PRACTICAL 7:THE KINETIC BEHAVIOUR OF ENZYMES

BIOL I 100-3

In this practical it was studied the effect of changing the amount of enzyme and substrate in an enzyme catalysed reaction. The enzyme used was trypsin, and BAPNA, which is an artificial substrate molecule, was used as the substrate.
The rate of the enzyme catalyzed reaction is expressed as amount of substrate converted to product per minute, in this practical a spectrophotometer was used, the amount of UV radiation absorbed is equal to the amount of substrate produced. Therefore, the unit of rate used is the change in absorbance per minute Abs/min. This will be calculated by subtracting the absorbance at 30s (A1) from the Absorbance at 90s (A2). It is important that our A1 is a value is in the first seconds of reaction (15 or 30 s) because this way the initial velocity will be obtained, the initial velocity is the instant before all of the substrate is converted into product and the curve levels off
The following table of results was obtained when the concentration of BAPNA was increased:

A plot could be drawn for each of the concentrations, time would be on the X axis and the absorbance on the Y axis, this way a curve would be obtained for each of the concentrations and the initial velocity would be calculated for each curve by drawing the tangent line for each curve and then drawing a vertical line through the point of initial velocity in the top most curve, wherever this vertical line intersects the other curves would be the initial velocity. These values would be used on the Y axis on a second graph together with the substrate concentration in the X axis to give a concentration plot. This would probably be a more accurate way of obtaining the initial velocity. However, for simplicity because otherwise 5 graphs would be needed, the Abs/min was calculated subtracting the absorbance value at 90s and the absorbance value at 30s, thus obtaining the initial velocity. The following concentration plot was obtained when the amount of substrate was increased:

It can be observed how the velocity increases with the increase in substrate, but as the substrate becomes larger the increase in rate becomes smaller. The substrate an enzyme have to collide forming an enzyme-substrate complex to form a product, at high substrate concentrations more enzyme-complex are found which increases the initial velocity. However, if the substrate concentration is increased at this point when most of the enzyme is present as enzyme-complex state, the reaction rate will level off as it reaches a maximal velocity.
The following table was obtained when the concentration of enzyme was increased:

The negative values obtained in the first measurements were probably due to the fact that the cuvette was introduced too quickly in the spectrophotometer and the reaction had not started.


The following concentration plot was obtained using the data on the table:

It can be observed how the initial velocity increases briefly to start decreasing rapidly. This is due to the fact that the same amount of substrate is present, but more enzyme is added, therefore, more enzyme-substrate complex are formed and the substrate is transformed into product rapidly. However, once the substrate is over only enzymes are present, thus the rate of reaction starts to decrease.