Module 7 - enzyme regulation, cell signaling pathways, and GPCR

Question 1

What is DFP?

Answer 1

irreversible enzyme inhibitor, forms covalent links with reactive serine residues such as chymotrypsin and phospholipases

Question 2

What two things regulate enzyme activity?

Answer 2

bioavailability of enzymes and catalytic efficiency

Question 3

Allosteric control involves…

Answer 3

the binding of small molecules called metabolites to regulatory sites on enzymes (not the active site)

Question 4

What processes affect enzyme bioavailability?

Answer 4

RNA synthesis, processing, protein synthesis, protein degradation, and protein targeting

Question 5

What affects catalytic efficiency?

Answer 5

inhibition, allosteric control, covalent modification, and proteolytic processing

Question 6

What is reversible inhibition vs. irreversible inhibition?

Answer 6

reversible - noncovalent binding of small molecules

irreversible - inhibitory molecule forms a covalent bond to enzyme active site

Question 7

Malonate is a —– —– of succinate dehydrogenase

Answer 7

reversible inhibitor

Question 8

What are the three types of reversible inhibition, and describe each

Answer 8

competitive inhibition:
binds in active site of E

uncompetitive inhibition:
binds outside active site on the ES complex

mixed inhibition:
binds outside active site, but to the E or ES complex

Question 9

How does the graph change when a competitive inhibitor is present?

Answer 9

same Vmax, but increased Km (higher substrate to reach 1/2 Vmax)

Question 10

How does the graph change when a uncompetitive inhibitor is present?

Answer 10

lower the Vmax and the Km

(uncompetitive inhibition is not overcome by increasing the substrate concentration)

Question 11

How does the graph change when a mixed inhibitor is present? What is a noncompetitive inhibitor?

Answer 11

can look many different ways

noncompetitive inhibitors are one specific type of mixed inhibitors, graph shows no change in Km but lowering of Vmax

Question 12

What is papaya enzyme called, and what is it good for? Why?

Answer 12

papain, good for meat tenderizing because it is a cysteine protease that degrades proteins

Question 13

How does a Vo vs. [S] graph change with the presence of allosteric effectors?

Answer 13

shifts right (>Km) with a negative allosteric effector and shifts left (<Km) with a positive allosteric effector

Question 14

Describe the regulation of ATCase and how the allosteric effectors would effect the graph of Vo vs. [S]

What is the quaternary structure of ATCase?

Answer 14

binding of ATP shifts ATCase to the R state (and shifts graph to the left)

binding of CTP shifts ATCase to the T state (and shifts graph to the right)

ATCase is a dimer with a catalytic subunit (where substrate binds) and a regulatory subunit (where allosteric effector binds), 3 dimers come together to form the C3R3 complex

Question 15

What are the three most common enzyme covalent modification? How do they happen?

What is an example of an enzyme that is activated by each modification?

Answer 15

Phosphorylation:
- of Ser, Thr, and Tyr residues by a kinase (removed by a phosphatase)
- adds an inorganic phosphate
- glucogen phosphorylase

Adenylylation:
- added and removed by an adenylyltransferase
- glutamine synthetase

Uridylylation:
- uridylyltransferase
- control of adenylyltransferase activity for glutamine synthetase activation

Question 16

What is a zymogen?

Answer 16

inactive precursor proteins that are synthesized with an active site that is inaccessible to protein substrates

Question 17

How does proteolysis regulate enzymes?

What is an example?

Answer 17

when a protein is synthesized as a zymogen, proteases can be used to remove segments and expose the active site, generating the active form of the enzyme
process is irreversible

ex: pepsinogen (inactive) has its N terminal cleaved to reveal the active site and generate pepsin (active)

ex: trypsin cleaves chymotrypsinogen to create p-chymotrypsin, which then cleaves other p-chymotrypsin molecules to generate the fully active a-chymotrypsin enzyme

Question 18

What are the 6 general steps of second messenger pathways?

What is the overall effect of a second messenger pathway?

Answer 18

1. first messenger (substrate)
2. receptor protein (conformational change)
3. upstream signaling proteins
4. second messengers
5. downstream signaling proteins
6. target proteins

effect: amplification through the activation of one or more downstream target proteins

Question 19

What percent of protein coding genes in the human genome are involved in signal transduction? How many signaling genes are there?

Answer 19

9%

about 2,000 signaling genes

Question 20

What are endocrine, paracrine, and autocrine?

Answer 20

endocrine - target tissue is far away

paracrine - target tissue is a nearby cell

autocrine - target tissue is the same cell

Question 21

What are examples of first messengers?

Answer 21

peptide hormones, lipids (like steroids), and small molecules like nitric oxide, Ca2+, and CO2

B-estradiol, epinephrine, acetylcholine, etc.

Question 22

How does insect saliva cause cGMP production?

Answer 22

At pH of 5, NO is bound to the heme of the nitrophorin protein.

At pH of 7, NO is released. A histamine binds to the heme where the NO was released.
The released NO binds to guanylate cyclase to stimulate cGMP production and cause vasodilation.

Question 23

How does sildenafil work?

Answer 23

prolongs NO mediated vasodilation by inhibiting cGMP phosphodiesterase (which breaks down cGMP)

Question 24

What enzymes cause ATP to generate cAMP and then AMP?

Answer 24

adenylate cyclase turns ATP to cAMP

cAMP phosphodiesterase turns cAMP to AMP

Question 25

Describe the second messenger signaling of DAG, IP3, and Ca2+

Answer 25

receptor activation of Phospholipase C (PLC) raises the intracellular levels of DAG and IP3

DAG binds to and stimulates protein kinase C (PKC) which targets downstream proteins

IP3 activates Ca2+ channels, releasing Ca2+ that forms complexes with calmodulin which bind to downstream target proteins

Question 26

What are the types of receptors?

Answer 26

neurotransmitter, GPCR, receptor tyrosine kinases, TNF receptors, nuclear receptors

Question 27

What do ligand-gated ion channels do?

Answer 27

control the flow of K+, Na+, Ca2+ ions across cell membranes in response to ligand binding

Question 28

How is the nicotinic acetylcholine receptor activated and what happens when it is?

Answer 28

acetylholine binds to it, opening the ion channel by inducing a conformational change that rotates the transmembrane alpha helices

Question 29

What is the structure of GPCRs like?

Answer 29

seven transmembrane alpha helices that transmit signals across the membrane through conformational changes
(amino toward the outside, carboxy toward the inside)

intracellular component of GPCR signaling is the GaBy heterotrimeric protein complex that co-localized to the membrane with the GPCR

Question 30

What is the sequence of the GPCR dissociation reaction?

Answer 30

1. ligand binds to GPCR
2. inactive GaBy (containing GDP in the Ga subunit) associates with the liganded GPCR
3. conformational change causes GDP to be exchanged for GTP and subunits dissociate into By and a
4. dissociated subunits activate downstream signaling

Question 31

What is rhodopsin?

Answer 31

a GPCR that detects light through a retinal molecule within a hydrophobic pocket formed by the 7 transmembrane alpha helices

Question 32

When B2-adrenergic receptors are binded by a ligand, what change occurs first?

Answer 32

ligands bind to the extracellular side and cause conformation changes on the cytosolic side (mostly in the bottom left)

Question 33

How does GaBy anchor to the membrane?

Answer 33

the C terminus and N terminus both have an anchor in the membrane

Question 34

What effects does the dissociated GBy subunit have?

What effects does the dissociated Ga subunit have?

Answer 34

GBy: phospholipase A, regulate ion channels, regulare receptor kinases

Ga: activate adenylate cyclase, inhibit adenylate cyclase, regulate neuronal signaling, stimulate phospholipases, stimulate phosphodiesterases

Question 35

How many different types of each subunit are there? How many complexes can be created that lead to different downstream effects?

Answer 35

17 Ga
5 GB
12 Gy

almost 1000 different complexes with different downstream pathways

Question 36

How exactly does Gsa subunit change with GDP or GTP?

Answer 36

Gsa has a subunit called switch II which exists in two different conformations depending on if GDP or GTP is present

Question 37

How does cAMP activate PKA?

Answer 37

2 cAMP molecules bind to each regulatory subunit of PKA, induce a conformational change that releases the catalytic subunits (that are now active)

Question 38

How is GPCR regulated?

Answer 38

guanine-nucleotide exchange factor (GEF) promote GDP to GTP exchange and activate signaling (encourage dissociation)

GTPase activating proteins (GAPs) stimulate the intrinsic GTPase activity of G proteins to inhibit signal transduction

Question 39

How is GPCR signaling terminated?

Answer 39

• GBy binds BARK and recruits PKA
• BARK and PKA phosphorylate serine and threonine residues in the GPCR cytoplasmic tail
• B-Arrestin binds the phosphorylated tail and the whole complex is endocytosed
• GPCRs are either degraded or recycled