Help! substrate specificity experiment: Trypsin and Chymotrypsin using BAPNA&NSLPNWatch
I was wondering whether anyone could help me understand why during a specificity experiment on the serine proteases Trypsin and Chymotrypsin using the synthetic substrates of BAPNA and NSLPN (n-succinyl-l-phenylalanine-p-nitroanilide) a product was formed when:
Trypsin was mixed with NSLPN and Chymotrypsin was mixed with BAPNA
when according to their individual specificites Trypsin should only be able to cleave the peptide bonds of BAPNA and Chymotrypsin should only be able to cleave NSLPN?
Any help would be greatly appreciated
I am not a biochemist, so my knowledge of the subject is only from studying biochemistry in medicine - please ignore my answer if it seems incorrect; however, I am going to mention a couple of points that will hopefully help you work out the answer to your Q:-
Firstly, the proteases [particularly the endopeptidases] secreted by the human gastro-intestinal system have predilections for cleaving peptide bonds next to certain types of aminio acids, but MAY NOT BE COMPETELY SPECIFIC TO ONE a.a., but rather prefer peptide bonds next to certain a.a.-s; trypsin has the greatest specificity; it cleaves peptide bonds on the carboxyl side of arginine and lysine [both of which have positively charged residues].
Chymotrypsin cleaves peptide bonds next to bulky hydrophobic residues; I suppose these would be aromatic a.a.-s like phenylalanine and tyrosine [with the water-hating benzene ring]. BUT IT ALSO CLEAVES bonds next to histidine, methionine and leucine, albeit more slowly.
Secondly, [and I have not heard of NSLPN or BAPNA], but the presence of a proline residue after the carboxyl end of the preferred a.a.-s of both trypsin and chymotrypsin prevents these enzymes from producing a cleavage. Could this be the case with either/both of these compounds that you are using (NSLPN and/or BAPNA)?
Hope this helps (apologies if you already know all this!).
Is there anything to indicate that BAPNA could have its hydrophobic moiety nestly into chymotrypsin’s hydrophobic pocket, facilitating the specific catalytic orientation required for nucleophilic attack on the carboxylic O?
What about the oxyanion hole that is housed by O during the formation of the tetrahedral transition state and its subsequent collapse and the formation of the acyl-enzyme thereafter?