Midterm 3 Flashcards

1
Q

What is the cell signaling pathway

A

linked set of biochemical reactions that are initiated by ligand-induced activation of a receptor protein and terminated by a measurable cellular response

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2
Q

Components of signaling pathways

A

●First messengers: extracellular ligands that bind to receptor proteins, cause conformational change
●Second messengers: intracellular signaling proteins

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3
Q

What is signal transduction?

A

●biochemical mechanism responsible for transmitting extracellular signals across plasma membrane and throughout the cell
●also involves second messengers and a variety of intracellular signaling proteins
●function together to transmit, amplify, and terminate signal

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4
Q

what do first messengers do?

A

●binds to receptor proteins, causing conformational change in receptor

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5
Q

Consequences of signal transduction

A

●changes information into chemical signal
●often ends with covalent or noncovalent modification of intracellular target proteins (phosphorylation and dephosphorylation)
●altered rate of protein expression (at transcriptional or translational level)

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6
Q

First messenger examples

A

●Insulin: first 1st messenger protein to be discovered, discovered as treatment for diabetes
●Hormones: biologically active compounds that are released into circulatory system and come into contact with hormone receptors in target cells (generally hydrophobic and nonpolar)
●Other soluble gases (NO: generate in cells via oxidative deamination of arginine, causes vasodilation and increased blood flow) (sildenafil -> viagra : know backstory)(Bugs use NO increase blood circulation)
●Neurotransmitters

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7
Q

How do hormones travel?

A

●Endocrine hormones: produced by secretory glands and are exported into the circulatory system. long distance
●Autocrine and Paracrine hormones: small peptides that function over short distances to activate receptors on nearby cells (paracrine hormones) or on same cell (autocrine hormones)

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8
Q

Endocrine Hormones

A

insulin, estrogen, testosterone

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9
Q

Paracrine Hormones

A

serotonin, histamine, growth factors

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10
Q

Autocrine Hormones

A

interleukins and growth factors

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11
Q

What are secondary messengers?

A

small intracellular molecules that amplify receptor-generated signal

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12
Q

Secondary Messengers examples

A

Cyclic GMP (cGMP), cAMP, Diacylglycerol (DAG), Inositol-1,4,5-triphosphate (IP3), Ca2+

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13
Q

Signal Amplification

A

process by which a signal that is initiated at the cell surface leads to multiple downstream events through the action of enzyme-mediated catalyzed reactions like phosphorylation

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14
Q

What are the receptor protein classes?

A

GPCRs, KLRs, tumor necrosis factor receptors, and nuclear receptors

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15
Q

GPCR

A

●G protein-coupled receptors
●involved in sensory perception (vision, taste, smell)
●Globular protein with 7 transmembrane regions (hydrophobic and helical in shape), known as 7-TM receptors
●Many are glycoproteins (help with cell recognition) that contain carbohydrate functional groups directly attached to the extracellular domain

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16
Q

GPCR signal transduction

A

●transmit extracellular signals to cytoplasm through direct interaction with membrane-bound protein complex called heterotrimeric G protein (has a G α , G β, and G γ subunit)
●Gα is a GTPase (active when GTP bound, inactive when GDP bound)
●When the trimeric G protein binds to activated GPCR, GDP -> GTP, activating the Gα subunit. Causes dissociation of alpha subunit
●Dissociation of heterotrimeric G protein complex is common to all GPCRs
●lipid membrane anchors (can move but stay on membrane)
●Several different α, β, and γ subunits- causes unique effects

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17
Q

Different G alpha subunit examples

A

●αt, αs, αq

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18
Q

Gαt subunit effects

A

αt activated by GTP -> cGMP phosphodiesterase (break down phosphodiester bonds) -> GMP
●regulates synaptic transmission in light-stimulated vision (just one example)

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19
Q

Gαs subunit effects

A

αs activated by GTP -> ATP turned into cAMP by Adenylate cyclase -> cAMP triggers PKA (phosphokinase A) -> activates numerous target proteins

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20
Q

Gαq subunit effects

A

αq activated by GTP -> PIP2 converted to DAG and IP3 by phospholipase C (cleave phospholipids in membrane) -> (IP3 -> Ca2+) and (DAG -> PKC) -> regulates many processes

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21
Q

GPCR Signaling in Metabolism

A

●multiple diff stimuli work together to accomplish common goal
● glucagon, glycogen, epinephrine
●makes sense because they (glucagon and epinephrine) signal for same phenotypic response (increase blood glucose levels)

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22
Q

PKA’s role in Epinephrine (β2 Bound) and Glucagon

A

●Gsα activation of adenylate cyclase, produce cAMP that will activate Protein Kinase A, ***Turns off glycogen synthesis, turn on glycogen degradation and glucose synthesis -> net glucose export

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23
Q

Epinephrine (α1 Bound)

A

●Gqα activation of PLC -> PIP2-> DAG and IP3 -> (DAG activation of PKC -> turn off glycogen synthesis) OR (IP3 mediated calcium release from ER -> turn on glycogen degradation) -> Net glucose export

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24
Q

Catecholamines

A

●1st messengers, include epinephrine
●derived from Tyr
●target α and β adrenergic receptors. Tissue distribution and physiological responses governed by these two receptors are diverse, controlling from metabolism in liver, skeletal muscle, and adipose cells, to relaxation and contraction of smooth muscles

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25
Q

Receptor Agonists

A

●Activate receptor signaling by mimicking the natural ligand
●some endogenous agonists: dopamine and norepinephrine
● recognize the two OH antennas

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26
Q

Catecholamines Gαs

A

●β-2 receptors mostly in airway smooth muscles, epinephrine binding to β-2 receptors causes changes in Gαs subunit
●GDP bound state: Gαs interacts with Gβy through switch II helix region
●GTP binding to Gαs induces conformational change in switch II helix region so it’s positioned to bind with adenylate cyclase (perfect binding site for adenylate cyclase)
●adenylate cyclase becomes active upon binding to Gαs
●Elevated cAMP levels activate PKA
●cAMP binds to regulatory subunit of inactive PKA to create active PKA monomers
●4 cAMP bind to inactive R2C2 complex and cause conformational change to activate PKA

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27
Q

Epinephrine stimulation of β-2 consequences

A

●cause dissociation of Gαs-> produce cAMP
●cAMP will activate PKA (functions to phosphorylate various enzymes contributing to relaxation of airway smooth muscles)
●cAMP reduces Ca2+ by inhibiting outflow (preventing muscle contraction)
●albuterol is agonist (treat asthma and COPD)

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28
Q

Receptor agonist and antagonist

A

●Agonist: activate receptor signaling by mimicking natural ligand
●Antagonist: bind to receptor with high affinity and block binding of physiological agonists without promoting structural changes needed for signal transduction

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29
Q

Termination of GPCR Signal

A

●desensitization of receptor after Gαβγ dissociation
●regulatory proteins called G protein-coupled receptor kinases (GRK) phosphorylate GPCR cytoplasmic domain on Ser and Thr residues
●phosphorylation by GRK provides docking site on GPCR for 2nd protein (β-arrestin), a protein that binds to receptor and prevents reassociating with Gαβγ complex
●β-arrestin binding “flags” the GPCR for endocytic translocation to cytoplasm where GPCR is dephosphorylated (not reversible until dephosphorylated)
●After GPCR dephosphorylation in endocytic vesicles, receptor is either degraded or returned to plasma membrane for another round of signaling

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30
Q

Receptor Tyrosine Kinase Signaling

A

●RTKs (subclass of KRT) transmit extracellular signals by ligand activation of intrinsic tyrosine kinase function found in cytoplasmic tail of receptor
●activated RTKs are dimers
●activated RTKs phosphorylate downstream signaling proteins that bind to RTK phosphotyrosines (include adaptor proteins- acts as molecular bridges to bring together other proteins- GRB2 containing SH2 domains)- and other kinases
●ex: Epidermal growth factor receptors (EGFR), insulin receptor (defect= type 2 diabetes)

31
Q

Growth Factors Binding to RTKs (general)

A

●ligand binding -> receptor dimerization and kinase activation
●phosphorylation of RTK cytoplasmic tails -> protein binding to RTK phosphotyrosines and phosphorylation of target proteins
●activation of downstream signaling pathways
●defects in EGFR can result in cancer (cell division= too much growth)

32
Q

EGFR signaling

A

●epidermal growth factor is serum growth factor that binds to EGFR and stimulates receptor dimerization on cell surface-> phosphotyrosine (pY) residues in RTK form binding sites for GRB2 (intracellular adaptor protein containing Src kinase homology-2 (SH2) domain )(necessary for phospho residue to bind) -> GRB2 binds to pY residues in EGFR and recruits GEF signaling (helps swap GDP to GTP) called son of sevenless (SOS), which binds and activates G protein called Ras -> phosphorylation causes cascade mediated by trio of related kinases (called mitogen-activated protein kinase (MAP kinase) pathway)
●SH2 examples: phosphoinositide 3 kinase

33
Q

Ras mutation

A

●dephosphorylator not active -> MEK stays active (cell proliferation)

34
Q

What are enzymes?

A

proteins

35
Q

Lock and key mechanism

A

●substrate binds perfectly to enzyme
●can’t explain enzyme regulation or how substrates were binding to sites deep within interior of protein

36
Q

NO as first messenger

A

●NO to guanylate cyclase
●GTP to cGMP and PP
●cGMP to protein kinase G and causes vasodilation
●cGMP phosphodiesterase breaks down cGMP to GMP

37
Q

Induced fit mechanism

A

●substrate binding to active site induces change in enzyme
●enzyme is flexible to accommodate ill-fitting substrate
●primary conformation is “trapped” by ligand binding -> induced fit to optimize interaction
●allows for larger amount of weak interactions between substrate and enzyme
●conformational changes can be minimal (lock and key mechanism) or drastic (fly trap ex)

38
Q

induced fit mechanism example

A

hexokinase, a metabolic enzyme
● conformational change blocks water from active site and promotes phosphorylation

39
Q

enzyme structure and function

A

●enzyme bind to substrate with high affinity and specificity
●active sites in enzyme bind substrates and promote catalytic reactions (chemical environment for rxns)
●enzyme activity is highly
regulated (dont waste energy)
●provide chemical environment that facilitate catalytic reactions by excluding excess solvent and bringing reactive functional groups of enzyme into close proximity to substrate

40
Q

Modes of enzyme regulation

A

●bioavailability: amount of nutrient or enzyme present in cell that is capable of participating in biochemical process
●catalytic efficiency: maintained by binding of regulatory molecules or covalent modifications (some molecules binding can increase efficiency)
●IMPORTANT FOR METABOLITE TO PREVENT DISEASES

41
Q

Enzymes as chemical catalysts

A

●alter rate of rxn without changing ratio of substrates and products at equilibrium
●decrease activation energy to speed up rxn
●DOES NOT CHANGE DELTA G

42
Q

Activation energy definitions

A

●energy barrier that must be overcome for reactants to transform into products
●some reactions can be energetically favored, but still must get energy to reach the products
●transition state: high-energy state molecules must go through to get from reactant to product (only there for picoseconds, can’t be isolated, at the top of the hill)
●activation energy: energy required to reach transition state
●higher activation energy= slower rate of rxn

43
Q

Activation energy collisions

A

●rxn between molecules are result of collisions
●Activation energy: minimum energy needed for collisions to lead to a rxn
●Collisions need enough energy and proper orientation to overcome activation energy

44
Q

How do enzymes increase rate of rxn (how is activation energy lowered)?

A

●stabilize transition state lowers activation energy
●provide alternate path for product formation, could involve formation of stable rxn intermediates that are covalently attached to enzyme (can be multiple steps)
●reduce by orienting substrates appropriately for rxn to occur
●ALL FACILITATED BY ENVIRONMENT OF ACTIVE SITE

45
Q

Active site contributes to catalysis

A

●enzyme active site contains binding sites to select substrates and align reactive groups correctly
●substrates have relatively high binding affinity for enzyme active site (NOT TOO STRONG THO BECAUSE IT MUST BE RELEASED AFTERWARDS)

46
Q

Chemical/Physical properties of active site that contribute to catalytic properties

A

●sequestered microenvironment: provides optimal orientation of substrate relative to reactive group, excludes excess solvent that could interfere with the rxn (ex: aldolase)
●binding interactions between substrate and enzyme to help create a transition state (or stabilize transition state)
●presence of catalytic functional groups: three most common catalytic rxn mechanism in enzyme site (acid-base, covalent, and metal-ion catalyst), cofactors and coenzymes are helper molecules that provide additional chemical groups to supplement protein when amino acids are insufficient to mediate a particular catalytic mechanism

47
Q

Aldolase example

A

●aldolase rxn in gluconeogenic pathway converts two phosphorylated three-carbon compounds into one bisphosphorylated six-carbon compound
●change causes more touch -> more interactions
●huge induced fit

48
Q

schiff base

A

●know mechanism
●primary amide to aldehyde or ketone (condensation rxn)
●more favorable way to form c-c
●electron sink: able to accept electrons, form c=c, more rxns

49
Q

Transition state stabilization model

A

●AA residues within enzyme active site make the most contacts during transition state of rxn, so breaking bonds during rxn requires no direct input of energy by protein

50
Q

Transition state analogs

A

●tight binding of transition state analogs to enzyme active site- support transition state stabilization model
●definition: stable molecules that mimic proposed transition state, bind tightly to active site
●gets enzyme stuck- no leaving groups
●can have stronger affinity of enzyme for transition state analog than enzyme for substrate
●transition state has an molecule for the inbetween of reactant and product

51
Q

Acid-base catalysis

A

●proton transfer that either involves water (specific acid-base) or a functional group (general acid-base)
●arrangement of residues at active site -> enzymes can often perturb pKa values of functional groups to increase proportion of group in correct ionization state needed for chemical rxn
●Histine-> pKa 6 -> can be weak acid or weak base

52
Q

Covalent Catalysis

A

●nucleophilic group on enzyme attacks an electrophile center on substrate to form covalent enzyme- substrate intermediate
●nucleophile donates electron pair
●electrophile accepts electron
●sulfur in the right environment to let go

53
Q

Metal-ion catalysis

A

●metals used to promote proper orientation of bound substrates and can aid in redox rxn
●enzymes with tightly bound metal ions are called metalloenzymes
●Zn2+ lower pKa of water and facilitates formation of nucleophilic hydroxyl group that attacks the substrate (through metal-ion coordination bonds)

54
Q

Cofactors

A

●small molecules that aid in catalytic rxn within active site
●include inorganic ions (Fe2+, Cu2+, and Mg2+)
●iron and cu irons are often required for enzymes that perform redox rxns
●magnesium, manganese, and zinc are frequently used to help bind substrates or stabilize an intermediate or transition state
●holoenzyme: enzyme with bound cofactor
●apoenzyme: removal of cofactor

55
Q

Coenzymes

A

●enzyme cofactors containing organic components
●include vitamin-derived species such as NAD+ and FAD

56
Q

Prosthetic groups

A

coenzymes that are permanently associated with enzymes

57
Q

What is glucagon

A

peptide hormone released by pancreas in response to low blood glucose

58
Q

What is glycogen

A

the way we store glucose until we need it to make ATP

59
Q

What is epinephrine

A

“fight or flight” hormone that can signal increased heart rate and mobilization of energy stores through glycogen breakdown and lipolysis (improve breathing)

60
Q

Parallel vs Shared Pathways

A

●Shared pathways: same pathway
●Parallel pathways: same end result, different pathways to end results
● glucagon and epinephrine beta 2 have shared pathway using Gs alpha
●epinephrine beta 2 and epinephrine alpha 1 have parallel because alpha uses Gq alpha
● when asking about either glucagon or epinephrine -> use shared pathway

61
Q

Epinephrine smooth muscle contraction

A

●PKA free to phosphorylate other proteins (in response to β-2 activation, PKA phosphorylates MLCK- enzyme crucial for smooth muscle contraction-, as it phosphorylates myosin light chains (MLC), enabling interaction with actin for contraction)
●PKA phosphorylating MLCK reduces its activity -> decreases MLC phosphorylation -> inhibiting smooth muscle contraction and promoting relaxation

62
Q

Classic examples of multistep reactions of enzyme reaction mechanisms and what do they reinforce

A

●chymotrypsin, enolase, HMG-CoA
●substrates bind to enzyme active sites through weak noncovalent interactions (orient AA functional groups within close proximity to reactive centers of substrates)
●enzymes use conventional catalytic reaction mechanism that follow basic organic chemistry

63
Q

good leaving groups

A

●weak base (br, cl, h2o, nh2)

64
Q

Chymotrypsin: serine protease

A

●covalent and acid-base catalysis
●catalytic triad (His, Asp, Ser) to form hydrogen-bonded network required for catalysis (close proximity but diff chains)
●ser converted to highly reactive nucleophile
●active site on protein surface (want water)
●AA in pocket decides what will be cleaved

65
Q

Chymotrypsin MOA

A

●Polypeptide substrate binds to active site
●His removes proton from Ser (nucleophilic attack by Ser oxygen on carbonyl carbon peptide
●His donates proton to amino group of substrate (peptide bond cleavage), caroxyl-terminal fragment released as first product
●Water enters active site, His acts as base and removes proton from water. Results OH acts as nucleophile and attacks carbonyl carbon on covalent acyl-enzyme intermediate
●His donates proton to Ser (cleavage of acyl-enzyme intermediate), amino-terminal fragment is released as second product and catalytic triad regenerated
●functional catalytic triad is regenerated within enzyme active site

66
Q

Enolase

A

●glycolytic enzyme also used in gluconeogenesis
●acid-base and metal-ion catalysis
●dimer
●active site has two divalent metal ions required for catalysis

67
Q

Enolase Dehydration

A

●Lys acts as base
●Mg2+ stabilize negative charge on intermediates
●Glu acts as general acid
●phosphoenolpyruvate product is formed
●PRODUCT HAS WATER

68
Q

Enolase metal ions important roles

A

●bind and orient substrate in active site
●make C2H acidic, facilitating removal by Lys (huge partial positive on metal makes oxygen more partially negative -> C2H more partially positive)
●stabilize negative O during catalysis

69
Q

HMG-CoA Reductase

A

●enzyme in biosynthetic pathway for cholesterol and other isoprenoids
●important pharmaceutical target as inhibitors directly and indirectly reduce serum cholesterol levels
●intracellular cholesterol reduce -> indirect benefit is more cell surface cholesterol receptors produce
●receptors bind cholesterol-containing particles in serum -> reduce serum cholesterol and risk of heart disease
●tetrameric protein containing four active sites located between monomer interfaces
●catalyze four-electron reduction of HMG-CoA to mevalonate and CoA using two NADPH molecules as cofactors
●mevalonate is precursor in production of terpenes and steroids (such as cholestrol)

70
Q

HMG-CoA Reductase MOA

A

●achieved by two hydride transfer steps involving two NADPH cofactors
●active site of protein is important for binding and orientating HMG-CoA and NADPH so hydride transfer can occur (provides functional groups to stabilize transition state)
1. Reduction of thioester: Hydride transfer from NADPH, Glu acts as acid (Lys stabilizes hemithioacetal product)
2. Cofactor exchange: (NADP+ -> NADPH)
3. Breakdown hemithioacetal: His donates proton to CoA and Glu acts as base
4. reduction of aldehyde: Hydride transfer from NADPH, Glu acts as acid

71
Q

Lovastatin

A

● first statin approved by FDA
●from fungus (aspergillus terreus)
●active form is beta-hydroxy acid
●activated in metabolism
●HMG-CoA: S-CoA is leaving group
● in drug: CH2 replaces leaving group and is not a leaving group -> inhibits -> stalls enzymes

72
Q

Where is epinephrine receptors found

A

liver cells

73
Q

Where is glucagon receptors found

A

liver and adipocyte cells

74
Q

G-protein’s activity controlled by 2 proteins

A

●Guanine nucleotide exchange factor (GEF): activate signaling, promote GDP-GTP exchange
●GTPase activating proteins (GAP): inhibit signal transduction, stimulate intrinsic GTP hydrolysis activity (inactivate GTP) (cleaves 3rd phosphate group of GDP)