Write the Michaelis-Menten equation of an enzyme displaying positive cooperativity and draw an associated v_S plot! Name a method to estimate the Hill coefficient! What is the Hill coefficient?
Explain the terms and provide units for catalytic constant, Michaelis constant and catalytic efficiency!
Km: Michaelis constant [µmol/L]
substrate concentration where v0 = vmax/2
“affinity” of the enzyme to the substrate (the higher Km, the more substrate is needed until vmax/2 is reached, the lower the affinity of the enzyme to the substrate)
typically rather high values (>1000) because too tight binding of substrate would inhibit the reaction
Km = k2+k-1/k1
kcat: catalytic constant/turnover number/frequency [1/s]
number of product molecules generated per enzyme and second
rate constant of the rate-limiting step (often similar to k2)
normal range: 10-1000 /s
fastest: carboanhydrase (600.000 /s)
kcat = vmax/[E]T ([E]T: total enzyme concentration)
kcat/Km: catalytic efficiency/specificity constant [1/sM]
combination of affinity of the enzyme to the substrate and the catalytic competence of the enzyme for the substrate
typical values: ~ 10^5 /Ms
Write the Michaelis-Menten equation and draw the corresponding v_S plot highlighting Vmax and KM!
v0: current reaction speed at current [S]
vmax: maximal reaction speed
[S]: substrate concentration
Km: Michaelis-Menten constant (substrate concentration at which vmax/2 is reached)
Name different types of bisubstrate enzyme reactions! How can these different mechanisms be distinguished by appropriate kinetic experiments?
Ordered sequential mechanism
(lactate DH)
Random sequential mechanism
(creatine kinase)
Ping-pong mechanism
(transaminase)
Differentiation by measuring v0 at different substrate concentrations (for both substrates A and B), then Lineweaver-Burk plot:
Explain how a competitive inhibitor is acting! Draw the corresponding v_S plot and the Dixon plot! Which kinetic constants are affected by a competitive inhibitor? Write the Michaelis-Menten equation for an enzyme reaction that is inhibited by a competitive inhibitor!
v-S plot:
Dixon plot:
-> used to estimate Ki (dissociation constant of the inhibitor)
Lineweaver-Burk plot:
At sufficiently high substrate concentrations, the effect of the inhibitor can be decreased.
Km is affected (it takes more substrate to reach vmax/2)
vmax is not affected (it takes more substrate, but vmax can still be reached)
Draw the reaction profile for an enzyme-catalyzed reaction and indicate the pre-steady state and steady-state phases! Highlight the concentration profile for E (free enzyme), ES (enzyme-substrate complex), S (substrate) and P (product)!
Definition of pre-steady state: The equilibrium of the ES complex is not reached yet (its concentration is still changing).
Explain how a non-competitive inhibitor is acting! Draw the corresponding v_S plot and the Dixon plot! Which kinetic constants are affected by a non-competitive inhibitor? Write the Michaelis-Menten equation for an enzyme reaction that is inhibited by a non-competitive inhibitor!
Km is unaffected
vmax is decreased
Name three different methods used for pre-steady-state analysis of enzyme reactions! Name the time regime that can be analyzed with each method, the type of reaction amenable for analysis and the signals/readout used! Explain one method in greater detail!
Method
time regime
type of reaction
signals/readout
Stopped-flow
dead time: 0.5-2 ns
UV-Vis
Fluorescence
CD
FTIR
Diode array
Quenched-flow
dead time: 2 s
NMR
EPR
Mössbauer
Mass
Flash photolysis
dead time: ns - s
Flash photolysis:
one of the substrates is inactive but can be activated using a laser flash
advantage: substrate and enzyme can be mixed properly before activation
Write the rate law for a first-order reaction and draw the corresponding progress curve.
(First Order Reaction Plot D9F (mavink.com))
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