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burst

by Dr. P. L. Birch (Submitted: 06/20/2005)

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WHAT IS IT?

This is a simulation of Hartley's "burst" experiment which provided evidence for the biphasic (ping-pong) nature of the catalytic reaction of chymotrypsin and the other serine proteases. Chymotrypsin catalyses the hydrolysis of peptide bonds in proteins using the following biphasic mechanism:

E-OH + R-CO-NH-R' --> E-CO-R + R'-NH2 (Acylation phase)
E-CO-R + H2O --> E-OH + R-COOH (Deacylation phase)

Using a normal substrate the first phase is slower than the second phase as it involves the cleavage of a very stable peptide (amide) bond. Hartley's experiment made use of an artificial substrate, p-nitrophenyl acetate (pNPA). The bond broken by the enzyme with this substrate is the very unstable phenolic ester bond and this reverses the rate balance between the two phases so that the acylation phase is faster than the deacylation phase.

On adding enzyme to a solution of pNPA every enzyme would very rapidly undergo the acylation phase, releasing one molecule of the product of that reaction (product 1) which is a yellow-coloured paranitrophenol, whose absorbance can be measured on a spectrophotometer. This produces a burst of colour whose absorbance is directly proportional to the enzyme concentration. The enzymes can only react with further substrates by releasing the second product which will take place at the much slower rate of the deacylation phase. The result of this is that on first addition of the reaction product 1 is generated, and absorbance due to it is observed, at the rate of the acylation phase. This occurs until all enzyme molecules have been acylated. Further generation of product 1 is dependent on the regeneration of free enzyme which occurs at the rate of the deacylation phase. There is an effective "separation" of the two phases showing a biphasic graph.

If a "normal" substrate is used, in which the acylation phase is slower than the deacylation phase, this biphasic effect will not be seen. The initial release of product 1 would be at the rate of the slow acylation phase. The acylated enzyme would then be relatively rapidly regenerated by deacyltion but this would not be observed directly as it is absorbance due to product 1 which is being observed. The regenerated enzyme can react with another substrate and generated another product at the rate of the acylation phase. The graph would show a single phase at the rate of the acylation phase.

HOW IT WORKS

Uses some of the basic ideas of the enzyme kinetics module in the library. More details are commented in the procedures page.

HOW TO USE IT

By default the model starts with p-nitrophenyl acetate (pNPA) as the substrate and the number of enzyme molecules to be added set to fifty. These can be changed by using the chooser button and the slider. I suggest that you leave at the default setting to start with. The output box, to the right of the screen lists some basic information about the chosen substrate, which will be changed, if you alter the substrate, after you press the Setup button.

Press the setup button. This will add 500 substrate molecules to the model. They'll appear as cyan coloured double blobs. You can hide them using the hide-substrate? switch, but this will not come into effect until you press the Go button.

Press Go. Nothing much will happen as there is no enzyme present. If you look carefully at the graph window you'll see the line moving along the bottom of the graph. Press the Add enzyme button. This immediately adds the requested number of enzyme molecules to the reaction mixture. Look at the graph. The red line, which represents the amount of enzyme mixture, rises immediately at the point of addition and then stays unchanged. The green line, the amount of product 1, almost immediately rises to the same level as the enzyme graph (the initial burst) and then continues rising more slowly ( the rate of deacylation. The size of the burst will be printed at the bottom of the output window. Compare this to the amount of enzyme used.

If you look at the graphics window you'll be able to see the enzyme's (red squares), product 1 molecules (small yellow circles) and product 2 molecules (small blue squares). You can slow the reaction down using the slider at the top left of the graphics window, and hide the substrate, if it's too obtrusive, using the hide button.

Change to the other substrate (a generic peptide), press Setup and repeat the above excersize. Compare the results with the different substrates.

THINGS TO NOTICE

THINGS TO TRY

Try varying the amount of enzyme added and look at the size of the burst.

Try using only one enzyme molecule so that you can concentrate on it. Run the graphic fairly slowly, using the slider, and hide the substrates. You'll be able to see the speed of binding to and release from, the enzyme of the substrate and the two products. Substrate bound to the enzyme is visible even when the hide-substrate button is on. Compare these binding/release activities using the different substrates.

EXTENDING THE MODEL

It would be easy to add more substrate variations with differing Ka/Kd values or perhaps introduce slider to let the user vary Ka/Kd values themselves.

You could set up a calculation of the rate of product 1 generation and compare these with Ka/Kd values.

NETLOGO FEATURES

RELATED MODELS

Enzyme Kinetics in the modules library

CREDITS AND REFERENCES

Copyright: Dr. P. L. Birch - University of Paisley

Permission is granted to copy, distribute and adapt this model for any non-comercial use as long as this copyright notice is maintained.

A Web version is available at:

http://www-biol.paisley.ac.uk/courses/mechenz/serprot/burst.html

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