Enzymes Flashcards
What groups make up enzymes?
Prosthetic group: Cofactor/coenzyme binds protein part of active enzyme
Holoenzyme: complete enzyme (even non-protein part)
Apoenzyme/apoprotein: polypeptide only (the protein)
What causes Phenylketonuria?
- Lack phenylalanine hydroxylase
* Can’t catabolise Phe, AA builds up, issues with brain development
What can hexokinase do?
• Couples phosphorylation of glucose and hydrolysis of ATP
What are the reaction intermediates in an enzyme reaction? What do they do?
• Multiple steps via transient chemical reaction intermediates
o ES, EP
o Lower the free energy of the transition state
What are the benefits of energy barriers?
o Energy barriers to reactions prevent spontaneous reversion
What don’t enzymes affect?
- Free energy change for S ↔ P
* Equilibrium constant
How do enzymes speed up reactions?
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
What is binding free energy?
- 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
How is enzyme kinetics studied?
- 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
Why does rate of reaction decrease over time?
o S depeleted
o Reaction reversible, more [P] = more reverse rate
o Pure enzyme unstable
What is the relationship between V0 and enzyme concentration?
• Rate proportional to concentration of enzyme (double enzyme, double rate)
What is Michaelis constant?
• Value of [S] at ½ Vmax = Km
What are the features of V as a function of [S] ?
- V0 at plateau region = Vmax
* Value of [S] at ½ Vmax = Km (Michaelis constant)
What is involved in the steady state assumption?
• 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)
What makes up the double reciprocal/Lineweaver-Burk plot?
- V0 = (Vmax[S]) / ([S]+Km)
- 1/V0 = 1/Vmax + Km/Vmax[S]
- Intercept = 1/Vmax
- Slope = Km/Vmax
What is Ka?
• Association constant = Ka = affinity for enzyme for substrate assuming equilibrium = 1/kd
What does low Km suggest about affinity?
High affinity
What is Kcat?
• 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
What is the specificity constant?
• Low [S], velocity of enzyme catalysed reaction proportional to specificity constant (Kcat/Km)
o Shows substrate affinity and catalytic efficiency
What is the mechanism of α chymotrypsin?
• 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
What is involved in the active site of α chymotrypsin?
- Catalytic triad
- Asp102
- His57
- Ser195
What are the steps of α chymotrypsin action?
- Form ES complex. Substrate binds, residue side chain goes hydrophobic pocket, position peptide for attack
- Cleave peptide bond. Asp forms H bond with N of His, deportonates Ser
- Ser side chain is nucleophile, binds electron deficient carbonyl carbon of main chain
- Ionisation of carbonyl O stabilised by H’s attached to N of Ser and Gly
- Form acylated intermediate. Bound to Ser. New formed amino terminus of cleaved protein dissociates.
- Deacylation. H20 activated by basic His and acts as nucleophile
- O of water attacks carbonyl carbon of Ser bound acyl group and serine OH group regenerated
- Remaining protein fragment of substrate release to regenerate enzyme
How does pH influence α chymotrypsin?
• 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
What are the types of reversible inhibition?
competitive, uncompetitive, mixed
What are the features of competitive inhibition?
- 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)
What are the features of uncompetitive inhibition?
- 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
What are allosteric enzymes?
- 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)
What is ATCase? What modulates it’s activity?
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
What are the differences between isolated catalytic and regulatory subunits?
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
