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)
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 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?
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)
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
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).
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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