EVIDENCE OF A CONFORMATIONAL CHANGE IN THE HUMAN CYTOMEGALOVIRUS PROTEASE UPON BINDING OF PEPTIDYL-ACTIVATED CARBONYL INHIBITORS

A series of N-gamma,N-gamma-dimethylasparagyl-L-alanyl-derived inhibit ors (trifluoromethyl ketone 1, pentafluoroethyl ketone, 2, methyl keto ne 3, and alpha-ketoamide 4, with respective K-I values of 1.1, 0.1, 2 100, and 0.2 mu M) of the human cytomegalovirus protease were used to study the effect of binding of peptidyl inhibitors on the intrinsic fl uorescence and CD properties of the enzyme. In the presence of saturat ing concentrations of compounds 1, 2, and 4, an identical blue shift i n the fluorescence maximum of the enzyme upon specific tryptophan exci tation was observed relative to that of the free protease. In the case of the methyl ketone 3, whose inhibition of the enzyme does not invol ve formation of a covalent adduct as evidenced by C-13 NMR studies of carbonyl-labeled inhibitors, the blue shift in the emission was also o bserved. For both compounds 1 and 2 which exhibit slow-binding kinetic s, the observed rate constants for the slow onset of inhibition of sub strate hydrolysis correlate well with the k(obs) values of the time-de pendent change in the emission spectra, Studies employing a double mut ant of HCMV protease Ala143Gln/Trp42Phe identified Trp-42 as the princ ipal fluorescence reporter. Taken together with information provided b y our recent elucidation of the crystallographic structure of the enzy me [Tong, L., Qian, C., Massariol, M.-J., Bonneau, P. R., Cordingley, M. G., & Lagace, L. (1996) Nature 383, 272], these observations are co nsistent with the inhibition of HCMV protease by peptidyl ketones invo lving a conformational change of the protease. A mechanism involving a k,, limited by dehydration of the hydrated species, followed by rapid ligand binding and a conformational change prior to covalent adduct f ormation, is proposed for activated inhibitors such as 1 and 2.