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BCMB20002 Biochemistry > Enzymes > Flashcards

Enzymes Flashcards

1
Q

What groups make up enzymes?

A

Prosthetic group: Cofactor/coenzyme binds protein part of active enzyme
Holoenzyme: complete enzyme (even non-protein part)
Apoenzyme/apoprotein: polypeptide only (the protein)

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2
Q

What causes Phenylketonuria?

A
  • Lack phenylalanine hydroxylase

* Can’t catabolise Phe, AA builds up, issues with brain development

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3
Q

What can hexokinase do?

A

• Couples phosphorylation of glucose and hydrolysis of ATP

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4
Q

What are the reaction intermediates in an enzyme reaction? What do they do?

A

• Multiple steps via transient chemical reaction intermediates
o ES, EP
o Lower the free energy of the transition state

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5
Q

What are the benefits of energy barriers?

A

o Energy barriers to reactions prevent spontaneous reversion

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6
Q

What don’t enzymes affect?

A
  • Free energy change for S ↔ P

* Equilibrium constant

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7
Q

How do enzymes speed up reactions?

A

Reduce activation energy of S at transition state
• Overall rate determined by slowest step (highest activation energy)
• Bind substrates in correct orientation relative to active groups
• Have catalytically active groups
• Stabilise transition state

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8
Q

What is binding free energy?

A
  • Difference between activation energies of uncatalysed and catalysed reactions
  • Maximum S and E interactions occur at transition stet (binding free energy ∆GB released and overcomes energy needed to reach top of hill)
  • ∆GCat net binding free energy plus ∆GUncat
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9
Q

How is enzyme kinetics studied?

A
  • Rate of reaction and how it changes in response to experimental parameters
  • Rate = V
  • Rate influenced by [S] (which decreases as reaction progresses)
  • Determine initial velocity, V0 defined as rate of reaction as time (t) reaches 0 before [S] has time to decrease
  • [P] vs t show V0 ( d[P]/dt )
  • Rate increases as [S] increases
  • Rate decreases with time
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10
Q

Why does rate of reaction decrease over time?

A

o S depeleted
o Reaction reversible, more [P] = more reverse rate
o Pure enzyme unstable

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11
Q

What is the relationship between V0 and enzyme concentration?

A

• Rate proportional to concentration of enzyme (double enzyme, double rate)

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12
Q

What is Michaelis constant?

A

• Value of [S] at ½ Vmax = Km

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13
Q

What are the features of V as a function of [S] ?

A
  • V0 at plateau region = Vmax

* Value of [S] at ½ Vmax = Km (Michaelis constant)

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14
Q

What is involved in the steady state assumption?

A

• d[ES]/dt = 0, K1[E][S] = k2[ES] + k-1[ES]
o rate of ES formation = removal rate
• [E][S]/[ES] = (K2 + K-1)/K1 = Km (Michaelis-Menten constant)
• [ES] = ([Et][S]) / ([S]+Km)

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15
Q

What makes up the double reciprocal/Lineweaver-Burk plot?

A
  • V0 = (Vmax[S]) / ([S]+Km)
  • 1/V0 = 1/Vmax + Km/Vmax[S]
  • Intercept = 1/Vmax
  • Slope = Km/Vmax
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16
Q

What is Ka?

A

• Association constant = Ka = affinity for enzyme for substrate assuming equilibrium = 1/kd

17
Q

What does low Km suggest about affinity?

A

High affinity

18
Q

What is Kcat?

A

• Kcat replaces K2
• K2: rate constant for rate limiting step
• Kcat = K2 = turnover number
o Number of molecules of substrate converted to product per unit time by one enzyme molecule

19
Q

What is the specificity constant?

A

• Low [S], velocity of enzyme catalysed reaction proportional to specificity constant (Kcat/Km)
o Shows substrate affinity and catalytic efficiency

20
Q

What is the mechanism of α chymotrypsin?

A

• Acylation (add acyl) of Ser residue
o Form acyl enzyme intermediate
• Deacylation
o Remove peptide, return enzyme to original form
• Acid base catalysis and covalent catalysis

21
Q

What is involved in the active site of α chymotrypsin?

A
  • Catalytic triad
  • Asp102
  • His57
  • Ser195
22
Q

What are the steps of α chymotrypsin action?

A
  1. Form ES complex. Substrate binds, residue side chain goes hydrophobic pocket, position peptide for attack
  2. Cleave peptide bond. Asp forms H bond with N of His, deportonates Ser
  3. Ser side chain is nucleophile, binds electron deficient carbonyl carbon of main chain
  4. Ionisation of carbonyl O stabilised by H’s attached to N of Ser and Gly
  5. Form acylated intermediate. Bound to Ser. New formed amino terminus of cleaved protein dissociates.
  6. Deacylation. H20 activated by basic His and acts as nucleophile
  7. O of water attacks carbonyl carbon of Ser bound acyl group and serine OH group regenerated
  8. Remaining protein fragment of substrate release to regenerate enzyme
23
Q

How does pH influence α chymotrypsin?

A

• pH7 transition related to changes in Kcat
• Less than pH 7, His protonated and can’t accept proton from Ser (low Kcat)
• Above 8.5, lose activity because changed in 1/Km due to loss of H+ from amino terminal of B chain Ile
o Lose salt bridge Ile/Asp, issues with hydrophobic pocket
• Max activity requires His to be unprotonated and Ile to be protonated

24
Q

What are the types of reversible inhibition?

A

competitive, uncompetitive, mixed

25
Q

What are the features of competitive inhibition?

A
  • Inhibitor competes for same binding site as substrate
  • I binds active site of E, form EI
  • Inhibition overcome at [S]
  • Vmax the same
  • Km increases in presence of I
  • Noticeable in double reciprocal plot of V0 Vs. [S] (Higher Km = lower 1/km)
26
Q

What are the features of uncompetitive inhibition?

A
  • Binds to ES complex at site distinct from active site
  • Often for enzymes with multiple substrates
  • I only binds ES complex (S binds E first)
  • Vmax decrease
  • Km decrease
  • Parallel lines on double reciprocal plot
27
Q

What are allosteric enzymes?

A
  • Regulatory, respond to changes in [molecule] in environment, regulated by allosteric modulators which bind reversibly/non-covalently
  • C- catalytic subunit
  • R- regulatory subunit
  • M- regulator
  • M binds, conformational change of active site, altered shape can cause S to bind with higher affinity (if M activator)
28
Q

What is ATCase? What modulates it’s activity?

A

Aspartate transcarbamoylase

• Positive modulator: ATP
• Negative modulator: CTP
• CTP end product of pathway, high levels inhibit its own formation by ATCase
• High ATP indicates cell growth
• Sigmodial kinetics
o Add CTP, inhibit ATCase, slow reaction, graph shifts right
o Add ATP, activate ATCase, less sigmoidal

29
Q

What are the differences between isolated catalytic and regulatory subunits?

A 
Isolated Catalytic Subunits
•	3 trimers
•	Catalytically active
•	Michaelis-Menten kinetics
•	Not regulated by ATP or CTP
Isolated Regulatory Subunits 
•	2 trimers
•	Catalytically inactive
•	Bind both ATP and CTP
•	Regulated the activity of catalytic trimmers in holoenzyme

BCMB20002 Biochemistry (7 decks)

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