Exam #1 Prep Flashcards
List the hydrophobic AAs (7)
Pro VAG PIL
- Proline
- Valine
- Alanine
- Glycine
- Phenylalanine
- Isoleucine
- Leucine
List the AAs with -OH side chains (3)
SHOTT -Serine (-OH) -Threonine -Tyrosine
List the basic AAs (3)
HAL
- Histidine
- Arginine
- Lysine
List the AAs with aromatic side chains
HTTP
- Histidine
- Tryptophan
- Tyrosine
- Phenylalanine
pH > pKa
- Give relative concentrations of A- to HA.
- Conceptual and mathematical reasoning.
[A-] > [HA]
- Conceptual: Because the solution is more basic than the R-group, and thus wants protons more.
- Mathematical: Given that pH = pKa + log ([A–]/[HA]), as A- increases, so does log ([A–]/[HA]). Thus, pH becomes greater than pKa.
pH = pKa
- Give relative concentrations of A- to HA
- Conceptual and mathematical reasoning.
[A-] = [HA]
- Conceptual: Since the solution is equally acidic as the pKa site, the protonated and deprotonated moities must be in equilibrium.
- Mathematical: Given that pH = pKa + log ([A–]/[HA]), and pH = pKa… [A-] = [HA], then [A-]/[HA] = 1. Log(1) = 0, thus, pH = pKa.
pH
[HA] > [A-]
Give the Henderson-Hasselbalch Equation
pH = pKa + log ([A–]/[HA])
pH + pOH = ____?
14
Give the dissociation of pure water.
[H+] [OH-] = 10^-14
Give the formula for the acid dissociation constant (Ka)
Ka = [A-][H+]/[HA]
The ratio of conjugate base to its acid varies by ____-fold ( _____ order(s) of magnitude) with each pH change of one unit.
10-fold (one order of magnitude)
The normal pH range of blood is _____ to ____, normal is ≈ ______
7.37 to 7.43
normal is ≈7.4
What pH and 1˚ increase/decrease constitutes respiratory acidosis?
pH
What pH and 1˚ increase/decrease constitutes respiratory alkalosis??
pH >7.45, PCO2 decrease
What is the pKa of the Bicarbonate/CO2 buffer in the blood?
pKa = 6.1
What pH and 1˚ increase/decrease constitutes metabolic acidosis?
pH
What pH and 1˚ increase/decrease constitutes metabolic alkalosis?
pH >7.45, HCO3- increase
What is the reference range and reference point for blood pH level?
7.35-7.45, 7.4
What is the reference range and reference point for blood PCO2 level?
33-44 mmHg, 40 mmHg
What is the reference range and reference point for blood O2 level?
75-105 mmHg
What is the reference range and reference point for blood HCO3- level?
22-28 mEq/L, 24 mEq/L
What is the reference range and reference point for blood Na+ level?
135-145 mmEq/L
What is the reference range and reference point for blood anion gap?
8-16 mEq/L, 12 mEq/L
What is the reference range and reference point for blood Cl- level?
98-106 mEq/L, 100 mEq/L
Give the Henderson-Hasselbalch Equation for the CO2/Bicarbonate buffer system
pH = 6.1 + log ([HCO3-]/(0.03xPCO2) i.e. [CO2])
Give the conversion for PCO2 to [CO2]
[CO2] = 0.03 x PCO2
List some causes for respiratory acidosis (9)
- Excess inhaled CO2
- Decreased alveolar ventilation:
a. central respiratory depression
b. nerve or muscle disorders (myasthenia gravis)
c. lung or chest wall defects (restrictive lung disease, COPD)
d. airway disorder (asthma)
e. external factors - Increased CO2 production
a. Hypercatabolic disorders (malignant hyperthermia)
List some causes for respiratory alkalosis (3)
- Increased alveolar ventilation via various drugs
a. Hypoxemia (acts via peripheral chemoreceptors)
b. pulmonary causes (pulmonary emboli) via intrapulmonary receptors
List some causes for metabolic alkalosis
- Loss of H+ via kidneys (diuretics) or gut (vomiting)
2. Gain of alkalai from exogenous sources
List some causes for metabolic acidosis
a. normal anion-gap (hyperchloremic)
b. high anion-gap (normochloremic)
Diarhhea, gain of organic acid (TITRATION OF BICARBONATE), loss of base.
a. renal (DIABETES), GI tract, other
b. ketoacidosis, lactic acidosis, renal failure, toxins
PCO2 should not rise above ____ mmHg in compensation.
55 mmHg
What is the formula for anion gap?
[Na+] - ([Cl-] + [HCO3-])
Define primary structure of proteins, how to read it, and where the AAs bond with one another.
Primary structure: Strictly the amino acid sequence; written/read from L to R starting with the N-terminal amino acid.
Joined covalently by peptide bonds between the alpha-carboxyl (-COO) group of one amino acid, and the alpha-amino (NH3) group of the next amino acid.
Are there any achiral, NOT optically active amino acids? If so, name them.
Only one: Glycine is the exception because its α-carbon has two hydrogen substituents.
All amino acids found in proteins are of the ___-configuration.
L-configuration
At physiologic pH, amino acids carry a ____ charge on their alpha-amino groups and a _____ charge on their alpha-carboxyl groups. What are their respective pKa’s?
Positive on amino (pKa ≈ 9.4), negative on carboxyl group (pKa ≈ 2.2).
The amino acids ____ and ____ are the reason proteins absorb UV light at ____ nm. Why is this?
Trp and Tyr.
Absorb at 280nm
Because they are AROMATIC.
#25
R groups in a peptide unit are almost always in _____ configuration, why?
trans configuration.
because of steric interference of the R groups when in the cis configuration (see lecture #4).
All amino acids are read from the _____ to the ______- terminal.
N- to the C-terminal
Alpha helices spontaneously forms when each carbonyl of a peptide bond forms a _____ bond with the -NH of a peptide bond _____ amino acid residues further along the chain.
The average length is ______ residues.
H-bond FOUR AA residues down the chain.
12 residues
List the nonpolar AAs (9)
VIP MALT Ph Glycine
- Valine
- Isoleucine
- Proline
- Methionine
- Alanine
- Leucine
- Tryptophan
- Phenylalanine
- Glycine
How many AA residues per turn of an alpha helix?
3.6 residues
Peptide bonds are resistant to conditions that ____ proteins, e.g. ______ and _______.
Denature proteins, e.g. heating, and high concentrations of urea
In ß-pleated sheets, which peptide bond components are involved in H-bonding?
All of them.
An alpha-helix is a _____-handed molecule, with its R-groups facing _____.
Right-handed molecule.
R-groups facing outward.
An alpha helix has _____ residues per turn.
3.6 amino acid residues per turn.
In a ß-pleated sheet, the H-bonds are _____ to the polypeptide backbone.
Perpendicular.
Regarding acid-base disorders, diarrhea will result in a change of what blood chemical and what acid-base disorder?
Loss of bicarbonate.
Metabolic acidosis.
If metabolic acidosis has an elevated anion gap, what four things could it be due to?
KULT: Ketoacidosis, uremia, lactic acidosis, toxins.
Intoxication with which OTC med could cause elevated ion gap acidosis?
Aspirin (salicylates)
What does anion gap tell us, i.e. what is its clinical significance?
It distinguishes between two types of metabolic acidosis:
- ) Lack of renal reabsorption or production of HCO3- (kidney failure), or…
- ) Excess of “other” anions, e.g. exogenous poisons, ketoacidosis (alcoholics, diabetics), lactic acidosis, etc.
Describe the general AA sequence (i.e. hydrophilic/-phobic) of the following types of proteins:
- Globular
- Fibrous
- Membrane
- Quasi random
- Repeats
- Blocks
L:5-6
Describe the 4˚ and 2˚ structure of Hb, and where it is found.
Tetrameric (four subunits).
Tetramer composed of two identical dimers: Two alpha (one dimer), and two beta chains (one dimer)
-Each chain has an Fe2+ prosthetic group.
Found exclusively in erythrocytes.
Describe the 4˚ and 2˚ structure of Mb, and where it is found.
Monomeric (i.e. a single PP chain that is structurally similar to the individual PP chains of the tetrameric Hb molecule).
Found in cardiac and skeletal muscle.
The R-form (relaxed) of Hb has a high or low affinity for O2?
Is it saturated or unsaturated?
High O2 affinity.
Saturated.
The Bohr effect reflects the fact that…
What type of shift does it cause?
The deoxy form of hemoglobin has a greater affinity for protons than does oxyhemoglobin (because of a higher pKa of side chains).
Right shift = release O2.
An increase in protons shifts the Bohr effect equilibrium to the _____, favoring which form of hemoglobin?
Right shift.
Favoring deoxyhemoglobin.
At lower pH, ____ is required to achieve any given O2 saturation.
Greater pO2.
Binding of CO2 favors the (R or T)/(oxy or deoxy) form of hemoglobin?
This results in an (increased or decreased) O2 affinity and a (right or left shift)?
T, deoxy.
decreased affinity, right shift.
p.67
Describe the O2-binding characteristics of transfused blood.
It tends to have lower levels of 2,3-BPG, and thus, a higher affinity for O2. Therefore, it does not want to release O2 to depleted tissues.
When CO binds to one or more heme sites, hemoglobin shifts to the ____ conformation, causing the remaining sites to bind O2 with _____ affinity.
What affect does this have on the O2 dissociation curve (2)?
R conformation.
High affinity.
Left-shift, and changes sigmoid to hyperbola.
p.67
What is characteristic of methemoglobin?
Oxidation of a heme iron in hemoglobin to the ferric (Fe3+) state; which cannot bind O2.
p.77
Can carboxyhemoglobin be reversed?
Yes. Can be reversed with hyperbaric oxygen therapy (100% O2 at high pressure), which facilitates the dissociation of CO from the hemoglobin.
How is the affect on BPG in transfused blood mediated?
Decrease of BPG in transfused blood prevented by supplementing stored blood with the nucleotide INOSINE (hypoxanthine-ribose).
-Ribose from the inosine is converted to BPG in RBCs.
Define Hb p50.
What does a decreased p50 indicate?
pO2 at which Hb becomes 50% saturated.
O2 affinity increase.
HbA synthesis starts in the ______ at about the ____ month of pregnancy and gradually replaces HbF.
Starts in the Bone Marrow at about the 8th month of pregnancy.
p.68
HbF has a _____ affinity for O2 relative to HbA, why?
What are the implications for fetal blood circulation?
- Higher affinity for O2 due to weak binding of 2,3-BPG.
- The higher oxygen affinity of HbF facilitates the transfer of oxygen from the maternal circulation across the placenta to the RBC of the fetus.
What are the chains that make up HbF?
Where do the other chains come from?
α2γ2
The two γ chains are members of the ß-globin gene family and come from chromosome 11.
Where do the α1 and α2 chains come from?
Chromosome 16
How many genes that form Hb come from chromosome 16?
2; α1 and 2
What chromosome do the ß globins in Hb come from?
Chromosome 11
Why does HbF weakly bind 2,3-BPG?
Because γ chains interact poorly with BPG.
A molecule of HbS contains two normal α-globin chains and two ______, in which _____ at position ____ has been replaced with ______.
- mutant β-globin chains (βS)
- glutamate at position six has been replaced with valine.
p. 74
During electrophoresis at alkaline pH, HbS migrates more _____ toward the _____ than does HbA. Why?
- migrates more slowly toward the anode (positive electrode)
- This altered mobility of HbS is a result of the absence of the negatively charged glutamate residues in the two β chains, thereby rendering HbS less negative than HbA.
How does HbS cause its symptoms?
At low oxygen tension, deoxyhemoglobin S polymerizes inside the RBC, forming a network of insoluble fibrous polymers that stiffen and distort the cell, producing rigid, misshapen RBC. Such sickled cells frequently block the flow of blood in the narrow capillaries. This interruption in the supply of oxygen leads to localized anoxia (oxygen deprivation) in the tissue, causing pain and eventually death (infarction) of cells in the vicinity of the blockage. The anoxia also leads to an increase in deoxygenated HbS.
Compared to normal RBC, sickled cells have a decreased ability to deform and an increased tendency to adhere to vessel walls and so have difficulty moving through small vessels, thereby causing microvascular occlusion.
What makes sickling worse, i.e. enhances the severity? List some variables (5).
- The severity of disease is enhanced by any variable that increases the proportion of HbS in the deoxy state (that is, reduces the affinity of HbS for O2).
- These variables include decreased pO2, increased pCO2, decreased pH, dehydration, and an increased concentration of 2,3-BPG in RBC.
Treatment for HbS includes transfusion and what else (and how does it work?)
Hydroxyurea: Increases expression of globin γ chains, which is not sticky.
- In normal adult HbA, the ß globin chains have a ____ (give amino acid name and 3-letter abbr.) at position 6.
- In HbS, ß globin chains have ___ at position 6, while HbC have ____ at position 6.
- Glutamic acid (Glu) at position 6
- HbS = Val at position 6.
HbC = Lys at position 6.
What is the formula for ∆E˚’ ?
∆E˚’ = E˚’(acceptor) – E˚’(donor)
How is ∆E˚’ related to ∆G˚’ ?
∆G˚’ = -nF∆E˚’
How does ∆E determine the magnitude of ∆G˚’ ?
The more positive the ∆E, the more NEGATIVE (favorable) the ∆G
What is the name for Type I Collagen?
What does it form?
- Fibril-forming
- Skin, bone, tendon, vessels, cornea
- For supporting elements with high tensile strength*
What is the name for Type II Collagen?
What does it form?
- Fibril forming
- Cartilage, IV discs, vitreous body
- Cartilagenous*
What is the name for Type III Collagen?
What does it form?
- Fibril forming (Reticular collagen)
- Vessels, fetal skin
- For distensible tissues*
What is the name for Type IV Collagen?
What does it form?
- Network forming
- Basement membrane
- Forms 3D mesh rather than distinct fibers*
What is the name for Type VII Collagen?
What does it form?
- Network forming
- Beneath stratified squamous epithelia
- Forms 3D mesh rather than distinct fibers*
What is the name for Type IX Collagen?
What does it form?
- Fibril associated (associates with type II collagen)
- Cartilage, IV discs, vitreous body
IX and XII link OTHER COLLAGEN fibrils together
What is the name for Type XII Collagen?
What does it help to form?
- Fibril associated (associates with type I collagen)
- Tendon, ligaments, skin, bone, other tissues
IX and XII link OTHER COLLAGEN fibrils together
- What is the 2˚ structure of mature collagen
- What forms this 2˚ structure?
- Left handed TRIPLE HELIX NOT ALPHA, alpha is right-handed
- INTERchain H-bonding (NOT within the same chains, that is alpha)
How many residues per turn in a collagen helix?
3.3 residues/turn
What is the general AA content of collagen?
Every THIRD AA is glycine
What are the important AAs facilitating the formation of a collagen triple helix (2)?
Proline and glycine
What is the general repeating AA sequence for collagen (3)?
Gly-X-Y-, where X is usually Proline, and Y is often hydroxyproline (but can be hydroxylysine).
What accounts for the characteristic “banding” pattern of collagen fibrils?
The regularly stacked packing/cross-linking (67nm periodicity) of individual collagen molecules (tropocollagen).
Describe the steps of collagen biosynthesis (8)
- ) Genes for pro-alpha-collagen transcribed into mRNA.
- ) mRNA translated into pro-alpha-collagen polypeptide.
- ) Some Pro and Lysine hydroxylated (requires Vit C, aka Ascorbic Acid) by hydroxylase (lumen RER).
- ) Some HyLys glycosylated with glucose and galactose.
- ) 3 pro-alpha-chains assembled into triple helix.
- ) N- and C- termini stabilized by disulfide bonds to produce procollagen (prevents unweaving).
- ) Procllagen secreted into ECM.
- ) Propeptides at N- and C-termini cleaved by procollagen peptidases to generate tropocollagen (mature collagen fibers).
What facilitates collagen fibril formation?
Crosslinks between Lys and oxidized Lys (Allysine) via lysyl oxidase (requires Cu).
Describe the pathology of Menkes disease –i.e. gene, enzyme, biological result.
-Symptoms
Lack of Cu due to a genetic defect in the ATP7A gene causes the ECM enzyme LYSYL OXIDASE (Cu-containing) to malfunction, thereby INHIBITING THE CROSS-LINKING OF COLLAGEN MOLECULES.
The lack of copper in some tissues results in defect in bone, skin, hair, blood vessels, and the nervous system.
-Symptoms include: COARSE KINKY HAIR, loose skin and joints.
Describe the pathology of Osteogenesis Imperfectica (OI) Type I
-Symptoms
Aka Brittle Bones Syndrome
Type I = OI tarda: Decreased production in collagen ALPHA CHAINS.
- NOT AS BAD AS TYPE II*
- Symptoms: Long bone fractures PRIOR TO PUBERTY, hearing loss, blue sclerae.
Describe the pathology of Osteogenesis Imperfectica (OI) Type II
-Symptoms
OI congenita (more severe): Substitution of Gly with AAs bearing a bulky side chains – substituted side chains caused by DOMINANT mutations that code for alpha-1 or 2 chains in Type I Collagen (prevents triple helix).
-Symptoms: Frequently lethal, multiple fractures at birth, kyphosis, blue sclerae, respiratory problems.
Where does the tensile strength of collagen arise? (3)
Triple helix structure, assembly of tropocollagen subunits into fibrils, chemical cross-linking into fiBERS.
Describe the pathology of Ehlers-Danlos Syndrome – EDS (Types III and V)
- Causes
- Symptoms
CT disorders that result from inheritable defects in the metabolism of FIBRILLAR collagen molecules (III and V).
-Caused by deficiency/decreased expression of collagen-processing enzymes LYSYL HYDROXYLASE, or N-procollagen peptidase. May also result from mutations in the AA sequences for collagen types I, III, or V
Symptoms: Type V (classic EDS) –stretchy skin, joint hypermobility. Type III (vascular EDS, most serious) –ARTERIAL RUPTURE (i.e. ANEUYSM).
What structural property of elastin allows for its characteristic stretch?
Hydrophobic side chains do not form H-bonds, rather, elastin molecules are cross-linked by desmosine
What AAs make up the bulk of elastin?
How are elastin fibers cross-linked (i.e. with what?)
Small, non-polar (hydrophobic) AAs (e.g. glycine, alanine, valine), and rich in proline and lysine (very little HyPro or HyLys).
Elastin fibers cross-linked with desmosine.
NON-POLAR in order to provide stretch
Give four examples of where elastic tissue is found
- ) Lungs
- ) Wall of large blood vessels
- ) Elastic ligements
- ) Skin
Describe to two main constituents of elastic fibers in the ECM.
Tropoelastin fibers deposited on a fibrillin (microfibrils) scaffold.
Describe the pathology/causes of Marfan Syndrome.
-Symptoms (8)
Mutations in the fibrillin-1 proteins creates defective elastic tissue. This impairs the structural integrity of the skeletal and cardiovascular systems.
Symptoms: Ectopic lens (dislocation of lens), myopia (nearsight), uncharacteristically tall, abnormally long limbs, scoliosis, stretch marks, AORTIC ANEURYSM, mitral valve prolapse.
Describe the four types of alpha thalessemia
(1) If one of the four genes is defective, the individual is termed a silent carrier of α-thalassemia, because no physical manifestations of the disease occur.
(2) If two α-globin genes are defective, the individual is designated as having α-thalassemia trait.
(3) If three α-globin genes are defective, the individual has hemoglobin H (β4) disease, a hemolytic anemia of variable severity.
•beta-tetramer aggregates form.
(4) If all four α-globin genes are defective, hemoglobin Bart (γ4) disease with hydrops fetalis and fetal death results, because α-globin chains are required for the synthesis of HbF.
Describe the two types of beta thalessemia
Individuals with β-globin gene defects have either:
(1) β-thalassemia trait (β-thalassemia minor) if they have only one defective β- globin gene. ○ Those individuals with β-thalassemia minor make some β chains, and usually do not require specific treatment.
(2) β-thalassemia major (Cooley anemia) if both genes are defective. ○ However, those infants born with β- thalassemia major are seemingly healthy at birth but become severely anemic, usually during the first or second year of life due to ineffective erythropoiesis. Skeletal changes as a result of extramedullary hematopoiesis also are seen. These patients require regular transfusions of blood.
• Because the β-globin gene is not expressed until late in fetal gestation, the physical manifestations of β-thalassemias appear only several months after birth.
What will cause misfolding of proteins and formation of beta-pleated sheets?
Improper 1˚ sequence (i.e. protein sequence)
What is Cruefeldtz-Jakob disease?
aka Mad Cow, aka Scrapie (sheep): Misfolding of PrP (alpha-helix) into PrSCc (ß-sheet), which aggregates.
PrSCs induce PrP to change into PrSCs.
What is amyloidosis?
Misfolded proteins assuming a ß-sheet conformation and forming aggregates known as Amyloid Fibrils
Describe the structure of elastin in terms of: •AA composition (6) •Side-chain characteristics •2º structure •Behavior in ECM.
Elastin:
- Composed of mainly Ala, Gly, Val, Lys, Pro + Hydroxy-Pro (only 10%, very little of this)
- Side chains of these hydrophobic AAs do not form H-bonds (allows core of elastin to separate and stretch)
- Beta-SPIRAL (helical structure with repeated ß-turns)
- Extremely stable and insoluble in ECM.
Describe how elastin cross-linking occurs (4)
- ) Some lysines on elastin are oxidized to allyl lysine by lysyl oxidase.
- ) DESMOSINE (cross linker) is made up of three allysines and one lysine.
- ) Elastin monomers are crosslinked using desmosine crosslinks.
- ) Desmosine crosslinks provide elasticity to elastin fibers.
Mutations of fibrillin affect the _______, and thereby its function in the ECM.
deposition of elastin
What inhibitory mutation leads to elastin degradation in the lung alveoli? What condition can this lead to ?
Glu –> Lys, Glutamate/Glutamic acid (Glu) toLysine (Lys): Causes lack of alpha-1-antitrypsin (AAT), an inhibitor of the protease Elastase, which is secreted by neutrophils in the lungs and degrades the elastin in alveolar walls.
This can lead to emphysema
What is another, non-mutation inhibitor of AAT?
Treatment?
Smoking oxidizes a critical Met residue in AAT and inhibits it.
-Treatment: Weekly injection of IV recombinant AAT.
Describe the elastin monomer, i.e. its composition.
Tropoelastin: Soluble monomer composed primarily of small, non-polar AAs (Gly, Ala, Val), also rich in Pro and Lys.
Define Cofactor
Metal ions that associate with an enzyme via non-covalent interactions.
Define holoenzyme
Enzyme + coenzyme/cofactor
Define apoenzyme
Requires a cofactor/coenzyme, but does not have one bound; and thus is inactive.
How do enzymes accelerate biochemical reactions?
Enzymes provide an alternate, energetically favorable reaction pathway different from the uncatalyzed reaction, i.e. THEY STABILIZE THE TRANSITION-STATE.
Define coenzyme and what are the two subcategories?
Organic molecules (mostly derived from vitamins) that associate with enzymes, either loosely (diffusible, cosubstrate) or tightly (non-diffusible, prosthetic group)
What affect do enzymes have on Keq? What is Keq?
None; Keq = [product]/[substrate] at equilibrium.
What ultimately gives the active site of an enzyme its specificity?
3D folding and 3˚ structure allows it to distinguish between one of two H’s for example.
How do enzymes accelerate reactions? What do and don’t they alter?
They lower the free energy of activation to make a substrate reach the transition state more easily.
They DO NOT alter ∆G (free energy) or Keq (eq. constant).
Describe the action of OXIDOREDUCTASES –give an example
Catalyze redox reactions (i.e. transfer of electrons), e.g. lactate –pyruvate
Describe the action of TRANSFERASES
Catalyze transfer of C-, N-, or P- containing groups.
p.108 Lipp
Describe the action of HYDROLASES –give an example
Catalyze the cleavage of bonds by the addition of water, e.g. BREAKING PEPTIDE BONDS.
•All end in -lase
Describe the action of LYASES (aka SYNTHASES) –
give an example
Adds or removes atoms to or from a double bond.
C-C Lyase: Breaks a C–C bond without using water.
•Also makes new bonds (synthase) without
directly using the energy of ATP.
Catalyze the cleavage of C–C, C–S, and certain C–N bonds, e.g. Pyruvate to Acetaldehyde, e.g. they make PEPTIDE BONDS.
Describe the action of ISOMERASES
Catalyze racemization of optical or geometric isomers.
Describe the action of LIGASES. What do they need in order to function? –give an example
Catalyze the formation of bonds between carbon and O, S, or N.
-Must be coupled to hydrolysis of high-energy phosphates, e.g. Pyuvate to Oxaloacetate.
Why are uncatalyzed reactions often slow?
Because of the high free energy of activation (∆E between the reactants and the high-energy intermediates).
For reaction to occur, molecules must contain sufficient ________.
energy to overcome the energy barrier of the transition state
In the absence of an enzyme, only a small proportion of ___________.
a population of molecules may possess enough energy to achieve the transition state.
Relate ∆G to ∆H, ∆S, and T
∆G = ∆H – (T∆S)
What is exergonic? Relate to ∆G
Net decrease in free energy of the system, i.e. rxn is spontaneous. Does not require energy.
∆G < 0 (negative)
What is endergonic? Relate to ∆G
Net increase in free energy of the system, i.e. rxn is NOT spontaneous. Requires energy.
∆G > 0 (positive)
What is ∆Gº’?
Standard Free Energy Change (∆Gº) is the energy change when reactants and products are at a concentration of 1 mol/L, 1atm, pH = 0, and 298K.
∆Gº’ = standard at physiologic (i.e. pH = 7), everything else standard.
Interpret ∆G = 0
Reaction is at equilibrium.
Rate foreward = Reverse rate (NOT [A] = [B])
If ∆G for the forward reaction is -5 kcal/mol, then the backward reaction is _______.
+5 kcal/mol.
∆G depends on the concentration of _______ at constant ________ and _______.
reactant and product.
temp. and pressure.
Compare ∆Gº to ∆G in terms of substrate and product concentrations.
∆Gº is not altered by substrate concentrations and, thus, cannot predict physiologic reactions because it is composed solely of constants (R, T, and Keq), where Keq = [B]eq/[A]eq.
Recall –Standard Free Energy Change (∆Gº) is the energy change when reactants and products are at a concentration of 1 mol/L, 1atm, pH = 0, and 298K.
Whereas…
∆G depends on the concentration of reactant and product at constant temp. and pressure.
Give the equation for ∆G
∆G = ∆G˚ + RT ln([B]/[A])
Remember, ∆G depends on concentrations of reactant and product at constant pressure and temperature
Relate ∆G˚ to Keq
∆G˚ = –RTlnKeq
Coupling only happens when…
the first reaction has a positive free energy change, and the 2nd reaction has a large NEGATIVE free energy change.
At equilibrium, ∆G = ?
Zero, no free energy change because fwd and rvs are equal rates
- The ratio of _____ to _____ is an indicator of the energy charge of a cell.
- How is this interpreted?
- ATP to (ADP + AMP)
2. If ratio is high, energy charge is high, i.e. energy rich. If ratio is low, energy charge is low.
The ∆G˚’ of a sequence of reactions is equal to ______.
The sum of ∆G˚’ values of the individual reactions.
That is how an unfavorable reaction can proceed if coupled with a favorable reaction.
CK2 appears approximately _____ hours following onset of chest pain, reaches a peak of activity (reflects its diagnostic usage period) at approximately _______, and returns to baseline after ______.
4–8 hours
24 hours
48–72 hours
How does Troponin I identify an MI and when? Describe its diagnostic capabilities, and when is its peak (and most useful clinically)
Troponin I appears in plasma within 4–6 hours after an MI, peaks in 8–28 hours, and remains elevated for 3–10 days.
- Compare CK2 and LDH
2. Where is CK3 found?
- CK2 peaks approx. 24hrs after an MI
LDH peaks 36-40 hours after. - CK3 found in skeletal muscle
How is LDH used as a diagnostic tool in electrophoresis?
Each of the four subunits of LDH exist as two isoforms. They can be separated from each other by electrophoresis.
How to interpret LDH isozyme test; i.e. what constitutes a normal test, and what will damage to the heart show, and what will damage to the liver show.
Normal = L1H3
Damage to heart will show L1H3 and H4 predominate.
Damage to liver will show L1H3 and L4 predominate.
Explain the basis for diagnosis of Vitamin B1 (TPP) deficiency.
Do assay with and without added B1 (coenzyme for transketolase).
If adding extra B1 with >15% activation of transketolase, B1 is deficient. You should already have enough B1, so adding B1 shouldn’t change activation of transketolase. However, if you add B1 and you get more activation of transketolase, that means you were B1 deficient.
How to interpret Vitamin B1 (TPP) deficiency assay, what is it testing for?
Testing for the presence/amount of Vitamin B1 (TPP), a coenzyme for transketolase, via its level of activation of transketolase
1.) Fluorescent spot test of blood samples incubated with G 6-P, 6-PG, and NADP.
2.) TPP (coenzyme for transketolase) added.
3.) B1 deficiency assessed by transketolase activation in hemolyzed erythrocytes.
4.) >15% activation by added TPP indicates B1 deficiency.
See lecture 9, #34
What is the difference between a heterotropic and a homotropic effector?
A heterotropic allosteric modulator is a regulatory molecule that is not the enzyme’s substrate. It may be either an activator or an inhibitor of the enzyme, e.g. 2,3-BPG on Hb.
A homotropic effector is a substrate for its target enzyme, as well as a regulatory molecule of the enzyme’s activity. It is typically an activator of the enzyme. For example, O2 is a homotropic allosteric modulator of hemoglobin.
Bohr effect concept: O2 is readily released in the tissues where [H+] is high due to ___________.
Production of CO2 by metabolic processes.
The binding of protons to HbA stimulates ______. Thus, H+ stabilizes the ______-form of Hb.
- The release of O2.
- T (taut)
- Stabilizes the DEOXY form of Hb
What is the inhibitor for complex 1?
Rotenone
Complex ONE – rotenONE
What is the inhibitor for complex 3?
Antimycin
What are the inhibitors for complex 4?
Cyanide and CO.
What is the inhibitor of ATP synthase? Can it be overcome?
Oligomycin. Yes, relieved by uncouplers.
Name an uncoupler
2,4-dinitrophenol
- ) How many electrons from one NADH? = ___ H+, = ____ ATP.
- ) How many H+ does it take for ATP synthase to produce 1 ATP?
- ) How many degrees does ATP synthase turn with one H+?
- ) 1 NADH = 2e- = 10H+ = 3 ATP
- ) 3H+ = 1 ATP
- ) 1 H+ = 120˚
What does cyanide do to ETC?
CN- binds Fe3+ in heme of cytochrome a3 and prevents O2 reduction.
How to treat CN- poisoning in Hb? (2)
- ) Nitrites convert Fe2+ in Hb to Fe3+ if caught early, thereby renewing a supply of Fe3+ that is not bound by CN- and allowing O2 reduction to H2O to continue.
- ) Thiosulfate allows enzymatic conversion of CN- to thiocyanate (nontoxic).
What competes for binding of CN- to complex IV?
Methemoglobin (Heme with Fe3+ instead of Fe2+) – Can’t bind O2.
What are the three types of ROS and how to detoxify
- ) Superoxide anion (O2 + e-), CoQ – O2: Superoxide Dismutase (SOD).
- ) H2O2, from peroxisomal enzymes: CATALASE.
- ) Hydroxyl Radical: From metal ion-catalyzed reaction of superoxide and H2O2: GLUTATHIONE PEROXIDASE.
What are Adenine Nucleotide and Phosphate transporters? Name TWO
ATP made is sent out of mitochondria, and ADP is brought in (similar to oligomycin). BLOCKS THE SUBSTRATE FOR ATP SYNTHASE.
- ) Atractyloside
- ) Meraslyl
Name the branched chain amino acids (3)
LIV – Leucine, isoleucine, valine
What is the average length of an alpha helix (in residues)?
12 residues (range 5-45)
How many amino acid residues per turn of an alpha helix?
3.6
What is the average length of polypeptide chains in a beta pleated sheet (in AA residues)?
5-8 residues
- Beta-bends connect successive strands of ______ beta pleated sheets.
- What stabilizes the beta-bend structure (2)?
- Which AAs create the beta-bends (2) and why?
- Antiparallel beta pleated sheets.
- H-bonds and ionic bonds
- Glycine and Proline:
- Glycine because it is the smallest.
- Proline because it creates a kink.
What determines the 3º structure of proteins (2)?
- AA sequence
2. Interaction between AA side chains
What are the five stabilizing interactions between R groups of tertiary protein structures?
- Disulfide bonds: Covalent bonding between –SH groups on two cysteine residues.
- Hydrogen bonding: AAs with R groups that contain oxygen-bound or nitrogen-bound H’s.
○ e.g. -OH groups in Ser and Thr bonding w/ carboxyl
or carbonyl group. - Ionic bonds: Negatively charged groups (e.g. Asp or Glu) bonding with positively charged groups (amino on Lys).
- Hydrophobic interactions: Between AAs with nonpolar side chains. Tend to be located within the interior of a polypeptide molecule (away from the polar environment) where they associate with other hydrophobic AAs.
Proteins in a nonpolar environment (e.g. lipid membrane) exhibit the reverse, i.e. gather on the surface (of the protein, not the membrane). - Metal-ion complexes (metalloproteins), e.g. zinc finger domain, copper binding domain.
- Which types of carbohydrates does salivary amylase digest (2)?
- Which doesn’t it digest?
- What is the optimum pH environment for salivary amylase, what provides that environment?
- Glycogen and starch
- No disaccharides
- Alkaline; bicarbonate
Where besides the mouth is starch digested and by what?
In the lumen of the small intestine by pancreatic amylase
- What are the products of starch digestion by salivary amylase, what does it cleave?
- What is/are the product(s) of starch digestion by pancreatic amylase? Where?
- Cleaves random ALPHA-1,4 bonds, leaving behind alpha-1,6 bonds that it cannot hydrolyze = Dextrins i.e. linear and branched oligosaccharides, which then head towards the stomach.
- Maltose and isomaltose (disaccharides); in the small intestinal lumen.
Where are monosaccharides absorbed into the blood stream (2)? How?
Duodenum and Upper jejunum; Membrane-bound transmembrane proteins of brush border on luminal surface of mucosal lining.
Give the carbohydrate monomers, types of linkages, and whether or not they are reducing sugars for the following: Maltose, lactose, sucrose, isomaltose
Maltose: Glucose x2 via alpha-1,4 –a reducing sugar.
Lactose: Glucose + Galactose via beta-1,4 – a reducing sugar.
Sucrose: Glucose + Fructose via alpha-1,2 –NOT a reducing sugar.
Isomaltose: Glucose x2 via alpha-1,6 – a reducing sugar.
What are the four disaccharidases?
Maltase, lactase (ß-galactosidase), sucrase/isomaltase (one protein, two enzymatic activities).
What is an oligosaccharide?
A saccharide polymer consisting of 2-10 monosaccharides
What is the activity of endoglucosidase (pancreatic alpha-amylase)?
What are it’s products (2)?
What can’t it hydrolyze (3)?
Hydrolyzes internal alpha-1,4 linkages of starch and glycogen at random intervals.
Produces a mixture of short, branched alpha-1,6 (disaccharides maltose and isomaltose), and unbranched oligosaccharides (mixed limited-dextrins).
It can’t hydrolyze disaccharides, alpha-1,6, or cellulose (ß-1,4)
Where is pancreatic amylase secreted and when?
Into the duodenum after chyme has been neutralized by bicarbonate (also from pancreas).
Where is glucose absorbed after digestion and by what means (2)? How (3)?
- Intestines via SGLT-1: Na+-dependent glucose/galactose CO-transporter on apical surface of enterocytes (small intestine).
•2º active transport with [Na+] gradient, against [glucose] gradient. - Kidneys: SGLT-2 and SGLT-1
•SGLT-2: In PCT; reabsorbs ≈90% of filtered glucose.
•SGLT-1: In PST, reabsorbs ≈10% filtered glucose.
Both 2º active transport via Na/K ATPase
#29
- Explain how to treat hyperglycemia with SGLT-2 inhibitors.
- What are benefits (4)?
- A reasonable ______ or _______ diabetes therapy.
- Give two medications.
- SGLT-2 inhibitors reduce renal tubular reabsorption of glucose.
- Reduction of blood glucose without stimulating insulin release, weight loss, reduced BP, increased HDL.
- 2nd or 3rd line DM therapy.
- canagliFLOXIN, dapagliFLOXIN
#30
Why are GLUT transporters necessary? How do they work?
Because glucose cannot cross plasma membrane without GLUT transporters.
•Work via facilitated transport WITH their concentration gradient.
#31
GLUT-1: Give locations (3), where it is at low concentrations, and one very important point about its physiologic activity.
-What is it primarily responsible for?
Locations: Fetal tissues, erythrocytes/RBCs (adults), blood-brain barrier (adults)
•Low concentrations in adult muscle
•NOT INSULIN SENSITIVE
•Primarily responsible for low-level basal uptake of glucose required to sustain cell respiration.
#33
GLUT-2: Give locations (4).
- What is one very important point about its physiologic activity.
- What is it primarily responsible for transporting (3)?
Locations: Renal tubular cells, small intestine (glucose transporting) epithelial cells, liver cells, and pancreatic ß-cells.
•High-capacity, low-affinity isoform.
•Transports glucose into/out of cells (sends glucose, via concentration gradient, into the portal system from liver cells)
•Can transport glucose, galactose, and fructose INTO PORTAL CIRCULATION based on [gradient] –> i.e. Glucose into and out of liver and kidney cell and INTO ß-pancreatic cells.
MAIN POINT: GLUT-2 in the basolateral membrane ensures sugar exits into the bloodstream when blood glucose levels are lower. •Flows with [glucose] •Glucose, galactose, and fructose are transported *into portal circulation* by GLUT-2 (to liver). #33-34
GLUT-3: Give locations (2)
•Give one very important point about its physiologic activity.
Locations: Mostly in NEURONS (main glucose transporter there) and the PLACENTA.
•High-affinity isoform
#33
GLUT-4: Give locations (3)
•Give one very, VERY important point about its physiologic activity, i.e. what is it primarily responsible for?
Locations: Adipose tissue, skeletal muscle, cardiac muscle. •*INSULIN-REGULATED GLUCOSE TRANSPORTER (uniport, w/ [glucose] gradient)* – Translocates from intracellular membrane to cell surface of adipocytes and skeletal/cardiac muscle in response to insulin --> Responsible for *insulin-regulated* glucose uptake and storage. #33
What is GLUT-5 responsible for transporting, where?
-Give one important physiological characteristic
GLUT-5: A facilitated FRUCTOSE transporter located on the apical surface (brush border) of intestinal cells (facing intestinal lumen). •It is ENERGY and SUBSTRATE INDEPENDENT #34
All carbohydrates are absorbed by the time they reach the _______, before material enters the ________.
- By the time they reach the lower jejunum
- Before entering the large intestine
Distinguish between 1º and 2º lactase deficiency
1º: Can increase with age –varies with ethnic population.
2º: Due to intestinal injury; lactase activity is first activity lost, and the last to recover (i.e. can last a while).
What is the most common carbohydrate enzyme deficiency? How is it acquired?
Lactase (ß-galactosidase) deficiency –Hereditary.
What medications can contribute to carb digestion enzyme deficiencies? How?
Augmentin and clindamycin: Destroy intestinal flora that help to digest carbohydrates that the body failed to digest and absorb.
How does kwashiorkor contribute to carb digestive enzyme deficiency? Cystic fibrosis (CF)?
- Kwashiorkor is protein malnutrition –can’t produce enough digestive enzymes.
- Lactase activity can be lower in CF mucosa.
What are the “activator” and “inactivator” proteins for GTPases involved in G protein-coupled signalling?
Activator: GEF –guanine exchange factor; swaps GDP for GTP (active) on the GTPase.
Inactivator: GAP – GTPase accelerating protein; activates inherent GTPase activity = GTP to GDP (inactive)
- G protein-coupled receptors (GCPRs) are _______, _______ proteins (2º structure and relation to membrane).
- But the actual G protein itself is _________.
- Alpha-helical, integral membrane proteins.
2. Membrane-associated
What produces second messengers?
Effector proteins
What is the name given to G proteins of GCPR that describes them based on their subunits?
Heterotrimeric
Name the four common second messengers and what they activate
- Adenylyl cyclase (effector protein) –> cAMP (2nd messenger) –> activates protein kinase A (PKA)
- Guanylyl cyclase (effector protein) –> cGMP (2nd messenger) –> activates protein kinase G (PKG) and opens cation channels in rod cells.
- Phospholipase C (effector protein) –> PIP2 –> a.) DAG (2nd messenger): activates protein kinase C (PKC), b.) IP3: opens Ca2+ channels in the endoplasmic reticulum.
- The alpha- and beta-globulin genes are on which chromosomes, respectively.
- What other genes share the same chromosome as the beta gene (3) and what types of Hb do they contribute to?
- Alpha: #16
Beta: #11 - Delta –HbA2, Gamma (x2) –HbF, Epsilon –Hb Gower 1
Glucagon receptors are present in what two tissue types?
Liver and adipose NOT in skeletal muscle
How is the activation of PKA reversed (and signal cancelled) in the Gs pathway?
cAMP is degraded to 5’-AMP by cyclic nucleotide phosphodiesterase.
What is the mechanism of cholera toxin? What is the result?
Cholera toxin ADP-ribosylates an Arg on Gs-alpha, freezing it in the active, GTP-bound state = increased cAMP, increased Cl-, H2O, and HCO3- = DIARRHEA.
What is the mechanism of pertussis toxin? What is the result?
ADP-ribosylates Gi-alpha on a Cys, freezing it in the inactive, GDP-bound state = increased cAMP = Whooping cough.
What is the net result of both cholera and pertussis toxins?
Overproduction of cAMP
Describe the affinity of a G-alpha subunit for the other subunits when G-alpha is GDP-bound
The affinity of G-alpha for beta and gamma subunits INCREASES when G-alpha is GDP-bound.
Give the four steps of RTK signalling
- Growth factor or hormone binds = TK domains of each chain activate one another and subunits DIMERIZE.
- Auto cross-phosphorylation of RTK tyrosine residues.
- SH2 domain-containing adaptor proteins bind phosphotyrosines.
- Docking proteins bind adaptor proteins on their SH3 domain.
- Activation of ______ helps in the translocation of GLUT4 to the cell surface in myocytes and adipocytes.
- The answer to #1 also inactivates ______ to stimulate _______.
- It also may activate ______ by phosphorylating its ________.
- PKB
- Inactivates glycogen synthase kinase-3 (GSK-3) to stimulate glycogen synthesis
- May activate protein phosphatase 1 (PP-1) by phosphorylating its G subunit.
- Which two substances promote the R Hb form?
2. Which three substances promote the T Hb form?
- Oxy – O2 and CO
2. Deoxy – H+, CO2, and 2,3-BPG
Which type of non-functional Hb cannot revert back to oxyhemoglobin?
Sulfhemoglobin
What is the primary modulator of hemoglobins O2 affinity? What type of shift does it cause and how?
2,3-BPG: Shifts the O2 binding curve to the right by stabilizing the T state.
- Describe the pattern of delta chain production in Hb and what type of Hb they are for.
- Describe the pattern of gamma chain production in Hb and what type of Hb they are for.
- Describe the pattern of beta chain productions in Hb and what type of Hb they are for.
- Begins at ≈ birth and are made at a very low level until +6 months past birth. Make HbA2.
- Begins at -9 months prior to birth, peaks at -6 months prior, begins to taper off at birth, ends at SIX MONTHS after birth. Makes HbF.
- Begins at -9 months at very low level, increases at birth, OVERLAPS WITH GAMMA (HbF) at ≈ +1.5 months, and peaks at +6 months. Makes HbA.
- How many total alpha-globin genes and on what chromosome?
2. How many total beta-globin genes and on what chromosome?
- Two copies of alpha-globin genes = FOUR TOTAL GENES.
•Chromosome #16 - One copy of beta-globin genes = TWO TOTAL GENES.
•Chromosome #11
How is HbA1c made (i.e. what is added and to where)?
Glucose added (glycation) to the amino terminal Val of ß-globin genes.
What are the qualitative Hb-opathies (3) and the quantitative Hb-opathies (3)?
Qualitative: Sickle cell anemia (HbS), hemoglobin C disease (HbC), and hemoglobin SC disease (HbSC).
Quantitative: Alpha thalassemia, beta thalassemia major, beta thalassemia minor.
- What is the normal adult Hb beta-globin AA sequence that is mutated in other versions (give AA and position)?
- Sickle cell?
- HbC?
- Glu at position 6
- Glu –> Val at position 6
- Glu –> Lys at position 6
Individuals afflicted with Sickle Cell Anemia have inherited ____ mutant genes.
TWO mutant genes –one from each parent.
What does Vmax reflect?
Saturation of all active sites of the enzyme with substrate.
Give equations for the following enzymatic qualities:
- Velocity (2; in terms of [ES] and in terms of Vmax)
- Km
- V = k3 [ES] = (Vmax [S])/(Km + [S])
2. Km = (k2 + k3)/k1
How does Km vary with enzyme concentration?
IT DOESN’T –Km reflects the affinity of an enzyme for its substrate.
- What does zero order with respect to [S] mean? When does this occur?
- How to increase v at that point?
- Means that [S]»_space;> Km, velocity is constant and equal to Vmax, and rate of reaction is independent of [S]. Occurs when reaction rate = Vmax
- Adding more enzyme is the ONLY WAY to increase rate (v) at zero order.
What are the two variables in the Michaelis-Menten equation?
Velocity, v, and substrate concentration, [S].
What is the optimum temp range for most human enzymes?
35-40º C (37º C) – human enzymes start to denature at temps above 40º, after which the rate falls rapidly.
- If you want to increase intracellular [substrate], what type of competition should a clinical drug utilize?
- If you want to decrease intracellular [product], what type of competition should a clinical drug utilize?
- Competitive and non-competitive; both inhibit utilization of substrate.
- NON-COMPETITIVE (or suicide) b/c as unused substrate accumulates, it would compete with a competitive inhibitor and eventually overcome its inhibition and continue to produce product. Increasing [S] in such a way would have no effect on non-competitive inhibition.
What do V-type and K-type alterations change in an enzyme?
V-type: Alters catalytic activity (velocity of reaction)
K-type: Alters affinity of enzyme for the substrate (K0.5)
- Suicide inhibition has what apparent effect on the enzyme?
- What are its effects on kinetics?
- Which type of inhibition does it resemble?
- Effectively lowers [E] because it makes it unavailable.
- Lowers Vmax (b/c [E] is directly proportional to Vmax), K0.5 unchanged.
- Non-competitive
- NAD+ = dissociable coenzyme or tightly-bound prosthetic group
- FAD/FMN?
- NAD = dissociable coenzyme (i.e. derived from vitamins)
* FAD/FMN = tightly-bound prosthetic group
What is the ∆Gº’ of ATP?
-7.3 kcal/mol
What competes with CN- for binding to complex IV? How is this principle utilized in medicine?
methemoglobin – heme with Fe3+ (ferric). Cannot bind O2
•Methemoglobin (Heme with Fe3+) binds free CN- and prevents it (CN-) from binding cyto a3 (a treatment for cyanide poisoning.
What two inhibitors block the supply of substrates for ATP synthase and what substrate are they blocking?
- Atractyloside: Blocks the transfer of ADP (into matrix) and ATP (out of matrix to intermembrane space).
•No ADP substrate for ATP synthase. - Merasyl: Blocks the transfer of H2PO4- and H+ into matrix.
•No phosphate substrate for ATP synthase.
Describe the following proteins involved in RTK signalling in terms of their analogous roles in G-protein signalling:
•Sos
•Ras
•Raf
•Grb2
•MEK
•MAPK•Sos: GEF that activates Ras (GDP –> GTP)
•Ras: G-protein
•Raf: Effector protein/Protein kinase (target of Ras; activates MEK)
•MEK: MAP/ERK Kinase – protein kinase that activates
MAPK
•MAPK: Mitogen activating protein kinase –translocates
to nucleus and activates transcription factors
What is the functional difference in the clinical usages of immobilized enzymes and ELISA? Give examples of each.
- Immobilized enzymes are used to measure the levels of metabolites in biological samples such as whole blood, serum, or urine, e.g. blood glucose (glucose oxidase), plasma triglycerides (lipase), or plasma cholesterol (cholesterol oxidase).
- ELISA is used to detect the presence of specific antigens, e.g. pregnancy tests (hCG) or infections (HIV).
Define the following terms and their parameters: ΔG, ΔG°, ΔG°′
ΔG: Actual free energy change of a biochemical reaction (not a constant) –depends on concentrations of reactants and products.
ΔG°: Standard free energy change where the initial concentration of each reactant and product is 1 M, pressure is 1 atm, pH = 0 and the temperature is 298 K (25° C) (constant for the reaction).
ΔG°′: Standard free energy change for
biochemical purposes where other conditions being standard, the pH is 7.0 (constant for the reaction).
Desmosine consists of which four amino acids?
Three allysine (oxidized lysine), and one lysine.
