Ackkerman Flashcards
Define the characteristics of enzymes
- Accelerate reaction rates but do NOT change the equilibrium;
- Decrease the activation energy of the reaction (stabilize the transition state)
- proteins or ribozymes (RNA molecules)
- 25% of human genes are coded for enzymes
- Highly specific in reactions they perform and in their substrates
What is the difference between the kinetic and thermodynamic parameters of chemical reactions?
For a given reversible reaction:
Kinetic: Activation Energy (energy from the reactant ground state and the transition state) determines the reaction rate; says nothing about the preferred direction. Note, Ea is the barrier in EITHER direction
Thermodynamic: only one direction releases free energy (G) and is the spontaneous reaction, and this occurs only if the difference in free energy between the reactant and the product is a negative value; SPONTANEITY IS INDEPENDENT OF THE PATH TAKEN TO COMPLETE THE TRANSITION IN THE RXN.
What is the transition state?
the activated species with the highest energy (T*)
What happens when the energy of the reactants is greater than the energy of the products?
the delta G is negative and so the reaction releases energy (exergonic) and spontaneous.. opposite for when the delta G is positive
What effect does an enzyme have on the kinetic and thermodynamic barriers?
Kinetic: enzyme decreases the activation energy, which in turn increases the reaction rate IN BOTH DIRECTIONS because the transition state is at a lowered energy level; speeds up the reaction (For each direction)
SINCE THE REACTION RATE IS INCREASED IN BOTH DIRECTIONS, the thermodynamic barrier is the same for the catalyzed and uncatalyzed reaction (Keq DOES NOT CHANGE)
Why is the MIchaelis Menten reaction plot of initial velocity vs. [S] for an enzyme hyperbolic and not linear?
Linear: without an enzyme the velocity (speed of reaction) increases linearly with the reactant concentration
Hyperbolic: in enzymes the hyperbola indicates saturation kinetics; at a fixed enzyme concentration a non-linear relationship between velocity and reactant concentration; because at a certain point the rate of the reaction is independent of the concentration of the [S] (at Vmax). This is because the rate of the reaction is limited by the intrinsic properties of the enzyme (its turnover rate essentially)
** NOTE: at VERY low [S] relative to the Km the velocity increases LINEARLY with [S] indicating maximal dependence; at VERY high [S]»_space;» Km the velocity approaches the point that is completely independent of [S] (occurs at Vmax)
What are the three assumptions of the Michaelis Menten equation?
- that substrate concentration is in molar excess over the enzyme concentration
- The initial velocity of the reaction is measured (thus k4 does not exist)
- That at steady state the rate of the enzyme substrate complex (ES) is equal to the rate of its dissociation (either into E + P or E + S)
Describe all of the terms in the Michaelis Menten equation
v = Vmax [S] / Km + [S];
velocity is proportional to the [ES] and if the enzyme is 100% saturated then [ES] = [E]t so the velocity is really proprtional to the total enzyme concentration
** reaction velocity indicates how much product is made in a define time period (mg per time or moles per time)
Vmax = maximal velocity = the fastes the enzyme catalyzed reaction can go
Km = the concentration of substrate and half max velocity
How is the velocity of the enzyme reaction described at vmax?
Vmax = kcat [E]total
where k cat = the reaction rate for ES –> E + P (first order rate constant)
what is the difference between turnover and specific activity? How are they each measured?
Turnover: the amount of product produced by an enzyme in a given amount of time: (how efficiently does the enzyme turn over the substrate into product); measure as sec -1 ( kcat)
Specific Activity: a measure of how “pure” a solution is; it tells you how much of the total protein present is the enzyme of interest at that specific time = SA; measure in units per mg protein (units = 1 umole/min); so its measured in umoles enzyme/ mg total protein * min
1 Unit = umole/ min
if you know the molar [enzyme] then you can calculate Kcat (turnover) = time -1
if you don’t know the molar [enzyme] but you can measure the total amount of protein, then you can calculate specific activity = U/mg of protein
What is Km in the michaelis menton enzyme equations?
Km = michaelis constant;
defines [ES] in the stead state (Km = [E] [S] /[ ES]
equals the substrate concentration that gives half maximal velocity thus if Km increases then more substrate is needed to achieve half maximal velocity (can be used to interpret affinity of an enzyme for substrate)
** if Km = [S] then the reaction is at i/2 Vmax
what is the concentration of a metabolite / S typically in vivo?
In vivo the concentration of a given metabolite in cells is typically in the range of the Km value of the enzyme ([S] ~ = Km)
Describe the Lineweaver Burke plot and where all of the values of the M - M equation can be found
Y axis = 1/ V X axis = 1/ [S] Y intercept = 1/ Vmax X intercept = -1/Km slope = Km/ Vmax
Is Km a dissociation constant?
** even though Km = same equation at Kd (dissociation constant) with respect to concentration of the substrate, Km IS NOT THE DISSOCIATION CONSTANT unless k 3 ««_space;k2 because then Kd = K2/ K1
Kd is defines by 2 rate constants (K2 /K2)
Km is defined by 3 rate constants (K2 + K3) / K1
what is the rate of an enzyme-catalyzed reaction dependent on? what is the relationship?
when the substrate is not limiting, the reaction rate is ALWAYS LINERARLY dependent on [E]
How is the rate of an enzyme -catalyzed reaction related to [S]?
the reaction rate increases hyperbolically with increases in [S] `
What are the 6 different classes of enzymes? describe each
- Oxidoreductases: transfer reducing equivalents (H2) from one redox system to another (AH2 + B + E –> A + BH2 + E)
- Transferases: transfer functional groups from one substrate to another (AC + B + E –> A + BC + E)
- Hydrolases: catalyze hydrolysis reaction (the addition of water to (AB + H2O + E –> HA + BOH + E )
- Lyses: catalyze the removal of groups of atoms without water (AB + E –> A + B + E)
- Isomerases: catalyse isomerization reactions by rearranging atoms in the molecules (A + E –> Aiso + E)
- Ligases : use ATP to catalyze the joining of two molecules (A+B+E + ATP –> AB + E + ADP + Pi)
How does a substrate interact with the enzyme ?
substrate binds to the active site of an enzyme via non-covalent interactions between the amino acids in the active site and the substate; interactions = H bonding, electrostatic, hydrophobc (active sites usually hydrophobic to prevent weakened interactions between ions due to waters high dielectric constant); NOTE: the amino acids involved in substrate binding and catalysis are NOT adjacent to each other in the linear protein
Two main methods of attachment
- Lock and Key model: enzyme substrate are perfect shapes for each other and form a tight bond
- Induced fit model: the enzyme and substrate are slightly off shape so either substrate or the active site needs to shift its shape so that they can fit.
also, some enzymes require a small molecules already located at the binding site (a cofactor) in order to bind the substrate
What are the main ways in which an enzymes lowers the energy of activation and decreases the energy of the transition state so the reaction goes faster?
- Orientation and Proximity: E binds S and holds it in the correct orientation for catalysis; can be intermolecular or intramolecular
- Substrate Strain: enzyme can impose strain or distortion on the substrate so that it binds the E
- General Acid Base Catalysis: side chains of aa in the active site can act as H donor/acceptor and facilitate rxn
- Metal ion catalysis: passive (ion involved in a stabilization of the coordination bond) or active (ion needed for the catalytic rxn)
- Covalent Catalysis: In the reaction BX +Y –> BY X the side chain of enzyme aa better attacker than Y and better leaving group than X
- Environmental Factors: an optimium for temperature and pH (proteins denature above a certain temperature and pH influences ionization of the E’s aa side chains and of the S leading to variations in stability, biniding, and rate of mechanism
What is unique thing about lysozyme?
- strain on substrate so one of the sugars is forced to assume half chair conformation
- pKa of Glu-35 is higher than measured for free aa, so at pH 5 the Glu -35 is abnormally protonated
What is interesting about the serine proteases with respect tot their active sites/protein structure?
They are basically the same exact thing except for that the active site is different;
- chymotrypsin Active site = large and nonpolar (A G)
- Trypsin active site has an aspartic acid residue
- Elastase : the active site is constricted
Describe the three types of reversible inhibition
- Competitive Inhibitor: binds to substrate binding site so it competes with the substrate; causes a DECREASE IN KM but does not change Vmax; it can be overcome by an increase in [S]
- Noncompetitive Inhibitor: binds at a site different from the binding site therefore it is INDEPENDENT OF THE SUBSTRATE (does not compete with it) causes a DECREASE IN VMAX no change in KM
- Uncompetitive Inhibitor: binds to the enzyme AFTER the substrate has bound; causes the KM TO decrease and the VMAX TO DECREASE; can be overcome by an increase in [S]
How does a a decreased affinity related to Km?
increased Km
which form of reversible inhibition is most similar to the irreversible inhibition? how?
noncompetitive because both of them cause a decrease in Vmax
What is a biochemical cofactor? What is an inorganic biochemical cofactor (ex)? organic biochemical cofactor?
cofactor = NON PROTEINACIOUS chemical compound that is BOUND TO A PROTEIN and is required for the biolofical activity of the protein;
(helper molecules that assist in the biochemical transformations)
inorganic cofactor = metal ions (help balance out charges on molecules they associate with), transition ions (they can exist in 2+ oxidation state)
organic cofactors = vitamin derivatives ( ie: fat soluble or water soluble)
what is a coenzyme? a prosthetic group? apoprotein?
coenzyme = cofactor that is loosely bound
prosthetic group = cofactor that is tightly bound
apoprotein = an inactive protein without its cofactor
What is ATP’s cofactor? why?
Mg 2+ because the 2+ is needed to balance out ATP’s negative charge
Describe the structural components of vitamins
Fat soluble: have a long hydrophobic polyisoprene chain; include vit KADE
Water soluable: polar molecules
What is the principal function of Vitamin A? How does it form other forms ? retinal?
retinal = signal transduction
retinoic acid = hormone
Beta-Carotene is a pro-vitamin; it’s center is cleaved to yield a group of molecules (retinoids) collectively known as vitamin A if the C terminal is an alcohol = retinol, acid = retinoic acid, aldehyde = retinal
Retinal is involved in signal transduction; its the cofactor for rhodopsin and when it absorbs a photon it changes to all trans which causes a change in opsin and triggers a G protein eventually leading to a nerve response.
What vitamins can act as hormones?
Vitamin A (retinoic acid) Vitamin D
what vitamins act as electron sinks?
Vitamin B1 (Thiamine) Vitamin B6 (pyridoxine)
What vitamins are involved in redox?
vitamin B2 (riboflavin) Vitamin B3 (niacin) Vitamin K (napthoquinone) vitamin C (ascorbate)
what vitamins act as antioxidants?
Vitamin C (ascorbate) Vitamin E (tocopherols)
what vitamins act as functional group carriers?
vitamin B5 (pantothenate) vitamin B7 (biotin) vitamin B9 (folate) Vitamin B12 (cobalamin)
What is the function of vitamin D? what’s it precursor and biologically active deriv?
7-dehydrocholesterol is the precursor of vitamin D and its present in high concentration in the epidermis; it absorbs sunlight an then rearranges into CALCITRIOL and VITAMIN D3 (D3); functions as a hormone
Describe the hormone action of vitamin D and A.
D3 and retinoic acid (vit A) are ligands for nuclear hormone receptors in the nucleus that are transcription factors that regulate expression of genes coding for proteins
Before a nuclear hormone receptor binds to DNA:
1. the receptor forms a complex with its specific ligand
2. two monoemeric receptor ligand complexes assemble a dimer
then the complex binds upstream to facilitiate transcription at that locus; vitamin D and A both regulate pathways of skin and bone development and metabolism; main target for vitamin D is calcium homeostasis
vitamin A and D deficiencies
vitamin A = dietary limitation or defective absorption of fat, can lead to night blindness, defective epitheliazation, excess vit A = GI discomfort and liver damage
vitamin D = less sensitive to diet /digestion, but can result from limited sunlight exposure ; hypocalcemia leads to defects in bone associated with rickets in kids and osteomalacia or osteoporosis in adults; excess vitamin D = renal failure
How do vitamins act as electron sinks? which vitamins?
Vitamin B1 (thiamine via TPP) and vitamin B6 (pyridoxine via PLP); they form a transient covalent bond with a substrate to stabilize new charges that are incurred in the transition state for the reaction and in turn provide an escape route for electrons (they stabilize the excess charge that is incurred by some reactions' transient state) TPP required for metabolic reactions with an alpha-keto acid or sugar; PLP forms a schiff base adduct
Vitamin B1 and Vitamin B6 deficiency
vitamin B1 (thiamine, TPP deficiency): leads to beri-beri: dry = periphery neuropathy, wet = added edema and cardiac failure; cerebral beri-beri (Wernicke Korsakoff) is associated with chronic alcoholism which inhibits intestinal absorption and conversion of thiamine to TPP
Vitamin B6 excess: temporary deadening of proprioception nerves causing unconscious perception of movt and spatial orientation
Explain the redox properties of vitamin B2 (riboflavin) and B3 (niacin), examples
vit B2 and B3 are the precursors of flavin nucleotide and nicotinamide nucleotide redox cofactors that participate in reaction in which 2H is removed from or added to a substrate (ex: lactate pyruvate, via FAD or FMN FADH2 or FMNH2, NADH NAD+) they are cofactors that catalyze hydrogen transfer because their rings can accept/release hydrogen easily;
NAD + (niacin deriv) is the substrate for ADP-ribosylases that attach ADP ribose to a protein in a posttranslational modification
What is the function of blue light treatment for jaundice and Vitamins? which vitamin?
Vitamin B2 has similar photochemical properties as bilirubin, and when a jaundice patient (buildup of bilirubin) is brought under blue light the bilirubin (and riboflavin) degrade, so pt are given vitamin b2 (riboflavin) supplements to avoid riboflavin from getting too low
What are the pathologies associated with Vitamin B2 and Vitamin B3?
Vitamin B2 deficiency (ariboflavinosis): causes irritation to skin and eyes, cracked red lips, mouth ulcers, sensitivity to BRIGHT LIGHT
Vitamin B3 deficiency (aniacinosis/aniacinamidosis): causes pellagra (4D’s: diarrhea, dematitis, dementia, death); B6 (PLP) is a cofactor for the reaction in which niacin is synthesized from tryptophan (so adequate vit B decrease the requirement for niacin in the diet)
excess Vit B3: niacin maculopathy: thickening of the macula and retina leading to blurred vision and blindness
Describe the redox function of vitamin K
Vitamin K is a redox cofactor for gamma-glutamyl carboxylase (makes GLA)
GLA proteins contain extra negative charges and this facilitates Ca2+ binding; thus GLA involved in Ca2+ regulated processes (blood coagulation, bone/EXCM hemostasis, Growth hormone regulation); By regulating the amount of active cofactor vit K , you can decreases in the [GLA] and thus decrease the GLA containing proteins ie: coagulation; thus inhibiting vit K reductase reduces the amount of active vit K to cofactor for GLA
Describe the vitamin K associated pathologies
vitamin K deficiency: GLA protein functions are impaired leading to massive, uncontrolled internal bleeding, cartilage calcification and severe malformation of developing bone, deposition of insoluble calcium salts in the arterial vessel wall
Describe the redox function of Vitamin C (ascorbic acid)
it is a very versatile cofactor (can exist in several different redox states); ex: for prolyl and lysyl hydroxylases of collagen synthesis pathway.
Stability of ascorbate as a radical is super important for its function as an antioxidant
Describe the antioxidant function of Vitamin C and E
free radicals are generated in vivo and are dangerous because they cause damage to cellular components;
Vit C and E have a tocopherol ring that is important in its ability to inactivate free radicals;
Vitamin C = POLAR; so antioxidant activity is in soluble portion of the cell (cytosol and intraorganelle compartments)
Vitamin E = hydrophobic tail (fat-soluable vitmain) makes it non-polar whose antioxidant activity occur in the membrane compartments
Describe the pathology associated with Vitaman C
Vitamin C deficiency: limited to humans and some gorillas, guinea pis, fruit bats;
= scurvy = defective collagen synthesis (vit C is important cofactor for functioning of hyroxyproline and hyroxylysine in collagen synthesis)
Describe the pathology associated with vitamin E
vitamin E is normally found in the vegetable oils and nuts/seeds;
Vitamin E deficiency = due to dietary limitation or defective fat absorption (similar to vit A/D); and causes impaired balance and coordination (ataxia), injury to sensory nerves (peripheral neuropathy), muscle weakness (myopathy), damage to the retina of the eye (pigmented retinopathy)
What vitamins are function as functional group carrier cofactors? what function groups? How do they function in general?
Vitamin B5 (pantothenate) = acyl groups Vitamin B7 (biotin) = CO2 Vitamin B9 (folate) = single-carbon units Vitamin B12 (cobalamin) = methyl adenosyl groups
General schematic of function:
Enzyme: cofactor complex binds 2 substrates
small group X originates on substrate 1 and is tranferred to the cofactor, and then product 1 leaves; then X is transferred to substrate 2 forming a transition state, then product 2 leaves (with X) and then the original enzyme: cofactor complex is regenerated
How does vitamin B5 act as a cofactor?
= pantothenic acid; it combines with cysteine and phsophate to make the biologically active vitamin derivative (phosphopantothenic acid) that carries ACYL GROUPS (functional group carrier) in a thioester linkage
How does vitamin B7 act as a cofactor?
= biotin; it is asstached to a PEPTIDE LINKAGE to the host enzyme for which it serves as a cofactor that TRANSFERS CO2
CO2 attachment to biotin requires ATP hydrolysis because the energy associated with the cofactor condensation reaction is used to drive CO2 condensation with the substrate in the reaction
Describe the pathology associated with vitamin B7
(biotin); human diet is rich in B7 and B5, but too much RAW EGG = biotin B7 deficiency (becasue Avidin a protein in raw egg binds biotin, so biotin cant bind to CO2)
symptoms include skin disorders, hair loss, neuropathy
Explain the function of Vitamin B9 and B12; how are they activated etc.
B9 (folate) is unreactive in oxidized form; it must be fully reduced to THF to react and form covalent linkages to 1-C group (reduced via NADPH as the REDOX donor, needs two NADPH linked reductase enzymes to completely reduce folate)
B12 (cobalamin) transfers a methyl group of andenosyl group; the core stucture of B12 is a corrin ring in which the central metal ion is cobalt (4 out of 6 coordination sites are by corrin ring nitrogens, 5th is by dimethylbenzimadazole, 6th site R is the functional group being transferred)
Why are humans co-dependent on vitamin B9 and B12?
= folate and cobalamin; because methionine synthase requires both B9 and B12 (and they cofactor some super essential enzymes like one that is essential for DNA replication, synthesis of purines de novo etc)
Describe vitamin B9 and B12 associated pathologies
homocyteinemia and megaloblastic anemias result from deficiency in either vit B9 or B12;
homocysteinemia results when methionine synthase activity is reduced due to vit B9 or vit B12 deficiency (needs to be kept in check! high levels of homocystein linked to heart and CNS pathologies)
magaloblastic anemias: B9 or B12 deficiency results in dTMP deficit and reduces DNA synthesis, since DNA synth lags behind cell growth, premature RBC overgrow
key observances in blood sample: large immature and dysfunctional red blood cells in the bone marrow sample and peripheral blood of hypersegmented neutrophils (5+ lobes)
What are the three main classes of non-covalent bonds? describe them
In general: DO NOT SHARE e-, strength of the bond is determine by the environment; depend on the forces that electrical charges exert on one another
- ionic (coulombic) interaction: aka salt bridges: interactions between permanently charged species or between the ion and a parmanent dipole (energy of the ionic interaction = kqq/Dr, D = dielectric effect of the intervening medium; the higher the D (ie water) the weaker the ionic interaction because the solvent highly interferes with the solute in the rxn
- dipole interactions: hydrogen: between uncharged but polar molecules (H, N, O ); found in W-C base pairs
- Van de waals: weak interactions between ALL ATOMS, independent of polarity (attractive = london dispersion: due to the movement of electrons in the interacting molecules as they instantaneously attract each other, or repulsive because the electrons are too close (sterics)
Describe the hydrophobic effect (behavior of non-polar molecules in water)
= a phenomenon associated with the ordering of water molecules around non polar species;
water wants to maintain the highest level of entropy (randomness) so when a non-polar molecule is brought into water, the water molecules are highly ordered around the hydrophobic alkly chains (form cages) which has a lowered entropy, but this allows the bulk phase of water to have lots of freedom with respect to orientation, so the overall result is that the entropy of water is maximized
NOT DRIVEN BY AN ATTRACTIVE FORCE BETWEEN NONPOLAR SPECIES; driven by waters desire to increase entropy (natures tendency to minimize ordering the structure of water)
Hydrophobic effect drives the association of amphiphilic (polar nonpolar) species to form lamellar and micellar structures, and the internalization of hydrophobic elements during protein folding.
END POINT: hydorphobic components stick together and sequester water minimizing order and maximizing entropy
Explain the relationship between pH and pKa. How is pH related to amino acids (ie: what form do they take at neutral pH, what are the pKa’s of groups)
pH = -log [H]; H+ can REVERSIBLY bind to many molecules, and pH determines the ionization state of functional groups (most biological activities are restricted to pH 7.2-7.5);
Ka = the extent of dissociation
pH = pKa + log (]A]/ [HA])
when pH = pKa [A] = [HA]
when the pH > pKa then the species is deprotonated
pKa of carboxy group = 2.2
pKa of amino group = 9.5
at neutral pH most amino acids exists as dipolar ions (zwitter) in which the amino terminus is protonated and the carboxy terminus is deprotonated
what is the function of a buffer? how does it function?
buffers resist change in pH; the buffering capacity of an acid/anion system is GREATEST AT pH = pKa;
buffer only works when pH = pKa +/- 1
What are the nonpolar amino acids?
GAP VILM
glycine alanine proline valine isoleucine leucine methionine
What is unique about the structure of Proline and its protonation?
the proline R group is covalently bound to the alph amino nitrogen such that the protonated form is NH2+ NOT NH3+, but the rind is not aromatic!! the side chain is still strictly aliphatic (nonpolar)
what is unique about the structure of methionine?
it contains S (only other amino acid that contains S is cysteine);
ATG is the only codon for methionine in the genetic code and it is also the start codon so translation of transcribed genes into proteins is initiated with methionine
What are the aromatic amino acids? what are their respective R groups? Other than being aromatic, what characteristic do all three share?
- phenylalanine: phenol group instead of one alanine methyl H
- tryptophan: indole group joined by methylene (CH2) = 5 member ring containing NH fused to a 6 member carbon ring
- tyrosine = phenyl ring with OH at para position
all 3 are hydrophobic, and tyrosine is also hydrophilic (H bonds)
What is unique about the UV absorption of Trp and Tyr?
they absorb UV light at longer wavelengths than other amino acids;
They are the only aa that absorb at 280 and molar extinction coefficient of Trp is 4x greater than Tyr at 280 nm
This is useful because the molar extinction coefficient for a protein at 280 nm can be calculated with the aa sequence is known (# of W and Y residues)
What are the polar uncharged aa?
Serine, Threonine, Cysteine, Asparagine, Glutamine
What is the functionality of the OH in Ser, Thr, and Tyr?
they are all target sites for covalent modification of proteins and are substrates for enzymes that catalyze reversible phosphorylation/dephosphorylation;
Ser / Thr OH is a site of sugar attachment
Tyr OH = acid: base rxns
What types of reactions does cysteine participate in?
acid/base, redox (oxidation = cystine), covalent bonds via disulfide bonds
what is the side group of asparagine and glutamine?
carboxamide (larger by one methylene (CH2) in Glutamine than Asparagine)
It participates in Hbonding but NOT in acid/base and the asp side chain is conjugated with sugars in N-linked glycosylation rxns
what are the basic amino acids?
+ charged; histidine, lysine, arginine
lys and arg are + charged at neutral pH (have a pKa»_space; than N-terminus)
His group has a pKa = 6 so its only + charged in acidic solutions
What are the acidic amino acids?
aspartic acid and glutamic acid; they have a deprotonated side group at neutral pH (pKa = ~ 4)
What do the pKa values of amino acids depend on?
temperature, ionic strength of the medium, microenvironment in macromolecules (so the pKa measure for a free aa might not be the same as it is for the aa in a solution.. ie: Glu is protonated in lysozyme even though it is usually deprotonated )
Describe the isoelectric point
the point when the overall charge on the amino acid is neutral (zwitter ion form) ; since the net charge is zero, the the aa is LEAST soluble in water and DOES NOT migrate in the electric field.
For non-ionizable species its found by pKa1+pka2/2 and for ionizable species its found by the average of the pKa values that define the acidic and basic form of the species
Define what are the primary, secondary, tertiary, and quaternary structures of proteins
1 = the linear polypeptide defined by the primary sequence that is a result of the codon sequence in genes; connected by peptide bonds that follow a unidirections N --> C terminus corresponding to 5' --> 3' 2 = microdomains (alpha Helix or B strands) that are a result of non-covalent elecrostatic interactions 3 = polypeptide folding into a 3D structure as a result of hydrophobic effect and electrostatic interactions 4 = a functional protein (native fold) made of multiple polypeptide subunits
Describe the features of peptide bonds
formed by the condensation (dehydration) reaction that releases water, consumes energy and pushes the equilibrium to the left;
peptide bond reaction is thermodynamically unstable (requires energy); but once the bond is formed, it is kinetically stabile (the rate of the hydrolysis is EXTREMELY SLOW)
peptide bonds have resonance due to:
- low reactivity
- rigid and nearly planar structure
- exhibit large dipole moment in the favored TRANS CONFIGURATION
what rotations about the peptide bond are permissible? what angles do these rotations form? what is the significance of this?
rotation around the peptide bond IS NOT permitted
rotation around bonds connected to the alpha carbon IS PERMITTED:
1. Phi = N amide –> alpha Carbon
2. Psi = alpha Carbon –> carbonyl carbon
they are dihedral/ torsion angles that are defined by 4 points in space;
some of the phi/psi conformations are UNFAVORABLE BECAUSE OF STERIC CROWDING and steric exclusion (the fact that two atoms cannot occupy the same space at the same time) is one of the main organizational principles for protein folding
some of the phi/psi conformations ARE STABLE BECAUSE THEY FORM FAVORABLE H-BOND INTERACTIONS along the backbone
Ramachandran plot shows some of the distribution of phi/psi and the common secondary structures
Describe the secondary structure of a protein and the major features of secondary structural elements
2 structure = local spatial arrangement of the polypeptide backbone
- alpha helix = stabilized by H bonds of aa nearby
- B sheets/strands = stabilized by H bonds between aa not nearby on chain
alpha helix = inner sruface = backbone and side chains project outwards; hydrogen bonds between CO of aa1 and NH of aa4 form a Hbond ;
each reside is related to the next by a 1.4 A rise along axis and rotation of 100 degrees thus 3.6 aa per turn; usually the helix is Right handed; Pro and Gly = helix breakers (pro cant rotate about N-C, and gly is too small); attractive or repulsive interactions between side chains affect formation
Beta strands: aa poitn in opposite directions and are 7 A apart; since the aa are wide apart, sterics is not a major
What are turns/loops
Turn: follow distinct pattern of interactions and involve 4 aa and typically involve a hydrogen bond between the CO of the most N-terminal residue in the turn with the NH of the amino acid 3 positions C terminal to the N terminal residue
Loops: connect secondary structural elements; they are well ordered even though they don’t follow a specific ordered pattern
What amino acids are typical of helix forming? sheet forming? turns/loops?
Helix = Q KERM LAND Sheet = SHIT CYF WIV Turns/loops = P G
Describe the factors that stabilize tertiary structure
3 = overall spatial arrangement of atoms in a protein; stabilized by numerous weak interactions between aa side groups, namely hydrophobic and polar interactions, also disulfide (interacting aa are not necessarily next to each other in the primary sequence)
Bsheets = stabilized by hydrogen bonding between 2 B strands; (NH –> OC bond) if the N to C terminal are in the same direction = parallel sheets, if they are opposite = antiparallel; antiparallel are more stable b/c H bonding is LINEAR;
Alpha helix bundles held together via VDW and H bonds (NOT HYDROPHOBIC!) the hydrophobic effect explains stability for transmembrane helices in the lipid species while ionic bonds drive stability of the soluble protein
what are the main ways that a protein is covalently modified?
- proteolytic cleavage of the polypeptide chain
- attachment of a small molecule
- attachment of macromolecule
How are vitamins provitamins?
Because they aren’t active cofactors in the form that they are consumed; include
Explain the significance of proteolytic cleavage for the generation of biologically relevant proteins
it is an IRREVERSIBLE MODIFICATION that involves:
- removal of a targeting signal (pre-proteins)
- removal of non-targeting amino acids (pro-proteins)
- Release of individual proteins from a polyprotein
- Removal of a targeting signal (pre-proteins)
occurs
A. CO-TRANSLATIONAL at the ER; exposure of a signal sequence of at the Nterm of the polypeptide triggers sorting the active ribosome to the ER membrane for a lumenal protease to cleave the signal sequence to generate a mature N-term in . Other sequence signals determine the fate of the newly synth protein (either soluble or transmem protein)
B. POSTRANSALTIONAL at the MITO: the first 15-20 aa = mitochondrial leader sequence MIL in mito matrix proteins; the MIL is cleaved by a matrix protease and then the protein folds and acquires the native structure (main diff from 1 is that it occurs entirely post translational even though same outcome)
- Removal of non-targeting amino acids (pro-proteins) = activation of zymogens via cleavage of prospecies non-targeting aa; the zymogen is activated by a protease at the location of action, but it is stored elsewhere (so you don’t have active enzyme 24/7) ex: proinsulin, coag cascade proteins, trypsin, GI enzymes
- Release of individual proteins from a polyprotein (cleave parts of a polyprotein to make little active protein bits)
What are the main ways attachment of a small molecule covalently modifies a protein
- Acetylation
- Methylation
- Carboxylation (gamma Glu = GLA)
- Phosphorylation
- Hydroxylation
- ADP-ribsoylation
Who does acetylation of an amino acid side chain covalently modify a protein?
- N-terminal acetylation = add acyl group to N-terminus via N-Terminus acetyl transferases (NATs) COTRANSLATIONAL + irreversible; most proteins are N-acetylated in order to prevent proteins from degradation in the cell (provides extra stability)
- Histone acetylation/deacetylation = POST-TRANSLATIONAL, REVERSIBLE modification to LYSINE residue; histone aceytyltransferase (HATs) add acetyl group which increases transcription activity vs. deacytelyation (via HDAC) decreases transcription activity
How does methylation of amino acid side chains covalently modify a protein?
similar to acetylation, but methylation of LYS = regulates transcription (and gene expression)
How does carboxylation of gamma-carbon in glutamic acid covalently modify protein?
adds a carboxy group onto GLU so then it increases the affinity for Ca2+ because it has a larger negative charge; its a vitamin K dependent enzyme super involved in coag cascade
How does phosphorylation of amino acid side chains covalently modify a protein?
addition/removal of a phosphate at SERINE, THR, TYR, is rapid and reversible; the reaction is ATP dependent (protein kinase adds Pi , phosphatase takes off Pi) and the end result is that the modification is altered by a NET CHARGE OF +/- 2 due to the addition/removal of a phasphate
How does hydroxylation covalently modify a protein? (examples)
hydroxylation of proline and lysine (to hydroxyproline/lysine);
hydroxyproline stabilizes the left handed alpha helices (secondary structure of collagen)
hydroxylysine stabliizes the quaternary structure of protein via inter-helix crosslinking
How does ADP-ribosylation covalently modify a protein?
important for the action of some bacterial toxins such as cholera toxin, diphtheria toxin, pertussis toxin, and heat labile enterotoxin; toxin proteins are ADP-ribosyltransferases that modify target proteins in human cels
Protein attachment as a covalent modification of proteins
- Protein attachment: ex = ubiquitination vs sumoylation; hypoxia inducable factor 1 alpha (HIF 1A) is a transcription factor that mediates the effects of hypoxia on the cell by promoting formation of blood vessels and migration of keratinocytes; in normoxia HIF 1A is not needed and hydroxyproline signals ubiquitination forming a proteosome that can be degraded (HIF1A degraded)
In hypoxia: HIF1A signaled and SUMO proteins attached (sumolyated) making HIF 1A stable and active;
^^ example of how two different proteins control activity (via attachments) of a 3rd protein
Lipid attachment effect covalent modification of proteins
- Lipid Attachment: modulates proteins association with the membrane (attachment at either N-term or an internal site):
A. Myristoyl anchor: 14C FA linked to Nterm of Gly, cotranslational, irreversible
B. Palmitate = 16C FA, linkage to Cys INTERNAL, Post Translational, REVERSIBLE
C. Faresyl Anchnor: farnesyl = 15C isoprenoid, linkages to Cys INTERNAL, Post Translational, Irreversible
Sugar attachement effect on covalent modification of proteins
- N linked (Asn attached) and O linked (Ser or Thr attached) sugar attachments forming glycoproteins (not the same as proteoglycans which are proteins attached to sugars that are usually sulfated giving them a large negative charge)
GPI -anchor proteins are attached to glycosylated phosphatidylinositol (a membrane phospholipid)
N-linked glycoproteins: to N of aspargine side chain and reaction takes place in the ER; forms an oligosach unit that is pre-assembled on a lipidic species in the ER called dolichol phosphate
O-linked glycoproteins: to Ser or THr hydroxy group; rxn takes place in GOLGI; no Dol-P (sugars are attached at one time)
Describe the protporphyrin IX ring
found in Heme and binds Fe2+; it has 4 pyrrole rings linked by methylene bridges; has 6 coordination bonds 4 in the plane of the flat porphyrin ring system and 2 perpendicular
Describe the role of the proximal His F8 and the distal His E7 imidazole side chains in the O2 binding pocket of Mb and Hb
In general the envirmonment around the Heme is nonpolar which favors the ferrous state (Fe2+) of the iron; EXCEPT the His F8 (proximal his) and His E7 (distal His), so in Mb His F8 gives the 5th coordination to Fe and leaves the 6th open to bind O2. The oxygen likes to bind to free heme at an angle, and the angle promotes H-bonding of E7 with the ligand, allowing for stabilization which is critical so that the oxygen DOES NOT leave the protein as the superoxide radical which would leave behind ferric iron because 1. the radical would cause cell damage 2. If the heme is left in ferric state it can’t bind O2
What is the relationship between CO binding to Mb (or Hb)
Heme has a MUCH MUCH higher affinity for CO than for O2, but CO cannot bind Heme at an angle (it prefers linear configuration) but since the E7 is there, it causes steric inference thus decreasing the affinity for CO by 100, but the affinity of Heme for CO is still 250x greater than it is for O2
What does the fraction of bound sites depend on?
the concentration of free ligand and the Kd (dissociation)
Differentiate between O2 binding curves for Mb and Hb and explain the features of each
Mb: rectangular hyperbola (still a saturation curve.. aka is indicative of specific binding interaction between two molecules); the P50 (half saturation pressure) is very very low (2.8), much lower than the pO2 of the tissues, so at the pressure of the tissues Mb still tightly holds onto Oxygen. Thus Mb is a good storage molecule but very ineffective in transporting oxygen to the tissue capillaries
Hb: has a sigmoidal curve (has a variation in O2 affinity for Hb over the range of physiologically relevant oxygen concentrations) indicates the versatility of the protein that is ideally suited for its functionas an O2 carries (its P50 is greater than the pressure in the tissue so it has a less affinity for the O2 than the tissue does thus O2 is transported into the tissue capillaries)
Both Mb and Hb are saturated with O2 at the pO2 levels of the lungs (have high affinity to bind O2)
Explain how ligand binding initiates a conformational change in globin heme proteins
two general conformers: T and R;
T is more stable because it has a greater number of interchain ion pairs (salt bridges), but the Hb affinity for O2 is greater for the R state (but O2 still binds to the T state);
Oxygen binding to Hb subunit in the Tstate triggers a change in conformation to the R state once all 4 subunits are in the R state there is not much change in tertiary structure, instead the subunits just slide past each other and rotate narrowing the pocket between the beta subunits
** in the deoxy state the iron is about 0.6 A out of the heme plane, but once the oxygen binds it pulls the iron back into the heme plane and since the proximal His F8 is attached to the Fe , this pulls the complex F helix like a lever on a fulcrum
What are the two models of O2 binding to Heme
The energy in the formation of Fe-O2 bond drives the first T–> R transition and then the other subunits change to R based on 2 different models:
- Concerted (Symmetry) model: = Hb can exists in either completely T state or completely R state, and with each additional O2 bound there is an increase probably that the Hb will transform all to R state
- Sequential model: some subunits are T some are R (transition occurs with each O2 that binds to the T)
Homotropic Allostery
= cooperativity
the effector and affected ligand are the same type of molecule such that the interaction is between duplicate structural domains in the protein (ie: O2 binding to one heme domain influences binding on the next heme domain)
positive cooperativtiy: first ligand increases affinity for the second ** sigmoidal curves = positive cooperativity
negative cooperativity: first ligand decreases affinity for the second
The Hill plot: what does it say about cooperative ligand binding? axis/slope?
Y-axis = log (theta/ 1-theta)
X-axis = log pO2
when log (theta/ 1-theta) indicates half saturation
sloe at half saturation point = hill coefficient (n)
if n = 1: no cooperativity
n > 1 : positive cooperativity
n< 1: negative cooperativity
For Mb: n =1
Hb : n =1 at low or high concentrations of oxygen, the acts like it has only a single site, so n = 1; at the mid rand n = 3
Is Hb more or less sensitive to small changes in pO2 in tissues than a non allosteric protein would be?
MORE sensitive (for a small change in pO2 the graph of Hb increases in saturation greatly compared to that of Mb) .. this suggests that is it effective in functioning in the changes in metabolism (if there is a small decrease in O2 Hb will drop off O2)
Heterotropic Allostery with respect to Hb?
Heterotropic allostery = effector and affected ligand are DIFFERENT TYPES OF MOLECULES (interaction between domains that are not structurally similar.. ie: between activity site and a regulatory site)
Case I: Negative allosteric = inhibitor
Case II: Positive allosteric = Actiavtor
ALL (below) ARE INHIBITORS (decrease protein affinity for oxygen) and thus promote the T-state
- 2,3 BPG
- protons (pH)
- CO2
- Chloride Cl-
What is the effect of 2,3 BPG on Hb? what are conditions with variations in its concentration
- 2,3 Biphosphoglycerate (2,3 BPG)
a highly ANIONIC compound present in RBC at the same conc. as Hb! the center of Hb is + charged and attracts the - 2,3 BPG which interact and causes a favoring of the T-state. This is an active promoter of the T-state and SUPER IMPORTANT IN O2 RELEASE (to the tissue) 2.3 BPG does NOT effect O2 loading, just increases its unloading
At high altitude: there is an increase in 2,3 BPG to overcome the higher pO2 barrier (which makes it harder to drop off O2 to tissue) so your body compensates by making more 2,3 BPG.
Fetal Hb = decrease in 2,3 BPG
Fetal Hb = 2 alpha, 2 gamma (not beta chain) which have a neutral Ser instead of positive His like in B chain; thus gamma have 2 less + charges and causes the affinity for 2,3 BPG to decrease (but a higher affinity for O2); this is important so net O2 flows from maternal red cells to fetal
What is the effect of protons (pH) on Hb?
Hb affinity for oxygen depends on pH; decrease in pH (increase in H+) stabilizes the T state because it favors the protonation of the His residue at the carboxyl terminus of the B globin chains (His C3); when protonated, His HC3 can make a salt bridge that interfers with the movement of Helix F which thus disfavors O2 binding to the heme iron (when no salt bridge with HC3 then His F8 (prox) can move when O2 binds to iron)
Explain the Bohr effect
pH effect on O2 binding to Hb;
When acidity increases (increase in H+ decrease in pH), the saturation curve of Hb for O2 is displaced to the RIGHT (thus affinity for O2 decreases at half sat.) this is super important because metabolizing tissues produce H+ and need more O2, which is possible because the affinity for O2 is decreased so more O2 is transported from the lungs to active muscles
What is the effect of CO2 on O2 binding?
Two main effects:
1. To increase [H+] in tissues (due to CO2 + H2) —> H+ HCO3- via carbonic anhydrase)
In tissue capillaries:
pO2 is low, pCO2 is high so it pushes the rxn to right generating more H+ which protonates Hb and favors the T-state causing O2 to unload
In Lungs:
PO2 is high, PCO2 is low: favors the deptronation of Hb increasing available H+ to drive the reaction to the left which generates CO2 which can be liberated as a gas
- via formation of an adduct (carbamate) by reacting with the free amino termini of the globin chains; the negative charge of the carbamate (NR2 – COO) favors salt bridges with vicinal positively charged amino acids and stabilizes the T-state of Hb (deoxy form)
What is the effect of chloride ion on O2 binding of Hb?
Chloride ion (Cl-) stabilizes one of the salt bridges that is formed in the T-state of Hb; in tissues Cl- enters RBCs in exchange for HCO3- that accumulates from the carbonic anhydrase reaction via an antiport such that a high concentration of CO2 directly proportional to a high concentration of Cl- Anion movement is reversed in the lungs
What are the key factors that stablize the Hb in the T-state? (deoxy Hb)
- Salt bridge between B-His HC# imidazole and B-AspFG1 side chain (vicinal F heliz that is involved in coordinating heme iron): this is favored by an increase in [H+] (pH decreased)
- Additional intra and inter chain salt bridges due to increased [CO2] and increased [Cl-]
- Expanded central cavity due to increased [2,3 BPG]
Compare alpha vs Beta Thalassemia and HbH vs Hb Barts
Alpha: loss of genes for alpha globin (there are 4 genes that typically code for alpha globin chain in Hb)
- if one alpha globin gene gone = asymptomatic
- if two alpha globin gene gone = mild alpha thalassemia (presents similar to anemia, except for that increasing iron supplements is ineffective)
- if 3 alpha globin gene gone = accumulation of B-globin; assembly into homo B tetramers called HbH which gives rise to HbH disease (defective for transporting oxygen) causing severe anemia
- if all 4 alpha globing gene gone = embryolethal because the Hb form in fetus is all 4 gamma chains (Hb Barts
Beta Thalassemia: the Beta gene is STILL present but it produces little Beta protein (degree produced varies) NOTE: B gene is still present even in B-0-Thalassemia
What is the causes and selective advantage of Hb S?
= sickle cell;
Caused by GluB6 –> Val mutation (polar –> nonpolar) this causes HbS to be sticky and clump together, distorting RBC shape to sickle, so then HbS aggregates into filaments aka rod shaped RBC;
Sickle RBC = severely compromised for passage through capillaries (blood flow can be completely blocked in some areas)
Heterozygous for HbS = resistance to malaria
