# Raman spectroscopic studies of acyl cysteine proteases.

 Titre: Raman spectroscopic studies of acyl cysteine proteases. Auteur: Doran, John D. Résumé: For a series of acyl cysteine proteases structure-reactivity relationships for the deacylation step have been established using a combination of Raman and absorption spectroscopies and enzyme kinetics. The simple chromophoric ligand 5-methylthienylacryloyl- (5MTA-) and three novel peptide based substrates labelled with the 5MTA- moiety, were used to create acyl enzyme adducts with papain, cathepsin B, cathepsin L, and protein engineered mutants of cathepsins B and L. The chromophoric specific substrates, 2-Ethoxycarbamido-3-(5-methylthienyl)acryloyl ethyl ester (NHCOOEt5MTAEt), 2-(N-acetyl-L-alanine)amino-3(5-methylthienyl)acryloyl ethyl ester (Ala5MTAEt), and 2-(N-acetyl-L-phenylalanine)amino-3-(5-methylthienyl) acryloyl ethyl ester (Phe5MTAEt), were designed to utilize hydrogen bonding and hydrophobic interactions which are known to promote catalysis in papain. For cathepsins B and L removing one of the hydrogen bonding groups making up the oxyanion hole reduces the deacylation rate 3-25 fold with the tour substrates. The deacylation rate constants for the acyl cysteine protease series span a 214 fold range, from 0.07 to 15 $\times$ 10$\sp{-3}$ sec$\sp{-1}$. Using $\sp$C=O substitution it is possible to detect the acyl C=O frequency, $\nu\sb{\rm C-O}$, for each acyl cysteine protease in the Raman difference spectrum. A correlation between the $\nu\sb{\rm C-O}$ frequency and the deacylation rate constant was established, where $\nu \sb{\rm C-O}$ increases with increasing reactivity. This trend is the opposite to that seen with acyl serine proteases. The opposite trend for acyl cysteine proteases is ascribed to the strong electron polarizing forces in the active site, due principally to an $\alpha$ helix dipole, which change the hybridization about the carbonyl carbon atom. A correlation was also established between the absorption maximum, $\lambda\sb{\rm max}$, and the deacylation rate constant. As the deacylation rate increases, 214 fold across the series, $\lambda\sb{\rm max}$ red shifts from 367 to 384 nm. It is proposed that differential interactions between the $\alpha$-helix dipole in the active site of the proteins and the $\pi$-electrons in the bound chromophoric substrate are responsible for these changes in $\lambda\sb{\rm max}$, with increasing red shifts being caused by more favourable $\alpha$-helix dipole-substrate interactions in the excited electronic state. It is also proposed that similar interactions occur between the $\alpha$-helix dipole and the transition state of the acyl enzyme on the reaction pathway, giving rise to the observed differences in deacylation rates. These results demonstrate the importance of $\alpha$-helix dipoles in cysteine protease active sites in modulating enzymatic activity, and provide the first experimental evidence for the role of $\alpha$-helix dipoles in catalysis. Date: 1996 URI: http://hdl.handle.net/10393/9624

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