Peptide amido groups active

The chymotrypsin-like activity of purified 20S proteasome was measured as follows 2 pg of purified recombinant 20S proteasome or 0.5 pg of eukaryotic 20S proteasome or 1 pi of eukaryotic 26S proteasome was incubated with 20 pM fluorogenic peptide substrate, Suc-Leu-Leu-Val-Tyr-AMC (for the proteasomal chymotrypsin-like activity), for 30 min at 37°C in 200 pi of assay buffer (20 mM Tris-HCl, pH 8.0) with or without the polyphenols (5 pM). After incubation, the reaction mixture was diluted with 200 pi assay buffer followed by a measurement of the hydrolyzed 7-amido-4-methyl-coumarin (AMC) groups using a VersaFluor Fluorometer with an excitation filter of 380 mn and an emission filter of 460 nm (Bio-Rad). 

One of the closest approaches so far developed is by Bizzozero and Zweifel (118) who tried to explain in 1975 why a proline residue involved in a peptide bond is resistant to a-chymotrypsin cleavage. The objective was to find if the unreactivity of the peptide bond results from an unfavorable interaction of the methylene groups of the proline ring with the enzyme active site or whether the steric hindrance occurs upon formation of the enzyme-substrate complex or during the subsequent bond-change steps, and whether this steric hindrance is related to the ring structure of proline or simply to substitution of the amido nitrogen. In order to answer these questions, the dipeptides N-acetyl-L-phenylalanyl-L-proline amide and iV-acetyl-L-phenyl-alanyl-sarcosine amide were synthesized and their behavior as model substrates of a-chymotrypsin studied. 

---