ES] is constant, steady state assumption
List the 7 classes of enzymes and what rxns they catalyze
1) oxidoreductases: transfer of e- (hydride ions or H atoms)
2) Transferases: group transfer (adding)
3) Hydrolases: hydrolysis (water)
4) lyases: cleavage of C-C, C-O, C-N, or other bonds by elimination, leaving double bonds or rings, or addition of group to double bond
5) Isomerases: transfer of groups within molecules to yield isomeric forms
6) Ligases: formation of C-C, C-S, C-O, C-N bonds by condensation rxns coupled to cleavage of ATP or similar cofactor
7) Translocases: mvmt of molecules or ions across membranes or their separation within membranes
How do pigs transmit viruses from birds to humans?
they have glycans making them susceptible to human and avian viruses
Competitive Inhibition
competes with substrate for active site, depends on [S]:[I] ratio. Can be overcome by increasing [S]
HIV Vaccine Development
There are a few rare antibodies from asymptomatic, HIV infected people that show a neutralizing responses (b12) —- read rest of HIV slides
Enzyme
-biomolecule catalyst
-specific chemical rxn
-increases rxn rate
-unchanged
-usually a protein, occassionally RNA
-does not change spontaneity of a process
The barrier to rxn may be due to a variety of factors, including: (4)
1) the entropy of molecules in soln (low prob of interaction)
2) solvation shell that surrounds and stabilizes biomolecules in soln
3) the distortion of substrates
4) need for proper alignment on enzymes
Adaptive immunity
Occurs only after exposure; delayed response, but specific; memory is rapid and effective
What monosaccharide is made from removing the C6 of glucose?
Xylose
What is the sugar in DNA?
2’ deoxyribose
Coenzymes
type of cofactor; complex organic or metalloorganic molecules that act as transient carriers. Usually derived from vitamines
Which statement is not associated with covalent catalysis by enzymes?
a) when the rxn is complete, the enzyme returns to its original state.
b) it never involves coenzymes.
c) a transient covalent bond is formed btwn enzyme and substrate
d) a new pathway from substrate to product is formed that is faster than the uncatalyzed rxn
B
What is an advantage of polyclonal over monoclonal?
They are easier to make
How do initial velocities change with increasing concentration?
Increase
How many times can inhibitor molecules act, and what are serpins?
Only once
Serpins: serine protease inhibitors (use large conformational change to trap protease)
Explain how IgE releases histamines and initiatives an allergic response
Through Fc region, to special receptors on mast cells, which leads to secretion of histamines. This causes dilation / increased permeability of blood vessels, facilitating movement of immune cells and proteins to site of inflammation
Example of small molecule antigen
Phosphorylcholine; review how it binds
General acid-base catalysis
transfer of protons to/from unstable intermediate to stabilize an unfavourable charge.
Base tilt normal to the helix axis in A, B, Z forms
20deg, 6deg, 7deg
What is the diameter of DNA in angstroms
20
True or False: if [S] is very high, we would expect v0 to approximately be equal to vmax
T
See mathematical derivation (L17 slide 14)
In samples of DNA isolated from two unidentified species of bacteria, X and Y, adenine makes up 32% and 17%, respectively, of the total bases. One of these species was isolated from a hot spring (64C). Which species is most likely to live in the hot spring?
Y
How do enzymes work?
Active sites
How to prepare polyclonal antibodies
Inject an antigen into a rabbit twice, 3 weeks apart, which stimulates the reproduction of cells producing antibodies that recognize it. Blood is drawn from rabbit, and the serum of the blood contains antibodies to all antigens the rabbit has been exposed, some of which recognize the injected protein.
What factors determine specificity and affinity of antibody for antigen?
1) shape of binding site: complementary, often concave
2) amino acid sidechains of binding site - should maximize noncovalent interactions
What of the typical role of carbs does not apply to glycans?
Acting as an energy source
Review structure diagrams of IgG
Are G:C or A:T base pairs stronger?
G:C
How do the number of polysaccharides for 3 different monosaccharides compare with the number of polypeptides for 3 amino acids
polysaccharides: 1056 possible structures
polypeptides: 6 possible structures
How many domains are there in Ig and how many different domain folds are there?
12 domains, all the same domain fold
Why is it important to control enzymes?
Some processes should only occur controllably
Rate of rxn
speed at which rxn takes place
Enzyme Commision
number that provides progressively finer classification
What does the label Fc correspond to in IgG?
F for fragment, c for crystallization, since it crystallizes readily
Which technique is best for determining the intracellular location of a protein?
Immunofluorescence
Why is DNA replication important?
To create new genetic info 4 cells to grow and divide
What are 2 main types of irreversible inhibitors?
1) transition state analogs
2) suicide inhibitors
How does a protein recognize another molecule?
It binds to it using a number of very specific noncovalent interactions
Problems with DNA damage
if damage is not repaired, it can lead to permanent changes to DNA, causing diseases such as cancer
Glycosyl bond conformation for A, B, Z forms
Anti, Anti, Anti for pyrimidines, syn for purines
How many polypeptide chains are in IgG? How are these divided?
4
2 heavy chains
2 light chains
What are the two important terminal sugars for glycans and what functional groups do they have?
1) Sialic acid: 6-Glycerol, 1-carboxyl, and 5-acetamido side chains
2) L-Fucose, a 6 - methyl group
What enzymes break glycosidic bonds?
glycohydrolases
Is the symbol for N-acetylhexosamine a square or a circle?
Square
What happens if DNA is mutated?
a) it can’t be replicated anymore
b) it gets degraded
c) point mutations arise in the protein
d) there are no consequences
C
What are the 3 parts of RNA and DNA structure?
1) pentose sugar
2) nitrogenous base
3) phosphate group
How do strands of DNA form double helix?
Hydrogen bonds
What enzyme helps make glycosidic bonds
glycosyltransferases
Key features of antibodies
Binding and recognition of foreign molecules
Do allosteric enzymes display Michaelis-Menten behaviour?
No, they diverge
Suicide inactivators
action requires enzymatic mechanism
reacts like substrate for first few steps, but is then converted into a new compound that binds irreversibly to enzyme
Which technique is best for determining the size of a protein?
Western blot
What is the diamond symbol used for?
Acidic Sugars
What is the complementary DNA strand of ATG?
CAT
DNA Melting, tm
when DNA is 50% denatured
What does an enzyme impact?
Reaction rate
Types of glycoconjugates (4)
1) glycoprotein
2) proteoglycan
3) glycolipids
4) glycoRNA
Induced Fit
conformational changes can occur upon ligand binding. Allows for tighter binding of the ligand
Glycoconjugate
covalent modification of another molecule with glycan
Transition state
highest E state; decay to substrate/product are equally likely
Phosphorylation of enzymes:
a) always increases their activity
b) is irreversible
c) is one of only five types of covalent regulation
d) generally occurs on Ser, Thr, Tyr side chains, His happening less frequently
D
Uncompetitive inhibition
Not very common
Binds at a site distinct from active site, and only binds to the ES complex
Increasing [S] does not overcome this
What 2 functions must the immune system possess?
1) recognition: recognize foreign material
2) defence: initiate a response to protect against foreign material
What is the diameter of A, B, Z forms of DNA?
A: 26Angstroms
B: 20A
Z: 18A
Immunoglobulin Diversity
Each Ig needs a unique sequence to allow it to bind a particular antigen; can produce more than 10^8 different antibodies with distinct binding specificities
DNA polymerase
works in 5’ to 3’ direction to replicate DNA
Role of the immune system
Defend the body against bacteria, viruses, fungi, and toxic materials
What does Km depend on
substrate and enzyme
T or F: vo is dependent on [S] when [S] is low
T
see derivation, L17 slide 15
What is different about immunoglobulins compared to other proteins?
a) they are composed of several structure domains
b) they can have different primary sequences
c) they can bind specifically to other proteins
d) they are composed of beta-sandwich type domain folds
B
IgG has a permanently attached carbohydrate, that means IgG is (choose as many as apply):
a) glycoprotein
b) hemoprotein
c) conjugated protein
d) allosteric protein
e) kinase
A and C
Immunofluorescence
an antibody specific for a specific protein is attached to a molecule that fluoresces.
Why is heterogeneity a drawback of polyclonal antibodies?
Makes them unnecessarily complicated
What Ig class(es) activates phagocytosis
G, M
What is the role of Fab vs. Fc in IgG?
Fab = recognition
Fc = effector
The best antibody raised against the peptide KRVLFK would have what mix of residues?
A mix of negatively charges and hydrophobic residues
Rate limiting step
highest activation energy step of a multistep rxn
What are active sites?
specific area in enzyme where reaction happens. Bind to specific substrates with specific amino acid residues
Nucleic acids
DNA and RNA
central dogma of biology
source of E (ATP)
cofactors (NADPH)
What is vmax in Lineweaver-Burk plot?
y-intercept = 1/vmax
2 types of general acid-base catalysis
1) specific: uses only H+ or OH- from water
2) general: mediated by weak acids or bases other than water, usually in the enzyme
Enzyme inhibitors and the 2 classes
Molecules that interfere with catalysis, slowing or halting rxns
1) reversible
2) irreversible
Practice Math examples: L17 slide 16,17,24, and 27
Cofactors
inorganic ions that binds to enzyme to assist in binding
Ground state
starting point for forward or reverse reaction
What are the 3 factors of complementary DNA strands?
1) antiparallel
2) complementary
3) major and minor groves (see diagrams on L20 slide 23)
Describe ELISA
Enzyme-Linked immunosorbent assay
used to measure if or how much of an antigen/antibody is in a sample.
Ex) HIV
Western Blotting
aka immunoblotting
for proteins that have been separated by gel electrophoresis
similar strategy to ELISA:
primary antibody, selective for protein of interest. Secondary antibody, binds primary antibody and catalyzes a reaction to produce a color change
True or False: tighter binding is achieved by less interactions that are more complementary
False; we want more interactions to have more complementary binding surface
Immunoglobulins
Proteins produced by the immune system
How do enzymes overcome the barrier of rxn due to entropy?
Productive collisions are rare; enzymes increase collisions by restricting relative motion of substrates and holding in proper orientation for rxn
Immunoglobulin G
the major class of antibody molecules; one of the most abundant proteins in blood
Which of the following statements is true?
a) glycoproteins have a higher % of carb than proetoglycans
b) glycolipids are inside the cell
c) synthesis of glycans controlled by enzyme regulation
d) glycans do not form glycoconjugates with RNA
C
What would we call a nucleoside with 1/2/3 phosphates?
1-Nucleotide
2- nucleoside diphosphate
3-nucleoside triphosphate
How many base pairs per turn in DNA
10.5
DNA ligase
fill in breaks where primers were used in DNA replication
Metal Ion Catalysis
1/3 of enzymes need metal ions for catalytic activity, can be prosthetic groups or cofactors. They help orient the substrate and stabilize transition states.
Metal ions mediate oxidation-reduction rxns via reversible changes in their oxidation state (LEO says GER)
Reactive amino acids
Found in active sites of enzymes
used in general acid-base and covalent catalysis
Glu, Asp, His
Lys, Arg, Cys, Ser, Tyr (less common)
How do allosteric enzymes work to regulate?
Undergo conformational changes in response to modulator binding; can be positive (activators) or negative (inhibitors)
Chargaff Rule
in DNA (not RNA), #A=#T, #G=#C
What is the symbol for hexoses?
Circle
Proteolytic Cleavage (zymogens and proenzymes)
Some enzymes exist as inactive precursors called zymogens; cleavage forms an active protease enzyme, often used to amplify a signal
Precursors where cleavage forms other proteins are called proenzymes
What is a coenzyme that is very tightly bound to the enzyme?
Prosthetic group
Allosteric enzymes:
a) are never regulated by substrate binding
b) have their activity changed by changes in intersubunit interactions
c) exhibit Michaelis-Menten kinetics
d) always have both inhibitory and activating modulators
B
What is the domain fold of Ig called?
B-sandwich/immunoglobulin fold
Two sheets of antiparallel B-sheets
What type of bonds link light and heavy chains of Fab fragment, and chains of Fc?
The disulfide bonds that link the light and heavy chains
What are the stop (3) and start (1) codons?
Start: AUG (Met)
Stop: UAA, UAG, UGA
What is an example of a macromolecule?
Lysozyme; read through lysozyme example
what is kcat and the formula?
kcat=vmax/[Etot]
turnover number; limiting rate of rxn at saturation
number of substrate molecules converted to product in a given unit of time on a single enzyme molecule, when the enzyme is saturated
What monosaccharide is modified to make N-acetyl-galactosamine and what is the modification?
Galactose; C2 N-acetyl
Km
Michaelis constant; [S] at 1/2 vmax
Approximates cellular substrate concentration
Topoisomerase
prevents supertwisting
Review free E diagrams for enzymes
Which type of inhibitor binds to the active site of an enzyme and undergoes the initial steps of rxn before combining irreversibly with the enzyme?
Suicide inactivators
What is the primary source of the energy enzymes used to reduce activation energies?
Noncovalent enzyme-substrate interactions
What is the most common secondary structure in RNA?
Hairpins
Consequences of DNA Damage
Damaged DNA leads to C*:G and C:G daughter strands
Damaged mutagenic DNA C:T base pair leads to CT and A:T daughter strands
No way to differentiate A:T from damage and natural A:T
Enzymes:
a) are always proteins
b) can be denatures and retain activity
c) may require multiple subunits for full activity
d) are also referred to as coenzymes
C
Is the interaction surface larger and/or flatter for small molecules or macromolecules?
Macromolecules
Stereochemistry of Carbs
carbs are chiral, with D form being biologically relevant (opp of proteins). Chair conformation; less hindered in equatorial positions
3 biochemical methods that make use of monoclonal antibodies
1) ELISA
2) Western Blotting
3) Immunofluorescence
Initial Velocity
Tangent to each curve at time t=0 (mM/min)
Why do C and A/T not form H bonds?
see L20 slide 18 for diagrams
What is a protein made by B lymphocytes?
Immunoglobulins, also called antibody or gamma globulin
True or False? DNA structure is more complex than RNA structure
False
How does induced fit in antibodies lead to tighter binding?
Allows complementary groups to interact fully
Pyrimidines
Pyrimidine, Cytosine, Thymine, Uracil
How would we identify a nucleic acid using a code for a phosphate group followed by NA with adenine, cytosine, guanine, thymine, and then adenine again?
pACGTA
Different classes of antibody
multiple effector regions to bind to antigens; each class has its own unique heavy chain, giving it a different and characteristic effector function
What are alpha and beta conformations of carbs?
beta on top (equatorial)
alpha on bottom (axial)
How do we name glycosidic bonds? (5 steps)
1) Name non-reducing left-most sugar
2) name anomeric configuration (alpha or beta)
3) name anomeric carbon number (i.e. where is the glycosidic bond)
4) name substituted carbon number
5) name substituted sugar
What is a problem with basing HIV tests on detecting anti-HIV antibodies?
Some people don’t have anti-HIV antibodies, if they are early in the infection
Purines
Purine, Adenine, Guanine
Types of DNA damage (not rxns, 6) and examples
1) SIngle base damage (oxidative stress, alkylation, hydrolysis)
2) mismatches (replication errors and stress)
3) pyrimidine dimers (T or C bases) (UV light)
4) Interstrand and intrasand cross-links, bulky adducts (environmental mutagens, chemotherapy)
5) single strand breaks (reactive oxygen species)
6) double strand breaks (ionizing radiation, endogeneous endonucleases)
What is Km in Lineweaver-Burk plot (2 eqns)
slope = km/vmax
x-intercept = -1/km
What do higher tm values mean
strands are held together more strongly, i.e. higher G:C content
Nucleophile vs. electrophile
Nucleophile: e- rich, donates e-, attacks bonds
Electrophile: e- deficient, accepts e-, pulls e- toward their side of the bond
How do some nucleotides act as signalling molecules?
Second messengers inside the cell; commonly the cyclic form is used
Hybridoma cells
a mixture of cells from the spleen fused with myeloma cells.
What two classes of Igs have similar structure to IgG?
IgD and IgE
What do the variable amino acid regions of IgG represent?
The regions that bind to the antigen; they are so variable because of the specificity
Many people with mild egg allergies are okay eating egg as long as its well cooked. Why?
a) cooking gets rid of the protein (lysozyme) that people are allergic to.
b) cooking disrupts the primary structure of lysozyme, so it cannot bind to the antibody.
c) cooking disrupts the tertiary structure of lysozyme, preventing the antibody from recognizing it.
C
In early studies of IgG, it was found that IgG was cleaved into how many fragments of what mass by papain?
3 50kD fragments
Roles of Carbohydrates (6)
1) Structural
2) Energy source
3) nucleic acid structure
4) lubricants (joints)
5) cell recognition
6) disease states
What statement about Ig effector functions is false?
a) Fc fragments that have been cleaves off of their Fab fragments can not interact with the molecules that are part of the effector systems
b) Fc fragments from different classes of antibodies often interact with different components of the immune system
c) Fab fragments bind to antigens but can’t mount an immune response alone
A
Where is DNA in the cell?
Nucleus
What form does IgM take?
membrane-bound, or a cross-linked pentamer
Ribozymes
RNA that catalyzes rxns
Roles in cleaving phosphodiester bonds and formation of bonds
found in ribosome; joins amino acids together
What monosaccharide is made from C6 acid of glucose?
Glucoronic acid
How do enzymes overcome the distortion of the substrate
Weak interactions with enzyme can compensate thermodynamically for the unfavourable energy from distortions. They can also induce strain from their active sites within a bound substrate to increase reactivity and favor formation of transition state
Catalysis by approximation
binding with multiple substrates and positioning them favorably for reaction, reduced rotational entropy and improves catalytic rate
100x faster
carbonic anhydrase
How many base pairs per helical turn in A, B and Z forms?
11, 10.5, 12
What is the coding RNA?
mRNA
Transition state analogs
mimic substrate transition state, and can bind very tightly to enzyme
ex) inhibitors to HIV protease (slide 17 L18)
True or False: sequences are written from 3’ to 5’, left to right
False
Reversible covalent modification
Add on a modifying group (enzyme catalyzed, conformational changes to alter amino acid interaction)
Second enzyme removes group
Carbohydrates
most abundant and diverse biopolymer in nature
heterogeneous in size and composition
hydrophilic
carbon hydrate
general formula Cx(H2O)y
What 2 forms of DNA are right handed and what form is left handed?
Right: A and B
Left: Z
Glycolipids
membrane components; found on the outside of the cell membrane; antigenic; 2% of membrane lipids
What does binding energy provide?
Specificity: specific matching between enzyme and substrate
What N base is present in uridylate?
uracil
What are 6 classes of DNA damage reactions?
1) oxidation
2) deamination
3) alkylation (can be natural)
4) cross-linking (UV)
5) minor groove adduction
6) major groove adduction
What is saturation in v0?
When further increases in [S] have no effect on the rate
Of A, B, and Z, what is most common DNA type?
B
What is DNA modification used for?
Plays a role in regulating and protecting genetic information
Which statement is False about the saturation effect?
a) the maximum initial rate of the catalyzed rxn is observed
b) virtually all of the enzyme is present as the ES complex
c) it is responsible for the linear increase of V0 with an increase in [S]
d) further increases in [S] have no effect on rate
C
General structure of purines
5 membered ring linked to 6 membered ring
Okazaki fragment
how DNA is replicated in the lagging strand
What is the glucose modification to make Galactose?
C4 Epimer
What are the 4 ways binding energy overcomes the initial barrier
reduces entropy
substrate desolvation
substrate distortion
aligns substrate with active site
Heterotopic allosteric enzymes
activators may cause curve to become more hyperbolic
inhibitor may cause curve to become more sigmoidal
True or False: IgG is a conjugated protein
True
Competitive inhibitors:
a) compete with other inhibitors for the active site
b) occupy the active site to exclude the substrate
c) are rarely similar in structure to the substrate
d) often combine with the enzyme to form a reactive EI complex
B
What sugars equilibrate between open chain and ring forms?
Free sugars / monosaccharides
T or F: determining Vmax is always easy.
F
Reversible inhibition and 3 types
rapid dissociation from enzyme
1) competitive
2) uncompetitive
3) mixed
What is the name of Nucleoside and Nucleotide of cytosine for RNA and DNA?
RNA: cytidine, cytidylate
DNA: deoxycytidine, deoxycytidylate
General structure of pyrimidines
6 membered rings with substituents
Enzyme classification / nomenclature
suffix -ase
7 classes based on type of rxn
systematic name describes function
trivial name based on function, location, rxn, etc
What are two types of immune system cells?
1) T Lymphocytes (cellular immunity)
2) B lymphocytes (humoral immunity, white blood cell)
