Enzyme kinetics Flashcards
Explain how enzyme activity may be measured using spectrophotometry
If the one of the product of a reaction has a significant absorbance at a wavelength where the substrate does not, then measuring the absorbance using spectrophotometry can be used to track the progress of the reaction
Explain how experimental values of reaction velocity at different substrate concentrations can be used to derive Km and Vmax for an enzyme
By plotting the absorbance against time for each concentration, the initial rate can be determined for each. After converting this to a rate involving volume instead of absorbance using the Beer-Lambert law, 1/S can be plotted on the x axis and 1/V0 on the y axis. The x intercept is equal to -1/Km and the y intercept 1/Vmax
Explain the effects of competitive and non-competitive inhibitors on Km and Vmax
Competitive inhibition: no effect on Vmax because if completely outcompeted by genuine substrate, maximum effect of enzyme unchanged, yet Km increased because genuine substrate must compete with inhibitor
Non-competitive inhibition: no effect on Km but decreased Vmax because some active sites have been denatured by allosteric inhibitor bindin
Describe the Lineweaver-Burk Plot
Since Vmax can be difficult to determine with accuracy directly, Lineweaver and Burk devised a method of plotting the data as reciprocals. The initial velocity (V0) of a series of reactions are calculated for a range of substrate concentrations [S]. As [S] increases 1/[S] tends towards zero, so that 1/Vmax is determined as the value of 1/V0 when 1/[S] = 0 (i.e. the intercept on the axis). Extrapolating the straight line graph until it crosses the other axis gives a 1/[S] value of –1/KM
What is meant by Km
According to Michaelis-Menten kinetics, Vmax is the maximum enzyme velocity which is approached asymptotically as [S] becomes large. KM, also known as the Michaelis constant, is the substrate concentration at which the rate of reaction is exactly ½ Vmax
How can you convert absorbance to concentration to rate
A = ε ́ c ́ l, where l = 1 cm (the length of the light path through the cuvette), you can simply rearrange the equation to give: c = A/ ε. For example, one group found that with the 4.0 mM GPNA solution the rate of change in A410 was 0.19 absorbance units per minute. This implies a rate of change in the concentration of p-nitroaniline (Vo) of 0.19/8.8 = 0.022 μmol.ml-1.min-1 and a value of 1/V of 46.3 μmol.ml -1.min-1.
What do the intercepts define
The intercept on the x axis defines –1/KM.
The intercept on the y axis defines 1/Vmax.
Describe the errors in this practical
In this experiment there can be quite a lot of error in determining the intercepts, and taking the reciprocal has the effect of magnifying errors in the measured rate, especially at the lower values of [S] such as the 0.5mM GPNA.
