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Flashcards in Unit 3 - Week 1 Deck (130):


small globular intracellular PRO with 132 AA abundant in vertebral muscle, with 8 alpha helices and 1 heme
-intracellular transport and temporary storage of O2 for aerobic metabolism of muscle


hemoglobin structure

2 alpha, 2 beta chains, each non-covalently bound to heme (that binds 1 O2 each), and connected to each other in tetrahedron



stable, rigid alpha-beta heterodimers that Hb monomers are first assembled into
-2 dimers come together to form loose/flexible tetramer, and rapid equiplibrium between them (favor tetramer)


T/R hemoglobin structure

quartenary structures with different O2 affinity/binding that are basis of cooperativity resulting in sigmoid binding curve
T: low O2 affinity
R: high O2 affinity (chain rotated 15 degrees)


heme structure

prosthetic group with 4 pyrrole rings (tetraphyrole/porphyrin) bound to central Fe (this bounds to O2)
-proprionate groups are on 2 adjacent chains
-if protoporphyrin IX, will have asymmetric vinyl substituents on other 2 adjacent chains


penta/hexacoordinate Fe (additional histidines)

originally has 4 ligands provided by 4 pyrrole rings of heme
-proximal histidine in deoxyHb and oxyHb coordinates Fe
-distal histidine only if OxyHb, since O2 will bind to Fe and DH; also bonds are shorter and Fe moves into plane of heme
--DH increases affinity for O2, while decreasing affinity for CO, and prevents oxidation of Fe by destabilizing linear intermediate in process


Coordination state VS oxygenation state VS oxidation state

CS: holo (heme present FeII, binds to O2) and apo (no heme, cannot bind O2)
OGS: Deoxy (dark red venous) or oxy (bright scarlet arterial) both have heme and can bind O2, but deoxy has FeII, and oxy is unknown
ODS: ferrous (heme has FeII to bind O2) and ferric (heme has FeIII and cannot bind O2; also methemoglobin metHB)


possible poisons that replace O2 on heme

CO, NO, and H2S bind heme with higher affinity than O2


O2 dissociation curves of Hb and Mb

Mb: hyperbolic curve (quickly saturated with O2)
Hb: sigmoidal curve (more slowly saturated with O2, shape from convolution of R and T state)
reveals cooperative interaction between O2 binding sites
-Mb binds under conditions in which Hb releases it, buffering O2 concentration and increasing transport rate via diffusion within cytoplasm


allostery VS cooperativity

A: something happening at another site (site on a multimeric PRO, different site, etc.) affects the active site
C: type of allostery where something happening at one site promotes the same thing at another identical site (O2 binding at one site increases affinity of other sites, mostly on multimeric PRO)


allosteric interaction of Hb binding

due to quaternary structure in equilibrium between T and R
-positive effectors: O2 (shift to left)
-negative effectors: BPG, CO2, H+, Cl- (shift to right)
(myoglobin has no allosteric effects b/c monomer)


BPG basics

2,3-bisphosphoglycerate; negative effector for allosteric O2 binding to hemoglobin
-stabilizes T state, reducing affinity and shifting curve to right
-doesn't change affinity as Hb moves from lungs to tissue, but sets midpoint affinity abount which it is varied by other effectors
-people in high altitudes have altered levels of BPG in blood


physiological role of BPG

binds deoxyHb in 1:1 molar ratio (per Hb tetramer) with Kd ~15 microM, binds only weakly to oxyHb, almost always bound to T-state
-must release BPG to become R state


torr and Hb saturation of aterial VS venous blood

Arterial: pO2 is 100 torr, Hb is 95% saturated (R state)
Venous: pO2 is 20 torr, Hb is 43% saturated (T state)
in vivo with BPG, Hb is efficient O2 carrier, unloading ~52% of O2 passing thru capillaries


BPG structural basis

1 molecule of BPG binds per tetramer of Hb, at tetramer interface where it interacts with lys, his, and B-chain N-termini in the center


Bohr effect

pH modulates affinity of Hb, but not Mb, for O2
High pH - low H+ - Hb has higher affinity for O2, more O2 is loaded; R-state
-in lungs
Low pH - high H+ - Hb has lower affinity for O2, more O2 is released (and CO2 is bound); T-state
-accelerated by carbonic anhydrase
-in active tissues



CO2 regulates O2 affinity of Hb, but not Mb
-CO2 combines reversibly with N-terminal amino groups of blood proteins to form carbamates
-H+ and CO2 synergize to unload O2 in capillary where it's needed


why does HbF has higher affinity for O2?

deoxy-HbF has lower affinity for BPG


possible causes of hemoglobinopathies

1. changes in surface residues (SCD)
2. changes in internally located residues (ustable Hb, causes hemolytic anemia)
3. changes in stabilizing methemoglobin (methemoglobinemia; not effective O2 carrier, and looks like R state due to Fe position, so prevents unloading)


the structure of HbS in SCD

Glu --> Val at Beta-6 causes aggregation and polymeration of HbS into rigid extended fibers spanning length of cell
-deoxyHbS fibers are helically twisted strands, and only one of two val6beta molecules contact each other


WT Hb VS common Hb variants

WT: HbA (a2B2, 95%), HbA2 (a2delta2, 5%), HbF (a2y2)
variants: HbS (a2B*2), HbC (a2 only), HbH (B4), HbBarts (y4)


HbS structuers in both oxygenated and deoxygenated states

O2: individual Hb tetramers
deO2: 14-stranded polymers


reversible sickle cells

cycle between biconcave and sickled shape, resulting in hemolysis (due to weakened membrane) and vaso-occlusion (sickle RBC and WBC stick to each other and endothelial cells)


irreversibly sickled cells

constitute 2 to 40% of circulating RBCs in homozygous sickle cell anemia
-stick to WBC to cause vaso-occlusion
-due to cysteine isoforms, b/c oxidative stress and decreased GSH creates DS bridge that closes an ATP-binding cleft and cannot depolymerize chains


what causes vasoocclusion

sticking together of sickle RBC and polymorphism'd WBC, endothelial cells, and plasma cells
-Xm 2, 6, and 11 are related
-will lead to ischemia


survival of patients with SCD crisis rates above 3 compared to below 1

15 year difference in survival rate


what factors lead to the variance in clinical severity and outcome?

1. genomic factors (more HbF will decrease severity)
2. varying inflammation, oxidative stress, vasculopathy, and hypercoagulation
3. changes in PRO expression and post translational modification (esp. cysteine isoforms)


which Xm the alpha and beta clusters are on

alpha - Xm 16
beta - Xm 11 (linearly arranged 5' to 3' with distal locus control region to direct expression of genes)


evolution from yolk sac to fetal liver to bone marrow blood cells

YS: epsilon
FL: gamma
BM: beta


3 Xmal loci associated with HbF expression and clinical severity

Xm 2 - trans acting BCL11A
Xm 6 - intergenic interval - trans acting HBS1L-MYB
Xm 11 - cis acting haplotypes of SCD



transcriptional repressor and gamma-globin silencer (trans-acting) on Xm 2
-binds ty C-MYB (hematopoeitic transcription factor that expresses gamma-globulin production)
-both bind to other transcription factors and complexes are responsible for switch from gamma to beta globin
-associated with sickle cell severity



influences expression of HbF (on intergenic interval of Xm 6)
-associated with sickle cell severity


relationship between HbS and SCD severity

increased HbF causes decreased severity
-altered transcriptional regulation of gamma to beta globin leads to increased HbF and decreased HbS
-if can raise HbF from 0.1-1% to ~20%, will cause less severe disease


4 haplotypes of beta-globin disease
(most severe to least severe)

(all have polymorphism cis to beta-globin-like gene clusters that regulate HbF expression)
-Bantu --> Benin --> Senegal --> Arab-Indian
-if in order of HbF, in reverse
-Bantu also has lowest response to hydroxyurea



burst of ROS production when blood flow is restored
-caused cellular metabolic changes caused by ischemia from vaso-occlusion
-cell xanthine oxidase (from endothelial cells and adherent leukocytes) converts O2 into superoxide



cells and tissues don't receive required O2
-caused by vaso-cclusion
-causes cells to increase expression of xanthine oxidase


what production of ROS leads to

-NFkB activation
-inflammation and cytokine release
-leukocyte activation
-increased expression of adhesion molecules on endothelial and WBC
-further vaso-occlusion
-decreased NO availability
-resulting abnormal endothelial dependent vaso-dilation


double jeopardy of sickle RBC

-contain 3x more O2 radicals compared to normal RBC
-low levels of reduced glutathione (GSH), especially in highest density RBC (most damaged)


ROS and antioxidants, starting from superoxide (O2-)

superoxide dismutase: O2- --> H2O2
catalase: H2O2 --> H2O + O2
GPX: H2O2 --> 2H2O
Haber-Weiss reaction: H2O2 --> OH. (most dangerous)


causes of increased ROS/RNS in SCD

1. increased autooxidation of HbS into metHb and O2
2. H2O2 in contact with metHbS causes release of heme and free Fe more readily than metHbA
-free heme and Fe are on cytoplasmic surface of RBC membrane and catalyze production of OH. by Fenton and Haber-Weiss
3. O2- binds to NO to form ONOO-
4. released HbS binds NO to limit vasodilatory, anti-inflammatory, and antithrombotic properties
5. ischemia-reperfusion injury leads to increased XO production and NADPH oxidase activity to make O2-, which is converted to OH.
6. PMNs make ROS in NADPH oxidase-dependent respiratory burst


reasons for decrease in antioxidant production in SCD

enzymatic and non-enzymatic anti-oxidant scavengers are reduced
-increased SOD activity leads to increased H2O2 and OH.
-glutathione and catalase activity are reduced in HbS, causing increased H2O2
-GSH is substantially reduced in HbS and intracellular GSH is inversely related to density


cysteine modifications related to oxidative stress in SCD

its thiol group that serves as redox-sensitive switch is a target of ROS/RNS
-oxidation states range from -2 to +6, and reversible, but the more ROS/RNS there are, the higher the oxidation until irreversible


RBC lipid bilayer components

-outer: mostly PC, SM
-inner: mostly PE, and exclusively PS (phosphatidyl serine)
-flipases use ATP to ensure any PS that accidentally goes to outer layer is returned to inner
-scramblases randomly move components from one bilayer to another, so if it takes more PS than flipases can save, will attract Ca and MP to kill the cells with PS on the outer leaflet


RBC spectrin-ubiquitin activity

RBC spectrin (heterodimer of alpha/beta chains) has E2/E3 ubiquitin conjugating/ligating activity that can ubiquinate itself
-these thio-ester linkages tru cysteine residues are on alpha-spectrin repeat 20, and target site on 21
-this effect is lost in SCD


2 step model for dense ISC formation

1. decreasing GSH levels in HbS create dehydrated cells (b/c K+ efflux from K+ channels, cation leaks, Mg++ loss, etc.) and oxidative damage to Gardos channel (also K+ efflux)
2. locking of ISCs due to oxidative damage to B-actin, lack of ubiquitination, less spectrin
all can be fixed by NAC antioxidant to raise GSH levels


NAC effects

antioxidant against SCD
-increase GSH
-decrease dense cells, ISC, and crises
-elminates scramblase moving phosphoserine to outer membrane


current SCD treatments

-blood and bone marrow stem cells


future SCD treatments

-stop K+ leakage from RBCs
-effect NO levels
-effect oxidative stress (NAC)
-effect adhesion
-effect inflammation
-effect HbF
-replace defective gene (gene therapy)


biomarkers for SCD severity

20 PRO whose levels are highly corolated w/ 5 year crisis rate
-want to ensure they are valid and predictive early in life


frequency and severity of SCD

1/400 African-Americans, often life-threatening
-lifespan: 50 females, 45 males
-<3% die in childhood


initial diagnosis

HbF in newborns is protecctive for first 3-4 months, but once fades away are susceptible to bacterial sepsis
-now are screened and treated with prophylactic antibodies


HbSC disease

one parent is HbS, and the other is HbC
-C is also defect of beta position (but lys, not val)
-C has no sickle, but increases viscosity
-milder disease than homozygous SS


bacterial sepsis

splenic vaso-occlusion inhibits bacterial clearance
-pneumococcus in previously unexposed children needs splenic clearance with penicillin prophylaxis, aggressive fever antibiotic, and immunizations
-present with fever that is common with everyone else, but 30-50% mortality (2/3 die w/in 8 hours)
--most common cause of death < 5 yrs


painful vaso-occlusive episode of SCD

most common complication throughout life, and not clear why episodic and unpredictable, lasting a few days to weeks
-hand/foot syndrome (swelling) in 1-2 yo
-excruciating pain needs narcotics (rarely get addicted)
-supportive care with fluids (decrease dehydration), fever control, but transfusions usually not helpful


stroke in SCD

10% develop, w/ unilateral weakness
-due to blocking of arterial vessels in children, hemorrhage in adults, but acute onset normally in children
-long term neurologic deficits --> death
-chronic RBC transfusions and prophylactic penicillin to prevent reoccurance


stroke prevention in SCD

use transcranial Doppler
-as arteries narrow, higher cerebral arterial flow, and higher risk
-prevent 80% of strokes by annual studies


acute chest syndrome in SCD

unique term for acute lung disease b/c hard to determine cause
-includes any/all: infection, vaso-occlusion, fat embolus
-most common cause of death >5 yo, can cause chronic lung disease in adults
-treat with antibiotics, oxygenation, incentive spirometry, and transfusions to ensure can breathe


acute splenic sequestration crisis

sudden enlargement of spleen due to acute vaso-occlusion (bleeding into spleen)
-occurs in first 5 years of life, rapidly results in hypovolemic shock
-acute treatment involves fluid resuscitation and transfusion to increase volume
-teach and trust parents


genetic counseling

let parents make their own decisions
-if at risk, use prenatal diagnosis via amniocentesis if termination is considered
-preimplantation genetics possible
-harvest placental cord blood from newborn sibling for safest possible transplant to cure affected sibling


overview of therapy

traditionally mainly supportive (hydration, pain Rx, oxygenation, RBC transfusions)
specific interventions (hydroxyurea is mild chemotherapeutic agent)


hydroxyurea treatment for SCD

mild chemotherapuetic agent
-use HbF and other mech
-serious toxicity uncommon
-risk of pain and acute chest syndrome decreases by 50%, and mortality decreases
-requries ongoing daily Rx


bone marrow transplant "cure" for SCD

only available cure
-currently needs HLA-identical sibling, since HLA-Ag play major role in rejection (graft-versus-host disease)
-best with umbilical cord
-use of unrelated HLA-identical donors is in


who should undergo bone marrow transplants for SCD?

risk/benefit decision is critical
-CVA or high-risk for CVA (via Doppler)
-frequent painful episodes
-recurrent/severe acute chest syndrome
-may be future risk factor analysis


what metals porphyrin chelates to

Fe, Mg, Zn, Ni, Co, Cu, Ag
-if binds to lead, causes lead poisoning


pyrrole VS porphyrin VS porphyrinogen

-5 member ring
-oxidized tetrapyrrole (what heme is)
-reduced tetrapyrrole


protoprophyrin and the isomer in heme

porphyrin (oxidized tetrapyrrole) with:
4 methyl groups
2 vinyl groups
2 propoionate groups
protoporphyrin IX is in humans chelated to FeIII


3 species of heme

a - like b, except modification of number 2 vinyl group
b - the original structure (M - V - M - V - M - P - P - M)
c - #2 V and #3 M are covalently bound to cystein residues of PRO thru 2 vinyl groups


axial liganding in heme a, b, and c

a/b - 2 histidines
c - 1 met + 1 his


Shemin pathway and enzymes involved

biosynthesis pathway for heme, that uses 8 succinyl-CoA molecules and 8 glycines for 1 heme
1. delta-aminolevulinate
2. ALA dehydrase
3. uroporphyrinogen synthase
4. uroporphyrinogen III cosynthase
5. uroporphyrinogen III decarboxylase
6. coproporphyrinogen oxidase
7. protoporphyrinogen oxidase
8. ferrochelatase


delta-aminolevulinate (ALA) synthase

first step to making heme, and thsu regulated
-uses cofactor pyridoxal phosphate
-made in cytosol, transferred to mitochondria
-gly + succinyl CoA --> delta-aminolevulinate
--delta-ALA looks like GABA, so accumulation from lead poisoning causes developmental changes (if too high, inhibits brain development)


ALA dehydrase

second step to making heme; inhibited by lead
-uses Zn cofactor
-delta-ALA is transferred from mitochondria to cytosol
-2 delta-ALA --> porphobilinogen (PBG) + 2H2O + H+


uroporphyrinogen synthase

third step to making heme; also called PBG deaminase
-happens in cytosol in 2 step process
-4 PBG --> 3 NH3 + uroporphyrinogen I precursors --> 3 NH3 + uroporphyrinogen I (tetrapyrrole ring)


uroporphyrinogen III cosynthase

fourth step to making heme, in cytosol
-catalyzes isomerization reaction from uroporphyrinogen I to III
-coenzyme is responsible for flipping D ring and insuring proper side group orientation
--instead of 7 A, 8 P, it makes it 7 P, 8A


uroporphyrinogen decarboxylase

fifth step to making heme, doesn't need ATP or prosthetic groups; in cytosol
-catalyzes decarboxylation of 4 acetyl side chains of uroporphyrinogen III to methyl groups to make coproporphyrinogen III (photosensitive) + 4 CO2


coproporphyrinogen oxidase

sixth step to making heme, doesn't use ATP or prosthetic groups
-COPRO is moved from cytosol to mitochondria
-catalyzes decarboxylation of 2 propionyl side chains of coproporphyrinogen to vinyl groups to form protoporphyrinogen IX (photosensitive) + 2 CO2


protoporphyrinogen oxidase

seventh step to making heme, doesn't use ATP or prosthetic groups; in mitochondria
-removes 6 H atoms from protoporphyrinogen IX to make protoporphyrin IX (photosensitive)
-basically makes double bonds



eighth and last step to making heme, doesn't use ATP or prosthetic groups; in mitochondria
-inhibited by lead b/c competes with Fe for insertion
FeII is added to protoporphyrin IX to make heme


regulation of heme synthesis in erythroid cells

synthesize heme once in lifetime, in vast quantities (makes 85%)
-heme stimulates the synthesis of enzymes in heme biosynthetic pathway
-use heme for hemoglobin


regulation of heme synthesis in liver

required in varying amounts throughout lifetime, so synthesis is tightly controlled
-main target of control is ALA synthase
-heme is used as prosthetic group for cytochrome P450 (oxidative enzyme involved in detoxification)


feedback-inhibition regulation of ALA synthase

1. repression of mRNA synthesis
2. inhibition of translation of ALA synthase mRNA
3. inhibition of import of ALA synthase PRO into mitochondria
4. direct inhibition of enzyme


drugs that induce heme synthesis

toxins or substances that induce cytochrome P450 synthesis (acetaminophen/Tylenol
-cP450 is used in liver for detoxification; if drink then take Tylenol, the Tylenol will become hepatotoxic!


porphyrias general

genetic deficiencies in heme metabolism
-enzymes partially defective, since deficiency is lethal
-occur at every step in the pathway
-some affect both liver and RBCs


2 types of hepatic porphyria and general facts

specific for liver heme synthesis; attacks induced by something else
-acute intermittent hepatic porpyria
-porphyria cutanea tarda


2 types of erythropoietic prophyria

specific for RBC heme synthesis
-chronic conditions, and commonly sensitive to sun and anemic, since prophyrins are highly photoreactive, giving off ROS
-congenital erythrpoietic prophyria
-erythropoietic protoporphyria


congenital erythropoietic porphyria (CEP)

deficiency in uroporphyrinogen III co-synthase (about 1/3 normal), autosomal recessive
-accumulation of uroporphyrinogen I and coproporphyrinogen I
-anemia, photoreactive skin ulcerates to scars, red urine, red-brown teeth, increased hair
-origin of werewolf legend


(erythropoietic) protoporphyria

(less severely in liver, always in erythroid)
-partial deficiency in ferrochelatase, autosomal dominant
-symptoms are similar to, but milder than, CEP
-anemia, photoreactive skin ulcerates to scars, red urine, red-brown teeth, increased hair


accute intermittent porphyria

most comon porphyria, in liver, caused by partial (50%) deficiency of porphobilinogen deaminase, autosomal dominant or haploinsufficiency
-buildup of delta-ALA and PBG, and usually asymptomatic until induced "attacks" from estrogen, barbiturate, low CHO, steroids, alcohol
-obscure symptoms, but include ab pain, vomit, diarrhea, neurologic dysfunction, red urine (since PBG is red and goes into urine)
--there are drugs that AIP patients must avoid, since they inhibit cytochrome c 450


porphyria cutanea tarda

in liver; deficiency of uroporphyrinogen decarboxylase; multifactorial or autosomal dominant
-asymptomatic until liver disorder imposed, causing photosensitivity
-sporadic is most common in NA due to alcoholism or contraceptives
-if deplete Fe stores, usually remission of symptoms


steps to heme breakdown (first 2 steps in reticular epithelial cells, last step in liver)

1. break heme ring
2. reduce broken ring
3. conjugate sugars to make it H2O soluble



carrier PRO that binds methemoglobin dimers (Hb with Fe in ferric state) if there is RBC destruction at a site other than spleen/liver (hemolytic anemia)



carrier PRO that binds free heme if there is RBC destruction at a site other than spleen/liver (hemolytic anemia)


heme degredation enzymatic steps

1. heme oxygenase
2. biliverdin reductase
3. glucuronyl bilirubin transferase


heme oxygenase

first step in heme degradation
-heme + O2 + NADPH --> NADP+ + FeIII + CO + H2O + biliverdin (linear chain)
-CO is the "bridging carbon" between pyrrole rings, so there will always be CO in blood circulation regardless of pollution
-done in the ER of spleen cells



first heme breakdown product (via heme oxygenase)
-poorly H2O soluble with green tint


biliverdin reductase

second step in heme degradation
-biliverdin + NADPH --> NADP+ + bilirubin
-done in spleen



second heme breakdown product (via biliverdin reductase)
-easily passes thru cell membranes (lipid soluble), diffuses into bloodstream to make soluble complex with serum albumin to liver
-one of the body's major antioxidants
-distinct yellow color (main reason behind yellow jaundice)


glucuronyl bilirubin transferase

third step in heme degradation
-2 UDP-glucuronic acid + bilirubin --> bilirubin diglucuronide (conjugated bilirubin)
-happens in the ER of liver
-UDP-glucuronic acid comes from UDP-glucose that is dehydrogenase'd with 2 NAD+


conjugated bilirubin

third and last heme breakdown product (via glucuronyl bilirubin transferase)
-soluble, so passes from liver into bile canaliculi to GB to intestinal tract
-bacteria digest it into urobilinogen and urobilins


Crigler-Najjar syndrome

due to deficiency of UDP-glucuronyl transferase
-cannot convert bilirubin to conjugated bilirubin
-varying degrees of severity from a little yellow (decreased atherosclreosis b/c increased bilirubin antioxidant), to severe jaundice


Neonatal jaundice and treatments

temporary condition due to production of insufficient levels of UDP-glucuronyl transferase
-phototherapy - irradiate w/ fluorescent lights to break bilirubin into excretable materials
-also may happen if Rh- mom has Rh+ baby, but doesn't present until a few days later



intestine bacterial breakdown product of bilirubin
-can be reabsorbed from intestine into portal system plasma, but re-excreted by liver into bile
-a small fraction is eliminated by kidney (1-4 mg/day)



oxidized urobilinogen
-contributes to color of normal urine and feces


prehepatic jaundice

massive hemolysis resulting in overproduction of free bilirubin
-liver cells cannot conjugate bilirubin at the rate it enters the liver
-buildup of unconjugated bilirubin in blood, absent conjugated bilirubin and urine bilirubin
-urine urobilinogen is present, and liver enzymes are normal


hepatic jaundice

diseased condition of liver (hepatitis, cirrhosis) that prevents uptake or conjugation of bilirubin
-both cannot conjugate fast enough, and also cannot put made CB into canniculi
-high conjugated bilirubin and liver enzymes, but normal urine bilirubin/urobilinogen


posthepatic jaundice

blockage of bile flow out of liver and into intestinal tract, causing buildup of conjugated bilirubin
-side effect may be pancreatic cancer
-high CB and ALP, but no urine urobilinogen


ferritin and hemosiderin

iron storage
-ferritin can contain up to 4500 Fe atoms reversibly
-hemosiderin is degraded form of ferritin
-if increased Fe entering tissues, increased ferritin content
-since RBC made in bone marrow and degraded in spleen, Fe must be moved back to BM for heme synthesis


where most of body's Fe reserve is present

liver, bone marrow, skeletal muscles, spleen
-if too much (life if frequent blood transfusions) causes Fe toxicity



intercellular glycoprotein Fe transporter, from one cell to another
-made in liver, localized in plasma
-transferrin and its receptor are recycled
-binding of free Fe in serum keeps levels low and acts as a microbial (anti-siderophiles)
-binds receptor in pH and Fe-dependent manner, and binds Fe in pH-dependent manner


steps of Fe absorption by receptor-mediated endocytosis of transferrin

1. Ferro-transferrin binds a receptor on the cell membrane at pH 7.0
2. receptor and ligand are taken up by endocytosis of clathrin-coated pits
3. pH in vesicle (CURL) is lowered to pH 5.0, causing dissociation of Fe from transferrin into cytoplasm
4. receptor and apotransferrin (Fe-less) are returned to plasma membrane, where pH is 7.0 and transferrin no longer binds to receptor



the transferrin receptor without Fe on it
-the Fe unbinds when in CURL (pH = 5)
-apotransferrin then unbinds to receptor when reaches cell membrane (pH = 7)



catalyze electron transfer



catalyze transfer of a functional group



catalyze cleavage of bonds by water



catalyze addition of groups across a double bond, or formation of a double bond



catalyze isomerization of molecules



catalyze formation of bonds between molecules


Gibbs free energy change equation

deltaG = Gproducts - Greactants
can predict whether a RXN is spontaneous or not
0 - cannot occur spontaneously (always needs Ea)

cannot be changed by enzymes, and does NOT predict the rate of reaction!


activation energy

unrelated to deltaG, and is what the rate of reaction depends on
-the lower the Ea, the faster the reaction
-can be lowered via enzymes (direct stabilization of transition state and/or creation of new reaction pathways)


how enzymes reduce energy at the transition state

transition state is least stable b/c highest energy
-directly stabilize transition state complex with enzyme AND/OR
-create a new pathway for the reaction (formation of new intermediates)
usually in "induced fit' state (so that reversible, instead of lock-and-key state)


common features of enzyme active sites

1. occupy a small part of total volume of most enzymes, usually in a cleft
2. 3D structure
3. bind substrates through multiple weak, non-covalent ineractions (same as PRO interactions)
4. water excluded unless reagent
5. highly specific binding of substrate
6. can include non-PRO prosthetic groups and cofactors


prosthetic group VS cofactor

PG: tightly and stably integrated into enzymes (covalently bound); usually vitamins
CF: loosely bound, come on/off enzyme (metal ions)
both provide active groups not present on enzyme


direct stabilization of transition state via enzymes

preferential binding of transition state (compared to substrate or products)
-basically "fits" better, and can form additional bonds to transition state
proximity and orientation effects to get reactants together for RXN


chemical assistance at active site via enzymes

acid-base catalysis (gives or takes H+ or both)
covalent catalysis (transient formation of covalent bond between E-S as new intermediate)
metal ion catalysis (usually cofactors, like for REDOX)
electrostatic catalysis (charge distribution in active site helps stabilize transition state)


4 major classes of proteases (and its regulation)

very highly regulated and highly specific, because incorrect cleavage is lethal and spontaneous (negative deltaG) with high Ea
-serine proteases
-aspartyl proteases
-thiol proteases
-Zn2+ proteases
each class uses a different set of catalytic strategies, and subclasses have different substrate specificities


serine proteases

catalytic triad of asp, his, ser
-separated in primary AA sequence, but brought into proximity by PRO folding
-H-bonded to each other when substrate not bound
-catalytic mechanism uses preferential binding to break down proteolytic reaction into 2 lower E steps (new pathway, with intermediate in the middle)


rate-limiting step (in serine proteases)

step that requires the highest E
-formation of acyl-enzyme intermediate (semi-stable dip between two transition peaks)
-since E to get thru tetrahedryl transition state I and form acyl enzyme intermediate > tetrahedyl II to form product


specificcity in serine proteases (3 types)

structural features near active site dictate specificity
1. chymotrypsin - cleaves after large non-polar side chain + large nonpolar pocket on enzyme
2. trypsin - cleaves after basic AA + negatively charged asp in pocket on enzyme
3. elastase - cleaves after small uncharged side chain + bulky AA on enzyme (val, thr) block pocket


suicide inhibitors

bind to enzyme because of resemblance to substrate and are converted to irreversible inhibitor by initial steps of enzyme's reaction mechanism
-like penicillin to glycopeptide transpeptidase, and nerve gases to AChE


acetylcholinesterase (AChE)

degrades ACh in synaptic cleft to control transmission of nerve pulses
-suicide inhibitors of nerve gases (sarin) inactivate active site serine, and bound substrate cannot be cleaved with water


irreversible VS reversible inhibitors of AChE

irreversible - nerve gas
reversible - Aricept, Cognex; used to improve cognitive function in Alzheimer's
-cannot reverse disease, but slow function
-boost concentration of free ACh