Previous Class - University of Windsormutuslab.cs.uwindsor.ca/vacratsis/lecture 7o6m.pdfThe Michaelis – Menten Equation v = Vmax [S] Km + [S] Km = Michaelis constant: Concentration

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•Michaelis – Menten equation

•Steady state vs pre-steady state

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Review derivation and interpretation

Graphical representation

Michaelis – Menten equations and parameters

The Michaelis – Menten Equation

v = Vmax [S]Km + [S]

Km = Michaelis constant: Concentration of Substrate needed to reach half maximum velocity – measure of substrate affinity

Vmax = maximum velocity – directly proportional to enzyme concentration

The Michaelis – Menten Equation

Km

Ef + S ES Ef + P k1 k2

k-1

[E][S][ES]

= k-1 + k2 = Km Eqn 1k1

Km is an apparent dissociation constant (Ks) and represents the [S] when v = ½ Vmax

Therefore, a lower Km value indicates a higher affinity for the substrate

The Michaelis – Menten Equation

The Michaelis – Menten Equation

Interpretation

Obtain kinetic behaviour of an enzyme

E + S ES E + P k1 k2

k-1

k2 = kcat Catalytic constant of the reaction (first order) when k2 is fast (saturating kinetics)

(When EP → E + P is fast)kcat is also known as the turnover number of the enzyme –defining the maximum number of substrate molecules converted to product per unit of time

kcat = Vmax/[ET]

Units

v0 = initial velocity of Product formation = moles Product formation (moles substrate loss)/litre x time

= mM s-1

Vmax = represents the maximum rate the enzyme reaction can achieve. Vmax occurs when all of the enzyme is in the ES complex.

Km = [S] at Vmax/2 = µM

kcat = first order rate constant = s-1

# of catalytic cycles active site undergoes per unit of time

The Michaelis – Menten Equation

kcat/KmAt any [S] including:

At very low [S] ([S]→0) : pre-steady state conditions

v0 = Vmax [S]

Km + [S]becomes v0 = Vmax[S] = kcat[E][S]

Km Km

The second order rate constant kcat/Km indicates the catalytic efficiencyof the enzyme:

A direct measure of the efficiency of the enzyme in transforming Subst.

kcat/Km combines: the effectiveness of transformation of bound product

the effectiveness of productive substrate binding

Unitsv0 = initial velocity of Product formation = moles Product formation (moles substrate loss)/litre x time

= mM s-1

Vmax = represents the maximum rate the enzyme reaction can achieve. Vmax occurs when all of the enzyme is in the ES complex.

Km = [S] at Vmax/2 = µM

kcat = first order rate constant = s-1

# of catalytic cycles active site undergoes per unit of time

kcat/Km = second order rate reaction of E and S = M-1 s-1

The Michaelis – Menten Equation

Lineweaver-Burke transformation of the Michaelis-Menton equation.

•Velocity vs substrate plots are useful for visually estimating kinetic parameters

•Hyperbolic curves cannot properly determine the upper limit of the curve (Vmax)

•Transforming the data to a form that can be plotted as a line.

Lineweaver-Burke transformation of the Michaelis-Menton equation.

v = Vmax [S]Km + [S]

Reciprocal of the equation is:

1

v Vmax [S]

Km + [S]=

Express reciprocal in the familiar form y = mx + b

1 V Vmax [S]

Km= + 1 Vmax

y = mx + b

Lineweaver-Burke transformation of the Michaelis-Menton equation.

Lineweaver-Burke equation represents a straight line with slope = Km/Vmax, y intercept = 1/Vmax, and x intercept = -1/Km

1 V Vmax [S]

Km= + 1 Vmax

1/[S] (mM)

1/v (mM/min)

0.3 0.25 0.20 0.15 0.10 0.1 0.15 0.2 0.25 0.30 0.35

0.6

0.5

0.4

0.3

0.2

0.1

A

B

Lineweaver-Burke transformation of the Michaelis-Menton equation.

•Most commonly used

•Magnitude of errors can become distorted

•Furthest point to the right (lowest [S]) influences where line is drawn

•e.g. small error in v = large error in 1/v

•Good for observing enzyme inhibition

Eadie-Hofstee Plot

•Multiply both sides of L.B. by Vmax

•Multiply both sides by v

•Rearrange for v

v0

[S]Km = -Vmax v0

Plot v0 vs v0[S]

Eadie-Hofstee Plot

v0 -Km = slope

Vmax

Vmax Km

v0/[S]

Eadie-Hofstee Plot

•Best for controlling slight deviations from linearity

•One disadvantage is that the least precise parameter (v0) is expressed in both sides of the equation and plot

Plotting kinetic data

Michaelis-Menten kinetics (steady state kinetics)

Measure Initial rates (v0) at different substrate concentrations by detecting the absorbance difference over time and obtaining the slope of the line

Obtain various initial rates at different substrate concentrations and plot

Use Lineweaver-Burke plots to obtain kcat, Km, kcat/Km

Plotting kinetic data

Measuring initial rates:

•The initial velocity is the amount of product produced per minute

•Assay involves adding all of the components including substrate first

•Once enzyme is added the absorbance is continually monitored andrecorded with respect to time

•Abs vs time can be plotted

•Determine slope of tangentA

bsor

.

time

Enzyme added

Plotting kinetic data

Measuring initial rates:

•The slope of tangent = ∆Abs/unit of time (min)

•Recall A = εcl, therefore ∆A = ε∆cl (change in conc of Product)

•When ∆c occurs in a known time period then ∆c min-1 = v0

•v0 = ∆A min-1/ εl = ∆c min-1

•Has M/min after multiplying by vol. of enzyme assay solution theunits change to mol/min.

Plotting kinetic data

Determine v0 at different substrate concentrations and plot