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Enzyme Regulation Flashcards

1
Q

what are enzymes?

A

proteins that:
catalyze CHEMICAL TRANSFORMATION of a substrate to a product
INCREASE RATE of rxn
lower Ea for the rxn

technically always reversible
form an enzyme substrate complex
substrate –> NEW PRODUCT

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2
Q

how does Ea change with enzymes?

A

lowers
two humps (transition state) on graph

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3
Q

what is the michehaelis-menten equation?

A

V0 = Vmax[S]/Km + [S]

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4
Q

what are regulatory enzymes?

A

multistep metabolic pathways, contain at least 1 rate limiting step

catalyst of rate limiting step (slow) is mediated by regulatory enzymes

regulatory enzymes ARE REGULATED

catalytic rate are controlled by “signals”

can be ACTIVATORS or REPRESSORS

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5
Q

what is feedback inhibition?

A

inhibition that occurs at the FIRST STEP of a pathway

no other products cause inhibition (JUST FINAL PRODUCT)

REVERSIBLE

ex. isoleucine does NOT bind to the active site –> separate allosteric binding site

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6
Q

what are the mechanisms of enzyme regulation?

A

allostery: REVERSIBLE, NONCOVALENT binding of regulatory compounds

reversible, covalent modifications: mediated by separate enzyme system

interaction w/ regulatory proteins

PROTEOLITIC cleavages (NOT REVERSIBLE)

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7
Q

what are allosteric enzymes?

A

regulatory enzyme with CATALYTIC activity modulated by the noncovalent bind of a specific compound at a site OTHER THAN the active site

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8
Q

compare non-comp inhibition vs allosteric modulators

A

both bind AWAY from the active site
both affect STRUCTURE of enzyme
non-comp are NON-PHYSIOLOGICAL
non-comp CANNOT be overcome by adding more substrate

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9
Q

compare allosteric enzymes to non-regulatory enzymes

A

structurally MORE COMPLEX –> usually multisubunit, different parts communicate w/ each other

have regulatory (ALLOSTERIC) sites

undergo CONFORMATIONAL CHANGES in response to modulator binding (can be subtle)

do NOT obey M-M kinetics

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10
Q

how does the binding of allosteric modulators influence K0.5?

A

Positive modulator: switch the T state –> R state

negative modulator: switches the R states –> T state

allosteric enzymes have a SIGNOIDAL shape

activity can be modulated by CHAGIN CONCENTRATION of modulator

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11
Q

what are the types of modulators?

A

homotrophic: both modulators and substrates

heterotrophic: NOT substrates –> do not bind to active site

NOT MM, bc not hyperbolic

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12
Q

what are the modulator effects?

A

negative –> reduce activity

positive –> increase activity

effet K0.5 or Vmax

both homo + hetertrophic modulators have one of these effects

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13
Q

what is the ATCase structure and function

A

ATCase catalyzes the committed step in the biosynthesis of pyrimidine nucleotides (eg. CTP)

REGULATED

forms N-carbamoylaspartate and Pi from carbomyl phosphate and aspartate

dodecamer –> 12 sites

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14
Q

what is the ATCase reaction?

A

transfers and activated carbamoyl group onto the amine group of aspartate

1st step for synthesizing a pyrimidine ring (eg. CTP)

carbamoyl phosphate + aspartate –> (aspartate transcarbamoylase) N-carbamoylaspartate

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15
Q

what is the subuint structure of ATCase

A

12 subunits

2 types: CATALYTIC (trimer) and REGULATORY (dimer)

6 of each

fully functioning enzyme: C6R6 made up of 2xC3 and 3xR2

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16
Q

how do subuints interact?

A

Zinc domains of the regulatory subuints –> cysteins coordinate a structural zinc ion

6 zinc domains on each R chain on the part of the dimer that touches C chain

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17
Q

how were the subunit structures identified?

A

cysteine modifying mercury compound and ultracentrifugation

graph shows two bumps –> smaller = r2, larger = c3

mercury DISRUPTS interactions

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18
Q

describe the allosteric modulation of ATCase

A

positive HOMOTROPHIC modulator

signoidal curve

substrate binding by ATCase is explained by allosteric cooperativity T–> R state transition

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19
Q

how do CTP and ATP modulate ATCase?

A

ATP –> +-ve modulator
indicates a lot of ENERGY in the cell
not the substrate, heterotrophic
low K0.5

CTP –> -ve inhibition
FEEDBACK INHIBITION (CTP is the product)
ATP and CTP bind to sites other than the active site on reg subunit

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20
Q

where do substrates and modulators bind?

A

substrates –> interface of C chains (center of complex)

modulators (ATP, CTP) bind to r chains (periphery)

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21
Q

describe the effect of changes in quaternary structures on allostery

A

T-state: ATCase has a more closed conformation –> blocks active site loop (240’s) from adopting a FULLY active conformation

R-state: ATCase is in a more open conformation –> allows active loop to adopt a more active conformation

cannot add substrate to study state b/c enzyme forms too quickly; adding ATP pushes into R-state w/o substrate

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22
Q

how can ATCase be pushed into the R-state?

A

add SUBSTRATE

add substrate ANALOG (PALA)

add ACTIVATOR ( ATP)

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23
Q

describe the difference between T and R conformations

A

T –> closed, less active, favoured by CTP (negative, heterotrophic allosteric) binding

R –> open, more active, favoured by substrate binding

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24
Q

what is PALA?

A

non-reactive bisubstrate analog that mimics the reaction intermediate of ATCase

cannot be consumed/form product –> allows us to study structure

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25
how does the addition of PALA change the structure of ATCase
SCISSOR trimers (vertical) rotate 15 degrees dimers (horizontal) rotate 10 degrees separate by 12A
26
what are the structural changes during the activation of ATCase?
Catalytic: flexing at interfaces b/w C units overall: catalytic trimers move apart 12A and rotate relative to each other regulatory dimers rotate changes in bond b/w r chains substrate analogs = artificial ligands
27
what mechanism does the T-->R state follow?
CONCERTED all subunits are either in T OR R state ATP binding can induce T-->R conversion in the absence of substrate
28
how can we measure K0.5 and Vmax
adding ATP decreases cooperativity ATP makes enzymes more efficiently, changes K0.5
29
what is the reaction in the journal article?
events in ATCase rxn occur in a fixed order: 1) carbamoyl phosphate (CP) binds to active site 2) aspartate then binds to active site --> large changes in quaternary structure 3) condenstation reaction occurs 4) product N-carbmoyl-L-aspartate is released 5) Pi is released as long as substrate is available, stays in R state
30
what is the key residue pair?
two charged AAs ion pairs Asp236 on the catalylic chain (D263c), Lys145 on the reg chain (K145r) interacts to STABILIZE T-state after mutation of either of these to ALANINE, enzyme CANNOT adopt T-state in the absence of ligands, WT and D236Ac ATCase have DIFFERENT structures in the presence of PALA, they have the SAME structure
31
what happens when scientists mess around with the pairs?
in the absence of the negatively charged R-group of D236 or the +-ve R group on K143, an ion pair that stabilizes the T-state is eliminated
32
what is the central question of the paper?
what can be learned by comparing the structures of D236Ac to that of the WT enzyme in the presence of analogs CP = substrate PAM = analog of CP (non reactive) malonate = analog of Asp (non-reactive)
33
describe the results of small-angle X-ray scattering
give idea of overall shape low resolution PALA puts ATCase into R state CP has little effect on WT ATCase, know from other studies it stabilizes the T state mutant looks more R-like in mutant, Cp mimics effects of PALA Asp pushes into R-state (ALLOSTERIC MODULATION), CP DOES NOT very different quaternary structures
34
describe the structural results
WT: catalytic subunits move 12A apart 10A difference in size (mutant bigger) when bound to PAM (analog of CP) mutant adopts the R-state this can happen even when only 1 catalytic trimer is bound
35
describe the difference in catalytic chains
X-ray structures of 2 catalytic chains are superimposed reveals local changes in conformation WT tubes are THICKER --> proportional to RMS deviation
36
describe the differences in C1
C1 of D236Ac bound to PAM alone is structurally very similar to WT C1 bound to both substrates
37
what was concluded on the ion pairs
Asp236Ac mutant eliminates the ion pair that forms between Asp236c and Lys143r this ion pair normally stabilizes the T state in the abscence of the Asp236c-Lys143r interaction (which connects the c and r subunits) CP can push ATCase into the R state direct interactions between the c and r subunits regulate the conformation of the enzyme by stabilizing the T state
38
what was the overall result of the experiment?
the mutant form of the enzyme never adopts the low-activity T-state always partially active trapped in a catalytic cycle
39
what is reversible covalent modifications?
enzyme activity can be altered by the COVALENT addition of modifying groups REVERSIBLE modifications and removals are mediated by a SEPARATE enzyme system
40
which amino acids are modified by phosphoylation?
Ser, Thr, Tyr, His have an -OH group
41
what is phosphorylation?
the addition of a (PO3)2- group REQUIRES ATP
42
what enzymes are involved in phosphorylation?
attachment is catalyzed by a PROTEIN KINASE SEQUENCE SPECIFIC removed by PHOSPHOPROTEIN PHOSPHOTASE
43
what are PKA sites?
phosphorylates at sites with: ---X-R-[RK]-X-[ST]-B [RK] = arg or lys [ST] = ser or thr B= hydrophobic AA
44
describe the PKA peptide binding site
R-X-[RK]-X-[ST]-B will be recognized by the catalytic site of PKA sequence will then be phosphorylated on the ser or thr residue
45
describe cAMP-dependant PKA regulation
regulatory subunit is smaller catalytic subunit is bound by a regulatory subunit regulatory subunit blocks the active site, inactivating PKA
46
can regulatory subunits of PKA be phosphorylated?
NO, just BIND contain sequence KRRGAI binds because ALMOST matches the catalytic subunit but Ala CANNOT BE PHOSPHORYLATED
47
describe activation of PKA by cAMP
many cellular processes trigger cAMP production regulatory subunits bind 2 cAMP molecules regulatory subunit then releases catalytic subunit, allowing it to phosphorylate the substrate
48
what does PKA regulate?
glycogen metabolism DNA condensation Fatty acid metabolism glycolysis cell surface ion channels
49
how long does cAMP last
SHORT LIVED hydrolyzed by cyclic nucleotide phosphodiesterase
50
what do phosphoryl groups introduce?
BULKY GROUP --> steric exclusion CHARGE --> electrostatic interactions O atoms --> HYDROGEN bomding possibly site for protein-protein interactions
51
describe glycogen phosphorylase activation
glucogen phosphorylase catalyzes the phosphorylysis (brea down of -(PO4)3-) of glycogen (glucose storage) 2 distinct cellular forms (a (active). b (inactive)) changed into an active "a" form by phosphorylation by physphorylase kinase reversible, does not regenerate ATP
52
describe the glycogen phosphorylase modification site
targeted to one specific residue: ser 14 stabilizes a structural state in glycogen phosphorylase phosphorylation of ser 14 favours the R-state (enzyme is active) because of salt bridges
53
describe glycogen synthase inactivation by phosphorylation
phosporylation can occur on many different sites of a protein and can produce different degrees of effect on protein function PKA phosphorylates glycogen synthase at some sites glycogen synthase kinase 3 (GSK3) phosphorylates GS at different sites effect of GSK3 is a potent inhibition
54
what do multiple phosphorylation sites allow for?
activity can be turned UP or DOWN (not just on/off)
55
describe the P-Ser binding domain
example of new protein-protein interaction provide enzyme substrate binding as well as other interactions phosphoserine binding domains mediate autoinhibition and substrate binding of GSK3
56
describe the Src-homolgy-2 (SH2) domain
binds to sites w/ phosphorylated tyr protein-protein interactions, mediated by SH2 domains are an important part of signalling processes ex. insulin SH2 is on the Gb1 protein
57
what is the SH2 domain function?
phosphorylation promotes kinase binding allows kinase domain to phosphorylate additional tyr residues kinase can also be phosphorylated --> SH2 then binds resulting in AUTOINHIBITION
58
describe regulation by proteolytic cleavages
enzymes can be synthesizes as PREACTIVE CURSORS --> do not always want active termed zymogen if activated by a protease OR a proenzyme if activated by a non-protease allows conformational changes in the enzyme to expose the active site IRREVERSIBLE
59
describe the activation of chymotrypsin
serine protease digestive system enzyme cuts itself to become fully active will cut up cell if active protein stays intact
60
describe the chymotrypsin structure
A, B, C chain catalytic triad disulfide bonds
61
describe the pH sensitivity of chymotrypsin
His 57 must deprotonate --> allows it to act as a proton acceptor and nucleophile a-amino group of Ile16 must be protonated --> forms ion pair with Asp194, stabilizing conformation
62
give a general overview of zymogen mediated blood clotting
activated by proteolytic cleavages should NOT happen spontaneously SERINE proteases --> fast, controlled process prothrombin --> thrombin (where all pathways converge, activates inhibition)
63
what is the clotting cascade?
INTRINSIC and EXTRINSIC --> converge activated ser proteases cleave different target serine proteases , activating them final protease is thrombin
64
what does thrombin do?
cleaves N-term end of fibrinogen, forming the hard clot
65
what are the important promoters of clotting?
vitamin K Ca2+ thrombin factor XIIIa (not a protease)
66
what is the role of vit K in blood clotting?
CARBOXYLATION of thrombin better to have 2 COO- groups of glu - not natural vit K covalently links COO- to Y-carboxyglutamate residue O2 is needed to activate Ch2 of glu Vit K is e- source regenerated by reductases
67
describe the carboxylation of thrombin
makes it a strong Ca2+ chelator (binder)
68
why is carboxylation required for prothrombin proteolysis?
many Glu residues at the N-term of prothrombin are converted to Y-carboxyglutamate this binds to Ca2+ prothrombin-Ca2+ is a substrate for ser protease factor Xa Xa converts thrombin to prothrombin
69
what reduces blood clotting
vit K antagonists ex. dicoumarol, warfarin
70
describe the fibrinogen structure
globular, B-strand structure 3 chains globular domains have a high affinity for motifs in the N-term ends of the a and B subunits ends are only exposed after cleavage
71
describe fibrinogen clotting
thrombin is a serine proteade thrombin cleaves fibrinogen --> fibrin ends interact w/ subunits billions of times many of these interactions result in a fibrin clot cleaved ends bind globular domains of adjacent fibrin molecules
72
what does factor XIIIa do?
CROSSLINKS --> COVALENT interaction XIIIa is a transglutaminase activated by protransglutaminase by thrombin forms and amide bond b/w gly and lys covalently links monomers converts soft clots to hard clots

BIOC 3560 (2 decks)

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