Chymotrypsin and trypsin specificity investigation - help please

[hlaurenc]

TL;DR

Trying to understand why the enzymes would have activity levels for synthetic substrate which contained amino acid where it does not cleave.

I recently carried out an investigation into the specificity of chymotrypsin and trypsin which I am in the process of writing up. In short, combined substrate with enzyme, incubated for 15 mins and measured at 430nm and used trendline equation to determine umol products formed per minute.

N-benzoyl-DL-arginine-p-nitroanilide (BAPNA) and N-succinyl-L-phenylalanine-p-nitroanilide (NSPLN) were the synthetic substrates used to be recognised by trypsin and chymotrypsin, respectively.

Results came back as expected; however, when doing the opposing combinations (chymotrypsin-BAPNA and trypsin-NSLPN), activity levels were recorded. I have been racking my brains and books for hours and hours and can't seem to understand how there would be any activity levels as trypsin doesn't cleave at phenylalanine, nor chymotrypsin at arginine.

I'm a first year 'mature' student undergraduate returning to university with no science background - this is way above me! If someone can help point me in the right direction, would be much appreciated.

Thank you in advance :)

 

[Ygggdrasil]

A few things to consider:
1) Enzymes are catalysts that accelerate reactions that occur slowly in the absence of enzyme (in this case, hydrolysis of the substrates). If hydrolysis of the substrate can occur in the absence of enzyme, surely it can also occur in the presence of the wrong enzyme. Did you perform a negative control reaction and measure the rate of hydrolysis in the absence of enzyme? How does the rate of reaction for the negative control compare to the rates measured for the wrong enzyme?

2) While enzymes are often highly specific, they can sometimes exhibit non-specific activities (e.g. cleavage of the wrong substrate). Furthermore, the substrates you are using in the experiment are not the natural substrates of the enzymes, so the enzymes did not evolve to distinguish between Arg and Phe in the context of these artificial molecules.

 

[hlaurenc]

A few things to consider:
1) Enzymes are catalysts that accelerate reactions that occur slowly in the absence of enzyme (in this case, hydrolysis of the substrates). If hydrolysis of the substrate can occur in the absence of enzyme, surely it can also occur in the presence of the wrong enzyme. Did you perform a negative control reaction and measure the rate of hydrolysis in the absence of enzyme? How does the rate of reaction for the negative control compare to the rates measured for the wrong enzyme?

2) While enzymes are often highly specific, they can sometimes exhibit non-specific activities (e.g. cleavage of the wrong substrate). Furthermore, the substrates you are using in the experiment are not the natural substrates of the enzymes, so the enzymes did not evolve to distinguish between Arg and Phe in the context of these artificial molecules.

Thanks for your reply - much appreciated.
1) we did not perform a negative control, no. This may be a silly question: how would hydrolysis occur without the presence of an enzyme? Surely, they'd be no reaction.
2) thanks - noted.

Just for information - the below figures are umol of product formed per minute. The two in question, are relatively low; however, I was under the impression it'd be zero.

Trypsin/BAPNA	0.038​
Trypsin/NSLPN	0.006​
Chymotrypsin/BAPNA	0.011​
Chymotrypsin/NSLPN	0.028​

 

[Borek]

how would hydrolysis occur without the presence of an enzyme? Surely, they'd be no reaction.

Nope. All hydrolysis needs to happen is the presence of water. Everything else is kinetics - low/high pH speeds the process up, enzymes speed the process up and so on.

 

[hlaurenc]

I knew that :) :) :)

 

[Borek]

Well, what you wrote suggested the opposite, so I preferred to clarify