SOLID-STATE C-13 NMR-STUDY OF A TRANSGLUTAMINASE INHIBITOR ADDUCT

We have used solid-state C-13 NMR to study the structure of the adduct resulting from the inactivation of the enzyme transglutaminase by 3-h alo-4,5-dihydroisoxazoles. These inhibitors were conceived on the assu mption that they would inhibit transglutaminase by attack of an enzyme active site cysteine thiol on the imine carbon of the dihydroisoxazol e 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 C-13 CPMAS spectra of th e chloro-, bromo-, and (ethylthio)dihydroisoxazole inhibitors, and the results indicate that the chemical shift of the C-3 carbon is sensiti ve to the nature of the heteroatom. Subtraction of the natural-abundan ce C-13 solid-state NMR spectrum of the enzyme from that of the enzyme inactivated by C-3-labeled chlorodihydroisoxazole reveals a broad pea k at 156 ppm. The chemical shift of this peak is very close to that ob served 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 thes e results with those obtained by solution NMR on an aqueous solution o f the enzyme-inhibitor complex. The C-13-labeled C-3 resonance was not observed in this case.