Enzymes Flashcards

1
Q

What are enzymes (overview)

A
  • proteins
  • catalyze the chemical transformation of substrate (S) to a product (P)
  • increase the rate of reaction

function by lowering the activation energy of the reaction

E + S <–> ES <–> EP <–> E + P

  • two intermeidates
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2
Q

What is the michaelis- menten equation

A

V0 = ( Vmax x [S] ) / (Km + [S] )

  • there is a max speed that chem rxn can occur, notice how simialr it looks to hemoglobin oxygen binding curve

as you inc concentration of substrate RXN goes faster

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3
Q

What are kinases

A

X-OH + ATP <—> X-OPO32- + ADP + H+

  • an enzyme that caalyses the transer of phosphate groups from higih energy phosphate donating moleucles to specific substances
  • this procuess is known as phosphorylation

substrate gains a phosphate group and high energy ATP molecule donate phosphate group

*addition of phosphate groups makes the molecule target for another enzyme

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4
Q

what is Phosphatase

A

X-OPO32- + H2O <—-> X-OH + HOPO32-

  • enzyme that uses water to cleave,a phosphoric acid monoester into phosphate ion and alcohol

bc phosphatase enzyme catalyzes the hydrolysis of its substrate its a subcategory of hydrolases

*kinase is energy dependent but phosphatease gets it from water

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5
Q

what is phosphorylase

A

X-O-Y + HOPO32- <—> X-POP32- + Y-OH

  • enzymes that catayse the addiiton of phsophate group from an inorganic phosphate (phosphate + hydrogen) to an acceptor

*outcoe basically same as kinase

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6
Q

What is Hydrolase

A

X-O-Y + H2O <—-> X-OH + Y-OH

commonly perform as biochemical catalyst that uses water to break a chemical bond

typically dividdes a larger molecule into two smaller molecules

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7
Q

what are regualtory enzymes

A
  • Multi-step metabolic pathways, contain at least one rate-limiting ste
  • catalysis of rate limiting steps is mediated by regulatory enzymes (these enzymes are regulated)
  • cataltic rate of these enzymes is controlled by specific signals

*adding another level of control over chemcial proces in a cell by regualteing the enzymes that regulate the reaction

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8
Q

explain feedback inhibitation

A

Inhibition occurs at the first step of the pathway

No other products cause inhibition

Isoleucine does not bind to the active site

Instead there is a separate allosteric binding site

Inhibition is reversible

*negative feedback, the product inhibits the enzyme that generates it

*inhibitor deosnt bind to active site it binds to a seperate allosteric binding site

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9
Q

what are the mechanisms of enzyme regulation

A
  1. allostery
    • reversible, non covalent binding of regulatory compounds
    • allosteric modulators
  2. Reversible colanet modifcation
    1. mediated by a separate enzyme system
  3. interaction with regulatory proteins
  4. proteolytic cleavage;
    • non reversible, chewing enzyme up and degrading it or putting into diff subunits
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10
Q

What is an allosteric enzyme

A

A regulatory enzyme with catalytic activity modulated by the noncovalent binding of a specific compound at a site other than the active site

* undergo confomational changes in response to modulator binding, think this is like when one oxygen molecule binds changing from T to R state and changed adjacent subunits

Properties:

  • larger, structurally more complex
    • usually multi subunit
  • have regulatory (allosteric) sites
    • bind modulatory
  • undergo conformational changes in response to modulator binding
  • do NOT obey michaelis- mentin kinetics
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11
Q

what does binding of allosteric modulator do

(how does it change a graph)

A
  • do NOT obey michaelis-mentin, can change K0.5 OR Vmax
  • get sigmoidal cruve of V0 vs [S]
  • influenced by cooperative substrate binding and /or binding of allosterid modulator
  • do not have Km use K0.5

Km = half saturation constant

K0.5 = [S] resulting in 1/2 Vmax

Positive modulator: switch the T to R state

Negative modulator: might switch R state to T state

*sigmoidal bc combination of T and R state

  • graph shows spepd at which reaction is going not occupancy, X axis is the same but Y axis different
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12
Q

what the the types of modulators

A

homotropic: both modulators and substrates, when expose enzyme to substrate you inc speed and affinity of reaction
heterotropic: modulatoes are not substracts, other bidning factors that dictates how the enzyme is able to execute the chemical reaction

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13
Q

what are the effects of modualtors

A

can have negative effects (reduce activity) or positive (inc activity)

-can affect K0.5 or Vmax

both homo and heterotropic modulators have one of these effects

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14
Q

what is ATCase

A

aspartate transcarbamoylase: ALLOSTERIC ENZYME

commited step in biosynthesis of pyrimidine nucleotides (ex CTP)

  • form N-carbamoylaspartate and Pi from carbamoyl phosphate and aspartate

*has both T and R state (inactive T and active R)

  • c nucleotides that this enzyme makes, CTP (ALLOSTERIC MODULATOR)
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15
Q

what is the aspartate transcarboamoylase reaction

A
  • ATCase transfers an activated carbamoyl group onto the amine group of aspartate

this is the first step for synthesizing puring rings (ex CTP)

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16
Q

what is the subunit structure of aspartate transcarbamoylase

A
  • ATCase has two distinct types of subunit: catalytic and regulatory
  • the enzyme contains 12 total polypeptide chains: 6 regulatory chains and 6 catalytic c chains

*subunits are individual peptides,

  • 3 c chains form each catalytic trimer (c3)

*we have 2 catalytic trimers bc 6 c chains total, 2 catalytic sites

  • 2 r chains forming regulatory dimer (r2)

*3 sites of regulation

  • subunits interact via zinc domains of regulaotry subunit: zinc domains have cysteines coordinating a structural zinc ion
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17
Q

how was the subunit structure of ATCase determined

A

via treatment with a cysteine modifying mercury compound and ultracentrifugation

  • run thorugh a size separating devise, if put on a lot of mercury to dissociate it we see two peaks, r2 and C3
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18
Q

describe the allosteric modulation of ATCase

A
  • substrate binding by ATCase is explained by allosteric cooperativity T -> R state transition
  • aspartate is a positive homotropic modulator
19
Q

What does CTP and ATP do in terms of being modulators

A

ATP = +ve heterotopic

CTP = -ve heterotropic

  • CTP functions in feedback inhibition
  • ATP and CTP binds to sits other than the active site (on the regulatory subunit)

*CTP is a negative modulator, binds to regualtory site and reduces the activity

*ATP is an activator and does opposite

20
Q

how are substrates and heterotropic modulatoes bound by ATcase different

A

*structurally very different

  • substrates bind at interfaces of c chains (center of complex)
  • modulators (ATP, CTP) bind to r chains (periphery of complex)
21
Q

explain the allosteric efects of ATCase

A

arise from changes in quaternary structure

  • Tstate: ATCase has a more closed conformation
  • this closed conformation blocks active site loop from adopting a fully active conformation

*key word is fully active, has some potential for activity

  • R state: ATCase is in a more open conformation
  • allows active site loop to adopt an active conformation
22
Q

Howdo we push ATCase into the R stae so we can characterize its structure and properties

A
  • add substrates (only gives short window to study in active state becuase substrate will be used up)
  • add substrate analogue that mimics both substrates PALA (mimics substrate and is non reactive, pushes ATCase into R state and keeps it there)

add activator ATP

23
Q

what are the changes in strucutre of ATCase

A
24
Q

what is PALA

A

N-(Phosphonacetyl)-L-aspartate

nonreactive bisubstrate analog that mimics the reaction intermediate of ATCase

  • binds in catalytic site but chemical reaction does not contiue to final product and does not get released
  • shoves ATCase from t to r state
25
Q

what is the strucutal change of ATCase when PALA binds

A

*dramatic changes in quaternanry strucuture

26
Q

what are the strucutral changes during activation fo ATCase

A
  • within catalyic subunits
  • flexing at interfaces between c chains
  • Overall
  • catalytic trimers move apart 12 Å and rotate relative to each other
  • regulatory dimers roate
  • change in bend between r chains`
27
Q

what happens when CTP bidns to ATCase

A
  • stabilizes T state

(substratte)

28
Q

what happens when you throw in activator in with ATCase

A

T -> R appears to be concerted

ATP binding can induce T -> R conversion in absence of substrate

*this change may not be permanent

29
Q

how can enzymes be regulated by reversible covalent modification

A

Enzyme activity can be altered by the covalent addition of modifying group(s)

Modification is reversible

Covalent modifications and their removal are mediated by a separate enzyme system

30
Q

what are the target residues for adenylation

A

Adenylylation = (Tyr)

31
Q

What is the target residue for phosphoryation

A

Phosphorylation = (Ser, Thr, Tyr, His)

32
Q

what is the target residue for ADP-ribosylation

A

ADP-ribosylation = (Arg, Gln, Cys, diphthamide)

33
Q

WHat is the target residue for ubiquitation

A

Ubiquitination = (Lys)

34
Q

what is the target residue for methylation

A

Methylation = (Glu)

35
Q

what is the target residue for acetylation

A

Acetylation = (Lys, amino-terminus)

36
Q

how does phosphorylation occur?

A

* ATP is the source of the phosphate

37
Q

how are enzymes regulated by reversible phosphorylation

A
  • Amino acids modified: Ser, Thr, Tyr, His
  • Attachment of the phosphoryl group is catalyzed by a protein kinase
  • Phosphorylation is sequence-specific
  • Phosphoryl group can be removed by phosphoprotein phosphatase
38
Q

what are the site of protein kinase A

A

Protein kinase A (PKA) phosphorylates proteins at sites containing the sequence:

  • X-R-[RK]-X-[ST]-B
  • e.g. F-R-R-L-S-I

Substrates of PKA contain an amino acid sequence that fits this pattern:

  • [RK] = Arg or Lys is acceptable;
  • [ST] = Ser or Thr is acceptable (and is the residue phosphorylated) X = any amino acid
  • B = any hydrophobic amino acid,

X-R-[RK]-X-[ST]-B will be recognized by the catalytic site of PKA
This sequence will then be phosphorylated on the serine or threonine residue

*PKA regualtes by phophorylates its target,

39
Q

how is PKA regulated

A
  • by regulatory subunit binding
  • The catalytic subunit of PKA (c; blue) is generally bound by a regulatory subunit (r; red)
  • This regulatory subunit blocks the catalytic active site, inactivating PKA
  • Regulatory subunits of PKA contain the sequence K-R-R-G-A-I
    This sequence binds the catalytic subunit because it (almost) matches the pattern X-R-[RK]-X-[ST]-B…
  • But it cannot be phosphorylated (Ala instead of Ser/Thr), Ser/Thr does nothitng to actually attract protein kinase A but is just wahts phosphorylated
40
Q

how is PKA activated

A
  • by cAMP
  • Many cellular signaling events will trigger cAMP production
  • The regulatory subunit will bind two cAMP molecules
  • The regulatory subunit then releases the catalytic subunit, allowing it to phosphorylate substrates
  • AKAP= A kinase anchoring protein, bidns to palsma membrane of cell, it binds regulatory subunits of PKA

*PKA in inactive form sits on plasma membrane, reacts to extracellular signalling

*once activated itll phosphorylate any targets

41
Q

what does PKA do?

A
42
Q

comment on the lifetime of cAMP

A
  • short lived bc hydrolyzed by cyclic nucleotide phosphodiesterase
43
Q

what do phosphoryl groups introduce

A

1) A relatively bulky group (can result in steric exclusion)
2) Charge – allowing electrostatic interactions (attraction, repulsion)
3) Oxygen atoms that can participate in hydrogen bonds
4) Site for protein-protein interactions