Module 5: V5 - V13 Flashcards
What is the equilibrium equation for the Michaelis Menten Theory?
E + S ⇌ ES -> E + P
What are the assumptions made in the Michaelis Menten Theory?
ES conversion to E + P is irreversible
steady-state conditions [ES] constant (rate of formation of ES = rate of breakdown of ES)
[S]»_space; [Et]
[S]»_space; [P] (initial conditions)
What is the equation for the Michaelis Menten Theory?
Vo = Vmax [S] / Km + [S]
What is the equation for the Michaelis constant (Km)?
[E][S]/[ES] = (k2 + k-1)/k1 = Km
What are some rules of the Michaelis Menten graph?
when [S]»_space; Km; Vo = Vmax
when [S] = Km; Vo = 1/2 Vmax
How is a typical Michaelis Menten plot converted into a linear graph?
by reciprocating both sides of the equation Vo = Vmax [S] / Km + [S]
When is Kd taken into account? When is Km taken into account?
under equilibrium conditions
under steady-state conditions (also takes account of the catalytic step)
What does a low Km value correspond to?
tighter binding of the enzyme to the substrate
What is the turnover number?
k2 which is also the rate constant for the rate-limiting step i.e. the number of molecules converted to product (S to P) per unit time per enzyme molecule saturated with substrate (i.e. when [ES] = [Et]
What does a larger turnover number correspond to?
a faster converting enzyme
What is the specificity constant?
the rate constant for the conversion of E + S to E + P (kcat/Km)
when [S] «_space;Km, Vo is proportional to (kcat/Km) i.e. kcat/Km reflects both substrate affinity and catalytic efficiency
What does a larger value for kcat/Km correspond to?
indicates a more efficient use of the substrate
What are irreversible inhibitors of enzymes?
molecules which bind covalently to the active site, destroy a functional group essential for enzyme activity, or form a stable noncovalent complex with the enzyme
What are reversible inhibitors of enzymes?
molecules which bind reversibly to enzymes and inhibit the enzyme either by competitive, uncompetitive or mixed modes of inhibition
How is a decrease in activity of an enzyme due to competitive inhibition shown in the Michaelis Menten equation?
using an ɑ factor which is placed in the denominator
e.g. Vo = Vmax [S] / ɑ Km + [S]
What would the double reciprocal plot of initial velocity (Vo) versus substrate concentration [S] look like with and without a competitive inhibitor? Why?
the gradient would get steeper as the ɑ factor increases in value
because competitive inhibitors bind to the free enzyme
How is a decrease in activity of an enzyme due to uncompetitive inhibition shown in the Michaelis Menten equation? Why?
using an ɑ’ factor which is placed in the denominator
e.g. Vo = Vmax [S] / Km + ɑ’ [S]
because uncompetitive inhibitors bind to the ES complex
What would the double reciprocal plot of initial velocity (Vo) versus substrate concentration [S] look like with and without an uncompetitive inhibitor? Why?
the gradient stays the same, however the y-intercept increases as the ɑ factor increases in value because uncompetitive inhibitors do not bind to the free enzyme and instead bind to the ES complex
What is mixed inhibition?
occurs when an inhibitor binds to both the free enzyme and the ES complex
How is a decrease in activity of an enzyme due to mixed inhibition shown in the Michaelis Menten equation? Why?
using an ɑ and ɑ’ factor
e.g. Vo= Vmax [S] / ɑKm + ɑ’[S]
because mixed inhibitors bind to the free enzyme and the ES complex
What would the double reciprocal plot of initial velocity (Vo) versus substrate concentration [S] look like with and without a mixed inhibitor? Why?
the gradient and y-intercept both increase as the ɑ factor increases in value because mixed inhibitors bind to the free enzyme and the ES complex
What is the role of allosteric enzymes?
often regulate metabolic pathways by changing activity in response to changes in the concentrations of molecules around them
What compounds are allosteric enzymes regulated by?
allosteric modulators and allosteric effectors
What do positive and negative modulators do?
positive modulators activate and negative modulators inhibit allosteric enzymes
How do modulators bind to an allosteric enzyme?
they bind reversibly and non-covalently to the enzyme
Which subunits usually exist in an allosteric enzyme and which subunit do modulators bind to?
catalytic and regulatory subunits
modulators bind to the regulatory subunit
How does a positive modulator affect an allosteric enzyme?
causes a change in conformation at the active site so the substrate can bind with higher affinity (stabilises the high affinity R state of the enzyme)
Do allosteric enzymes show M-M kinetics?
no
What happens when S (e.g. for ATCase, S is Asp or carbamyl phosphate) binds?
there is a transition from the T state to the R state to give a sigmoidal Vo versus S plot
What process is ATCase involved in and what are positive / negative modulators of ATCase?
ATCase catalyses the first step in the E. coli pathway to produce the nucleotides UTP and CTP
CTP is a negative modulator of ATCase when at high levels and ATP is a positive modulator at high levels
How many subunits does ATCase have and what type of subunits are they?
ATCase has 12 subunits
6 catalytic subunits (2 trimeric complexes) and 6 regulatory subunits (3 dimeric complexes)
How does CTP and ATP affect the Vo vs [S] plot of ATCase?
as ATP increases, the plot becomes more hyperbola-like (R state) and as CTP increases, the plot shift to the right (T state)
What are some structural characteristics of α-chymotrypsin?
5 disulfide bonds within and between the A, B and C chains
hydrophobic pocket that binds the side chain of Phe, Tyr or Trp in the substrate protein
an active site
What is the role of chymotrypsin?
chymotrypsin is one of several proteases that cuts peptides at specific locations on the peptide backbone
Which location in a peptide is chymotrypsin able to cleave?
the peptide bond adjacent to aromatic amino acids
Why does the sharp increase in activity within chymotrypsin correspond to changes in kcat?
because below pH 7, His57 is protonated and cannot accept the proton from Ser195, so kcat decreases
above pH 8, His57 is all deprotonated, so kcat does not change
Why does chymotrypsin activity decrease above pH 8.5?
because H+ is lost from the N-ter NH3+ of B chain (Ile16)
this results in loss of the Ile16-Asp194 salt bridge which changes the hydrophobic pocket where substrate binds, so 1/Km decreases
What does a big value of alpha mean? What does a big value of alpha’ mean?
a big value of alpha results in a steep gradient on the double reciprocal plot and a big value of alpha’ results in a large y-intercept
How is it possible that some allosteric modulators can activate and some can inhibit - the same enzyme?
because modulators can either be positive or negative
