Spectroscopic Probes Incorporated into Specific Substrates and Inhibitors of Papain for the Study of Acylenzyme Formation

α‐β‐Unsaturated aromatic amino acids such as β‐phenyldehydroalanine (PDA) or β‐styryldehydroalanine (SDA) can serve as spectroscopic probes of acylpapain formation. The spectral changes observed upon acylation of the enzyme are a large red shift of 49 nm (277 nm to 326 run) in the case of PDA, and of 59 run (318 nm to 377 nm) in the case of SDA. Using these chromophores, the following specific substrates were synthesized: Ac‐Phe‐PDA‐OEt, Ac‐Phe‐PDA‐NH 2 , Ala‐Ala‐Phe‐SDA‐OMe, and Ala‐Ala‐Phe‐SDA‐NH 2 . Similarly, the specific competitive inhibitors Ac‐Phe‐PDA ( \documentclass{article}\pagestyle{empty}\begin{document}$$ {\rm (\bar K}_{\rm i} = 1.9 \times 10^5 {\rm M}^{ - 1} ) $$\end{document} ) and Ala‐Ala‐Phe‐SDA ( \documentclass{article}\pagestyle{empty}\begin{document}$$ {\rm (\bar K}_{\rm i} = 3.5 \times 10^4 {\rm M}^{ - 1} ) $$\end{document} ) were prepared. By this method acylenzyme formation by inhibitors and substrates is demonstrated qualitatively. To make possible the independent observation of the physical binding of substrates and inhibitors to papain, an additional chromophore p ( p ′‐dimethylaminophenylazo)‐phenylalanine (DAP) was introduced as a second probe into the peptide. The spectrum of this chromophore is known to depend on its environment. Thus, binding to papain of the competitive inhibitor Ala‐Ala‐DAP‐SDA ( \documentclass{article}\pagestyle{empty}\begin{document}$$ {\rm (\bar K}_{\rm i} = 4 \times 10^5 {\rm M}^{ - 1} ) $$\end{document} ) and its corresponding amide and methylester can be observed qualitatively and quantitatively by following either the increase in absorption at 480 nm or the decrease at 550 nm. The simultaneous formation of the acylenzyme can be followed at 377 nm. The extent of acylenzyme formation was found to decrease in a sigmoidal way with increasing pH, with a transition point around pH 5.5.