How do you find Km and Vmax from a Lineweaver Burk plot?

The Lineweaver–Burk plot was widely used to determine important terms in enzyme kinetics, such as Km and Vmax, before the wide availability of powerful computers and non-linear regression software. The y-intercept of such a graph is equivalent to the inverse of Vmax; the x-intercept of the graph represents −1/Km.

Also know, how do you calculate Vmax?

You can estimate KM and Vmax from the graph of initial velocity versus [S].

Run a series of reactions with constant [Etot], varying [S], and measure Vo.
Graph Vo vs. [S].
Estimate Vmax from asymptote.
Calculate Vmax/2.
read KM from graph.

Additionally, what is the unit of Vmax? Vmax "represents the maximum rate achieved by the system, at maximum (saturating) substrate concentrations" (wikipedia). Unit: umol/min (or mol/s).

Hereof, how can a Lineweaver Burk double reciprocal plot be used to determine km?

The double-reciprocal (also known as the Lineweaver-Burk) plot is created by plotting the inverse initial velocity (1/V₀) as a function of the inverse of the substrate concentration (1/[S]). The V_max can be accurately determined and thus K_M can also be determined with accuracy because a straight line is formed.

What is the unit of KM?

Kilometre (km), also spelled kilometer, unit of length equal to 1,000 metres and the equivalent of 0.6214 mile (see metric system).

33 Related Question Answers Found

What are the units of kcat?

Kcat is equal to K2, and it measures the number of substrate molecules "turned over" by enzyme per second. The unit of Kcat is in 1/sec.

What are the units of Km and Vmax?

The units of Km are those of concentration i.e. mM, mM or Km is the concentration of substrate at which half maximal velocity is observed. Vmax can be expressed in a variety of units depending on what information is available.

Can km be negative?

If you follow the disappearance of something, the velocity should be "negative" and hence you need to invert it to get a positive reaction velocity. Only when the reaction rate is positive will you find both Michaelis-Menten parameters to be positive. Also, make sure your reaction rate is faster as [S] increases.

Is kcat the same as Vmax?

Kcat is equal to Vmax/[Enzyme]. Because the concentration of enzyme is taken into account in this equation, Kcat does NOT vary with the amount of enzyme used and is therefore a constant for an enzyme. Kcat is equal to the number of molecules of product made per enzyme per unit time.

How do you solve for KM Michaelis Menten?

The equation that defines the Michaelis-Menten plot is: V = (V_max [S]) ÷ (K_M + [S}). At the point at which K_M = [S], this equation reduces to V = V_max ÷ 2, so K_M is equal to the concentration of the substrate when the velocity is half its maximum value.

What is the slope of a Lineweaver Burk plot?

A plot of 1/V₀ versus 1/[S], called a Lineweaver-Burk or double-reciprocal plot, yields a straight line with an intercept of 1/V_max and a slope of K_M/V_max (Figure 8.36). The intercept on the x-axis is -1/K_M.

What Vmax means?

Vmax is the reaction rate when the enzyme is fully saturated by substrate, indicating that all the binding sites are being constantly reoccupied.

What is Lineweaver Burk equation?

The Lineweaver-Burk equation is a linear equation, where 1/V is a linear function of 1/[S] instead of V being a rational function of [S]. The Lineweaver-Burk equation can be readily represented graphically to determine the values of K_m and V_max.

How do you plot a graph Michaelis Menten?

Plotting the Michaelis-Menten Curve

Label the x-axis mM of [S] or concentration of substrate. Label the y ax- sec/micro-mole of V or velocity of reaction. Insert different values of [S] into the Michaelis-Menten equation, along with the values found for Km and Vmax, to solve for V.

Why is Lineweaver Burk plot more accurate?

It is also more robust against error-prone data than the Lineweaver–Burk plot, particularly because it gives equal weight to data points in any range of substrate concentration or reaction rate (the Lineweaver–Burk plot unevenly weights such points).

What is kcat km?

Kcat/Km represents the rate of the reaction at negligible substrate concentration. Or in other words, Kcat/Km is the (pseudo-)second order rate constant between the enzyme and the substrate, when [S]≪Km[S]≪Km.This still leaves the issue of why Kcat/Km is often referred to as the "specificity constant" of the enzyme.

What is the Km and Vmax?

The rate of reaction when the enzyme is saturated with substrate is the maximum rate of reaction, Vmax. This is usually expressed as the Km (Michaelis constant) of the enzyme, an inverse measure of affinity. For practical purposes, Km is the concentration of substrate which permits the enzyme to achieve half Vmax.

How do you calculate kcat?

Note: the enzyme concentration is the same for all of the test tubes; only the substrate concentrations vary in the assay. Divide the Vmax (from Section 2, Step 4) by the enzyme concentration (from Section 2, Step 5). The result is the value of Kcat.

Why is Hanes Woolf more accurate?

Hanes-Woolf plot is the most accurate of the three; however, its major drawback is that again neither ordinate nor abscissa represents independent values: both are dependent on substrate concentration.

What are the advantages of the Lineweaver Burk plot?

For instance; Lineweaver-Burke plot, the most favoured plot by researchers, has two distinct advantages over the Michaelis-Menten plot, in that it gives a more accurate estimate of V_max and more accurate information about inhibition. It increases the precision by linearizing the data.

How do you calculate the velocity of a reaction?

The reaction velocity (v) equals (V_max [A])/(K_m + [A]) as described by the Michaelis-Menten equation where V_max is the maximal velocity, [A] is the substrate concentration, and K_m is the Michaelis constant, or the substrate concentration at half maximal velocity.