Chymotrypsin absorption spectra

The initial evidence for the formation of an acyl-enzyme ester intermediate came from studies of the kinetics with which chymotrypsin hydrolyzed analogs of its normal polypeptide substrates. The enzyme turned out to hydrolyze esters as well as peptides and simpler amides. Of particular interest was the reaction with the ester p-nitrophenyl acetate. This substrate is well suited for kinetic studies because one of the products of its hydrolysis, p-nitrophenol, has a yellow color in aqueous solution, whereas p-nitrophenyl acetate itself is colorless. The change in the absorption spectrum makes it easy to follow the progress of the reaction. When rapid-mixing techniques are used to add the substrate to the enzyme, an initial burst of p-nitrophenol is detected within the first few seconds, before the reaction settles down to a constant rate (fig. 8.8). The amount of p-nitrophe-... 

When racemic 173 (R = Me) was hydrolyzed in the presence of chymo-trypsin, the resulting optically active acid 173 (R = H) exhibited an ORD absorption spectrum characteristic of L-phenylalanine the starting ester possessed an axially oriented carbomethoxy group.338-362 Atropisomerism and conformational asymmetry of a precisely definable nature in a substrate are therefore recognized by chymotrypsin. X-ray diffraction studies confirmed that the chymotrypsin-active isomer has an axial ester moiety in the solid state, and that the ester mutarotates in solution to a CD-inactive isomer, whose ester group is in the equatorial position.363... 

The second modified flavin of natural origin to be discovered was 8a-S-cysteinyl-FAD, the coenzyme of monoamino oxidase from liver and kidney outer mitochondrial membranes. Taking their departure from investigations of Yasunobu (8J) and Hellerman (SO), which indicated the presence of covalently bound flavin in preparations of this enzyme, Singer and his group (85, 185) isolated the flavinyl peptide by degradation of MAO with trypsin-chymotrypsin and identified cysteine as the amino acid residue bound next to the flavin moiety (184). The absorption spectrum of the flavin peptide from monoamino oxidase is readily differentiated from that of riboflavin by a hypsochromic shift of the second absorption band (360 nm, compare with 372 for riboflavin), in the neutral oxidized state (44, 184). It is similar to that of 8a-histidyl-riboflavin in the cationic state in that the band centered around 400 nm (abs. max. 375 nm, shoulder at 410 nm) is partially resolved. The fluorescence emission (4, 30) is only 10% of that of riboflavin, but oxidation with peracids raises it to 90% of riboflavin emission. 

Detection of the intermediate is possible if it has a spectrum sufficiently different from that of the enzyme. The cinnamoyl chymotrypsin intermediate is characterised by a UV maximum at 292 nm the acyl papain intermediate JV-benzoylaminothionacetyl papain has a UV maximum at 313 nm. The UV absorptions of the reactions catalysed by papain and chymotrypsin wax and wane in the presence of substrate giving rise to these intermediates. 

---