Enzyme Kinetics

[Ladeira]

Hello people

 

I found myself dealing with this experiment...

 

In my school, we've set 7 tubes with the following quantities of sucrose at pH 5.0 (umol):

 

T1 = 0

T2 = 5

T3 = 10

T4 = 25

T5 = 37.5

T6 = 45

T7 = 50

 

So after climatization step at 37ºC, we added same quantities of invertase to each tube and took it to water bath at 37ºC for 15 minutes. Then we added DNS and took the tubes to boiling water bath so the enzyme could denature. After their cooling off, we took the tubes to spectofotometer so that T1 was the blank and the absorbance data was taken from the others.

 

For the ones who don't know, point is that this enzyme forms reductor sugars (glucose and fructose) which reduces DNS, darkening it, so we can measure the concentration of sucrose using DNS spectofotometric method.

 

I made a calibration curve using quantities of sucrose (umol) x absorbance data, so it is ok till here.

 

But my BIG problem is basically making a curve for speed of reaction x concentration of sucrose, cause I'm not being able to keep a clear connection between 'concentration of sucrose before reaction' and 'concentration of glucose after reaction' as I don't know how this enzyme works exactly. I mean, I still didn't get how I can calculate the speed of reaction (mg glicose/min) and why it is connected to concentration of sucrose on the graph.

 

Help help

 

Thanks

[Greippi]

The enzyme takes sucrose and converts it to glucose and fructose. In this reaction, one mole of sucrose produces one mole of glucose and one mole of fructose.

So therefore the rate at which the sucrose disappears (goes down in concentration) and glucose appears is indicative of the rate at which the enzyme is working.

[Ladeira]

Thanks Greippi!

 

But how can I calculate the speed of reaction and the Km (Michaelis constant which means 'concentration of sucrose' when speed of reaction is half the maximum speed)?

[Greippi]

Do you know how to read off maximal velocity (Vmax) from a michaelis-menton graph (plot rate against sucrose concentration)? Once you have Vmax, you can easily calculate Vmax/2 and thus read off Km.

or, if you're familiar with it, it's easier to do this with a lineweaver-burke plot (plot the reciprocal of the rate and substrate concentration).

 

To find the velocity (speed) of reaction for each sucrose concentration, you just need to work out how many mg of glucose are being produced per minute. Because the rate of glucose formation is the same as the rate of sucrose disappearance for the reasons I outlined above.