MCP Flashcards
1
Q
pH and Bohr Effect of CO2
A
- In active tissues where [CO2] is high, pH decreases as does affinity for O2
- promotes efficient unloading
- In the lungs where [CO2] is low, pH is raised which increases the affinity for O2
- promotes efficient loading
2
Q
carbamylation of Hb due to CO2
A
CO2 combines reversely with N-terminal amino to form carbamates; stabilizes the T state, reducing affinity of Hb for O2
3
Q
BPG (2, 3 DPG)
A
stabilizes the T state, reducing affinity for O2 and shifting the curve right
- increases efficiency of unloading in uscle by moving the steepest part of the curve
- in absence of BPG, Hb would be a poor O2-delivering system
- people in high alt. have altered BPG levels
4
Q
sickle cell anemia (HbS)
A
- form of hemolytic anemia due to Glu→Val mutation in ßchain
- HbS polymerize into extended fibers that span RBC
5
Q
a and ß thalassemia
A
insufficient a or ß chains leads to insufficient/nonfunction Hb in RBCs
- major: complete lack of function chains vs. minor: decreased production of functional chains
6
Q
methemoglobinemia
A
- caused by
- mutation in Hb that stabilizes oxidized form
- defect in enzyme (CYB5R) that normally reduces MetHb to Hb
- chemical agents (Na+ or K+)
- unable to combine reversibly with O2 or CO2
- cyanosis occurs because MetHb is greenish/black due to oxidation of heme Fe (2+ to 3+)
7
Q
myoglobin
A
- intracellular O2 transport and storage protein
- predominantly a-helix with heme moiety
- hyperbolic O2 binding curve, O2 binds under conditions in which Hb releases it
8
Q
hemoglobin (Hb)
A
- predominant O2 carrier in circulatory system
- tetrameric protein (a2ß2)
- stable, rigid aß dimers (protomers) come together to form loose tetramer
- each a and ß chain is noncovalently found to heme
- **cooperative allostery **results from structure of tetramer being in equilibrium between T state (low affinity for O2) and R state (high affinity for O2)
9
Q
heme group
A
- 3 different species, all contain a porphyrin ring structure with Fe as the chelated metal
- Protoporphyrine: 4 pyrolle rings that create binding site for Fe; isomer in heme is protoporphyrin IX
- Enzymes bind heme by ligating axially to Fe with amino acids
- deoxyHb: Fe(II) is 5 coordinate
- oxyHb: Fe(I) is 6 coordinate, O2 binds to distal histadine
10
Q
Hb O2 binding curve
A
- sigmoidal curve due to convolution of high affinity R state and low affinity T state
- At high [O2]: dissociation curve is close to R curve, as [O2] decreases, it approaches T curve
- negative effectors (pH, BPG, CO2) cause right shift of curve
11
Q
effect of pH on Hb O2 binding curve
A
- oxygenation of Hb makes it a stronger acid
- high pH (low H+): Hb has higher affinity for O2, more O2 is loaded
- low pH (high H+): Hb has lower affinity for O2, more O2 is released
12
Q
5 steps of heme synthesis
A
- ∂-aminolevulinate (ALA) synthesis from glycine and succinyl CoA in mitochondria: **committing/regulating step
- Porphobilinogen synthesis: ALA dehydrase Zn cofactor can be inhibited by Pb
- Uroporphyrinogen synthesis
- Protoporphyrin IX synthesis: no energy input needed
- Protoheme IX (heme) synthesis: no energy input needed, ferrochelatase enzyme in mitochondria can be inhibited by Pb
13
Q
porphyrias
A
diseases associated with inability to synthesize heme
- hepatic: come as induced attacks
- erythropoietic: chronic conditions
14
Q
Congenital erythropoietic porphyria (CEP)
A
deficiency in uroporphyrinogen III co-synthase that flips D ring
- build up of photoreactive heme precursors
- origin of werewolf legend
15
Q
Protoporphyria
A
partial deficiency in ferrochelatase (last step of biosynth), similar but milder symptoms as CEP, occurs in erythroid cells and less severely in liver
16
Q
Acute intermittent porphyria
A
most common porphyria, caused by deficiency of porphobilinogen (PBG) deaminase in liver, attacks usually occur following use of an agent that induces heme synthesis
17
Q
Porphyria cutanea tarda
A
deficiency in utoporphyrinogen decarboxylase, asymptomatic until liver disorder is imposed (e.g., HepC); attacks usually occur following use of an agent that induces heme synthesis
18
Q
HbSS
A
homozygous or doubly heterzygous state of mutation in RBCs that causes polymerization of Hb upon deoxygenation→hemolytic anemia and blockage of capillaries
- most common life-threatening genetic disorder; 1/400 African-Americans
- most severe form of sickle cell disease
- clinical variations in severity are poorly understood
19
Q
Sickle Cell Trait (HbAS)
A
silent carrier state of sickle cell disease; >55%HbA, the rest is HbS
- parents of most newly diagnosed infants with SS didn’t know they were carriers
20
Q
HbC
A
does not sickle but increases Hbß viscosity
21
Q
HbSC Disease
A
AS x AC; results in a milder disease
22
Q
Sickle ß-thalassemia
A
AS x ß-thalassemia; can be as severe as HbSS if there is no HbA production
23
Q
how does HbF affect sickle cell disease?
A
don’t develop signs and symptoms until 3-4 mo when production of HbF switches to HbA after birth
- the higher [HbF] relative to [HbS] the milder the disease because HbF blocks polymerization of HbS
- different ethnicities have different levels of HbF throughout life (Arab-indians and Senegalese have higher HbF, most severe disease is related to the Bantu and the Benin)
- research aimed at changing transcriptional switch so levels of y-subunit are maintained
24
Q
why is overwhleming infection such a risk for HbSS patients?
A
spleen clear encapsulated bacteria (e.g., pneumococcus) for which you don’t have antibody, slow circulation making it sluggish and hypoxic→sickling
- bacterial sepsis: 30-50% mortality, 2/3 of dsease within 8 hours of 1st symptoms; most common death <5
- prophylactic penicillin and aggressive treatment of fever
25
painful vaso-occlusive episodes
most common complication of sickle cell disease
* pain is excruciating and can be anywhere (hand-foot syndrome in children)
* impairs ability to attend school regularly/be employed
* **pain management is essential**, basiased and inappropriate therapy based on race and assumptions they are drug-seeking
26
what are the common co-morbidities of sickle cell disease?
* bacterial sepsis
* **painful vaso-occlusive episodes**
* stroke
* **acute chest syndrome**
* **acute splenic sequestration crisis**
27
stroke in sickle cell disease patients
* 10% incidence; classic hemiparesis usually from major vessel obstruction
* prevent with transcranial Doppler
* treat with exchange transfusion; maintain chronic transfusion program to prevent reoccurance
28
acute chest syndrome
unique term of acute lung disease in sickle cell patients; difficult to determine cause so treat all bases
* antibiotics, O2, incentive spirometry, transfusion
* major cause of mortality in adult sickle cell patients
29
acute splenic sequestration crisis
sudden enlargement of speel due to acute vaso-occlusion→hemorrhaging
* occurs in 1st 5yrs of life and can rapidly result in shock
* treat with fluid resuscitation and transfusion, teach parents splenic palpation
30
what is the role of genetic counseling in sickle cell disease?
empower patients to make their own reproductive decisions; harvest cord blood from newborn if they do not have sickle cell disease because they could be a donor if next child has sickle cell disease
31
hydroxyurea
milke chemotherapeutic agent used to treat sickle cell disease
* results in 50% reduction in the incidence of complications
* long-term toxicity is very rare
32
what is the only cure for sickle cell disease?
bone marrow transplantation; candidates: have had/at risk for stroke, debilitating pain, recurrent acute chest syndrome
* requires HLA-identical siblings (only 25% of full siblings are HLA identical, available in only 14% of families
33
what is the mutation that results in sickling of RBCs?
single amino acid substitution (A→T) in HbB on chromosome 11 (val instead of glutamic acid)
* causes dramatic effect in Hb; deoxyHbS forms 14 stranded polymers
34
reversibly sickled cells vs. irreversibly sickled cells
**reversibly sickled:** switch back and forth based on polymerization status of HbS
**irreversibly sickled:** locked into sickle shape even when HbS is depolymerized and oxygenated
* Oxidative stress changes membrane skeleton composed of spectrin and actin interactions
* Disulfide bridge creates actin filament that cannot depolymerize; spectrin with diminished ubiquitination→association between spectrin and actin
* Oxidative stress causes reduced flippase activity and increased scramblase activity so phosphatidyl serine appears on outside of RBC bilayer→hypercoagulation and thrombosis because it makes sickle RBC adherent to blood vessel wall and it enables recognition by macrophages→anemia
35
what is the pathophysiology of sickle cell vaso-occlusion?
* activation of endothelial cells associated with blood vessel; WBCs are more adhesive and adhere to vessel endothelium, RBCs also adhere; WBCs and RBCs adhere to each other→hypoxia
36
ischemia-reperfusion in vaso-occlusion crises of sickle cell disease
* **ischemia:** metabolic response to hypoxia→xanthine oxidase to covert O2 to superoxide ROS
* **reperfusion: **burst of ROS when blood flow is restored; activated NFkB transcription factor→expression of inflammatory molecules→inflammation→new expression of adhesion proteins on cells that interact with adjacent cells
* sickle RBCs have 3x as much oxygen radicals compared to normal RBCs and very low levels of reduced glutathione (GSH)
* increased activity of SOD→elevated H2O2 but decreased activity of GPX and SCD so no conversion of H2O2→water and oxygen, instead H2O2→OH-
* No antioxidant enzyme binds up OH- so oxidative damage occurs to proteins and lipids on membrane skeleton of RBCs→sickling
37
molecular basis of dense ISCs
* induced cation leak channel due to damage from sickle cycling, K+ and Mg2+ leave cell; Ca2+ in
* activates other channels (including Gardos channel); more K+ leads and water follows→dense cells
* active oxidation diminished spectrin ubiquinilation→locked sickle cells→dense ISCs
38
potential therapies for sickle cell disease
* Effect oxidative stress (NAC is an antioxidant that easily enters cells AND increases GSH levels)
* Effect K+ leakage from RBCs (Gardos channel inhibitor)
* Effect inflammation
* Effect HbF
* Gene therapy: viral vectors to insert normal ß-chain via benign illness without immunosuppression of transplant
39
Km
substrate concentration that gives you half Vmax
* catalytic rate of enzye most sensitive to substrate when [S] is \< Km
* approximately the same as the dissociation constant when K2\>\>K3 (substrate binds and dissociates many times before going to product)
40
Vmas
maximal velocity achievable for specific concentration of enzyme
* at this point, enzymes are saturated with substrate; increase Vmax by increasing [E]
41
Kcat
intrinsic constant that shows how fast one enzyme could convert substrate (Vmax/[E]); higher Kcat implies higher efficiency of the enzyme
42
lineweaver-burk plot
linearized michaelis-menten, use to estimate constants
* -1/Km= x-int
* 1/Vmax= y-int
43
quantitative properties of inhibitors
* Ki: strength with which an inhibitor binds to an enzyme; dissociation constrant for enzyme-inhibitor complex; *higher Ki implies weaker inhibition*
* **competitive:** compete with substrate for binding to active site; overcome by increasing [S]
* *curves intersect at y-axis*
* **noncompetitive**: bind to a site other than active site
* *curves intersect at x-axis*
44
how do enzymes affect energetics of chemical reaction?
do not change ∆G, *lower activation energy by direct stabilization of transition state and/or creation of new reaction pathways*
45
common features of enzyme active sites
* small part of total enzyme volume
* 3D structure
* bind substrates through multiple weak, noncovalent interactions
* water is excluded unless it is a reagent
* highly specific binding of substrate (induced fit)
* can include non-protein prosthetic groups/cofactors
46
6 basic strategies for enzymatic catalysis
Direct stablization of transition state
* **preferential binding of transition state** with greater affinity than substrate or products (better fit)
* **proximity and orientation effects;** enzymes immobilize substrates
Chemical assistance at active site
* **acid-base:** weak acid on enzyme provides partial proton transfer/weak base accepts (histidine usually involved)
* **covalent:** transient formation of covalent bond
* **metal ion catalysis**
* **electrostatic catalysis**
47
suicide inhibitors
bind to enzyme because of resemblance to substrate and are converted into an irreversible inhibitor
* ex. penicillin: suicide inhibitor of glycopeptide transpeptidase, crtical for bacterial cell wall biosynthesis
* ex. AChE (degrades ACh in synaptic cleft) is *irreversibly inhibited* by nerve gas, *reversibly inhibited* by aricept/cognex (used to improve cognitive function)
48
structure-based drug design
best inhibitors mimic transition state conformation (enzymes show preferential binding of the transition state)
* most drugs are reversible inhibitors: turn down function of target enzyme but don't completely inhibit activity
49
HIV-protease inhibitors
* developed using knowledge of *aspartyl protease mechanism* and protein structure
* resistance: high error rate by RT and large number of virus particles synthesized daily; use drug combinations
* resistant virus is insensitive to drug (high Ki) and normal catalytic activity (wt Kcat/Km)
50
allosteric regulation
frequently operate at control points (rate-limiting steps\_ in metabolic pathways; activity of enzyme is modulated either by levels of substrate or byactivating inhibitory modulators
* feedback inhibition: end product acts at allosteric inhibior of first enzymatic step
* does not follow michaelis-menten
* e.g. ATCase catalyzes first step in CTP synthesis
* CTP is negative regulator
* ATP is positive regulator
51
reversible covalent modificaiton
causes conformational change that affects catalysis; alter celular location of enzyme, alter interactions with other proteins
* ex. phosphorylation: kinase activates enzyme, phosphatase dephosphorylates to inactivate
52
irreversible covalent modification
synthesize enzyme in inactive form and active in the time/place where they are needed
* ex. zymogen: inactive precursor form of protease that is activated by specific proteolytic cleavage by another protease (irreversible by activated proteases can be turned off through inhibitors)
* **digestive enzymes:** enteropeptidase cleaves trypsinogen to trypsin in small intestine
* **coagulation cascade**
* **protein-protein interaction: **enzyme activity can be regulated reversibly by interactions with other proteins (PKA regulated by cAMP which releases inhibition; calmodulin shows Ca2+ depedent interactions with multiple enzymes)
*
53
coagulation cascade as an example of irreversible covalent modification
* *rapid activation of blood coagulation:* cascade of zymogen activations allow sequential activation of series of serine proteases
* *localization of clot to injury site*
* *rapid termination after clot formation to prevent thrombosis: *reverse zymogen cascae hydrolyzes close
54
deficiencies to protease inhibitors
* mutant pancreatic trypsin inhibitor inhibits inappropriately activated digestive proceases in pancrease by protein-protein interactions
* **antithrombin deficiency:** excessive clotting
* **a1-antitrypsin deficiency:** too much active elastas in lung eventually causes COPD
55
Diagnostic measurement of enzyme levels
* **measure rate of product formation** in presence of excess susbtrate because product formation with time will be linear
* **coupled enzyme system:** one of the products of the first reaction is stoichiometrically converted to a detectable product by second reaction
56
using enzymes clinically to measure of substrate
e.g., blood glucose: used coupled enzyme assay, NADPH is detected by ability to reduce colored dye to form colored product
57
using enzymes to diagnose tissue damage by isozyme distribution
* isozymes: different forms of enzyme that carry out same reaction; different amino acid sequences and specific expression in different tissues or a specific pattern of expression during development
* non-plasma specific enzymes can appear in serum because of damage to tissue of origin or because "spillover" due to overproduction in tissue of origin
* e.g., following MI: CK-2, AST, LDH-1 appear and have characteristic rise and fall (vs. DMD long term rise in CK-3)
58
list of tissue-specific isozymes
* **ALT: **viral hepatitis
* **amylase:** acute pancreatitis
* **lipase: **acute pancreatitis
* **lactate dehydrogenase 5:** liver disease
* **alk. phos:** various bone disorders, obstructive liver disease
* **creatine kinase: **muscle disorders and MI
* **phos, acid: **metastatic carcinoma of prostate
