Peptidyl acyloxymethyl ketones, previously established as potent inactivators of the lysosomal cysteine proteinase cathepsin B, were evaluated against smooth-muscle calpain, a member of the family of Ca(2+)-dependent cysteine proteinases. Only modest rates of time-dependent inhibition could be achieved, even with peptidyl affinity groups optimized for calpain and linked to a carboxylate leaving group of very low pKa [2,6-(CF3)2PhCOO-, pKa 0.58]. Selective inactivation of cathespin B versus calpain was consistently observed with this type of inhibitor. Examination of other potential inhibitors revealed a rank order of potency against calpain to be: peptidyl sulphonium methyl ketones > fluoromethyl ketones, diazomethyl ketones >> acyloxymethyl ketones, an order which differs sharply from that found for cathespin B.

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We have used solid-state 13C NMR to study the structure of the adduct resulting from the inactivation of the enzyme transglutaminase by 3-halo-4,5-dihydroisoxazoles. These inhibitors were conceived on the assumption that they would inhibit transglutaminase by attack of an enzyme active site cysteine thiol on the imine carbon of the dihydroisoxazole ring. The tetrahedral intermediate formed could then break down with the loss of the halide group and the subsequent formation of a stable imino thioether adduct. We have compared the 13C CPMAS spectra of the chloro-, bromo-, and (ethylthio)dihydroisozazole inhibitors, and the results indicate that the chemical shift of the C-3 carbon is sensitive to the nature of the heteroatom. Subtraction of the natural-abundance 13C solid-state NMR spectrum of the enzyme from that of the enzyme inactivated by C-3-labeled chlorodihydroisoxazole reveals a broad peak at 156 ppm. The chemical shift of this peak is very close to that observed for a model 3-ethylthio compound and suggests the formation of a stable imino thioether enzyme adduct. Similar results were obtained for lyophilized enzyme adducts and for frozen solutions of the enzyme adduct in the absence and presence of Ca2+. We have also compared these results with those obtained by solution NMR on an aqueous solution of the enzyme-inhibitor complex. The 13C-labeled C-3 resonance was not observed in this case.

The resonance Raman (RR) spectra of six transient dithioacylpapains have been obtained. For five of these intermediates the dithioacyl group comprises an N-acylglycine dithioester, RC(=O)NHCH,C(=S)S--, while the sixth is a benzoyloxyacetic acid dithioester, C6H5C(=O)OCH2C-(=S)S--. The R R spectra indicate that, for the great majority of molecules in each dithioacylpapain population, the vibrational properties of the dithioester center undergoing catalytic attack are markedly perturbed. By analogy with the R R spectra of models (the corresponding dithioacyl ethyl esters) and by noting the effects of amide hydrogen-deuterium exchange, the source of the perturbation is identified as an intramolecular interaction between the acyl's amide (for the five N-acylglycine dithioester groups) or ester (for the benzoyloxyacetic acid dithioester group) moiety and the dithioester that forms the dithioacylpapain linkage. The R R and ab-I t was demonstrated recently (Storer et al., 1979) that the resonance Raman (RR) spectrum of a dithioacylpapain enables us to monitor the vibrational spectrum of the bonds undergoing catalytic transformation in the enzyme's active site. During catalysis, a transient -C-C(=S)S-linkage is formed in which the substrate and the enzyme contribute the -C-C-(=S)-and -S-moieties, respectively (Lowe & Williams, 1965). The dithioester group has a A , ,near 315 nm, and by use of laser excitation in the 330-nm region, R R bands can be detected that contain contributions from the stretching motions of the C=S bond, which is undergoing nucleophilic attack in the enzyme's active site, and the C-S bond, which is being cleaved during enzymolysis. The R R spectrum of a dithioacyl-enzyme can be used to form a detailed understanding of the structure of the labile group in the active site. However, spectral interpretation was hampered initially by the lack of background spectroscopic information concerning dithioesters. Thus, we instigated a series of infrared, Raman, and RR spectroscopic studies aimed at providing the material required to interpret the R R spectra of dithioacyl-enzymes. The present paper can only be understood by reference to the spectroscopic work and we summarize the important conclusions from these studies as follows:( 1 ) Simple dithioesters of the type CH3C(=S)SCH3 have an intense H -H* electronic transition near 307 nm, and laser excitation within this transition gives rise to intense RR features near 590 and 1195 cm-l. By means of extensive isotopic substitution and a normal coordinate treatment (Teixeira-Dias et al., 1982), these bands are assigned to modes which, for the 590-cm-' feature, have high contributions from stretching motions of the C-C, C=S, and C-S bonds about the C-C-(=S)-S group and, for the 1195-cm-I feature, have a major contribution from C=S stretching coupled to a smaller contribution from C-C stretching. The vibrational spectral regions sorption spectra of the dithioacyl-enzymes are unchanged between pH 4 and 9, but below pH 4 there are marked spectral alterations which o...