Evidence of a conformational change in the human cytomegalovirus protease upon binding of peptidyl-activated carbonyl inhibitors.
A series of N-tert-butylacetyl-l-tert-butylglycyl-l-Ngamma, Ngamma-dimethylasparagyl-l-alanyl-derived inhibitors (trifluoromethyl ketone 1, pentafluoroethyl ketone, 2, methyl ketone 3, and alpha-ketoamide 4, with respective KI values of 1.1, 0.1, 2100, and 0.2 microM) of the human cytomegalovirus protease were used to study the effect of binding of peptidyl inhibitors on the intrinsic fluorescence and CD properties of the enzyme. In the presence of saturating concentrations of compounds 1, 2, and 4, an identical blue shift in the fluorescence maximum of the enzyme upon specific tryptophan excitation was observed relative to that of the free protease. In the case of the methyl ketone 3, whose inhibition of the enzyme does not involve formation of a covalent adduct as evidenced by 13C NMR studies of carbonyl-labeled inhibitors, the blue shift in the emission was also observed. For both compounds 1 and 2 which exhibit slow-binding kinetics, the observed rate constants for the slow onset of inhibition of substrate hydrolysis correlate well with the kobs values of the time-dependent change in the emission spectra. Studies employing a double mutant of HCMV protease Ala143Gln/Trp42Phe identified Trp-42 as the principal fluorescence reporter. Taken together with information provided by our recent elucidation of the crystallographic structure of the enzyme [Tong, L., Qian, C., Massariol, M.-J., Bonneau, P. R., Cordingley, M. G., & Lagacé, L. (1996) Nature 383, 272], these observations are consistent with the inhibition of HCMV protease by peptidyl ketones involving a conformational change of the protease. A mechanism involving a kon limited by dehydration of the hydrated species, followed by rapid ligand binding and a conformational change prior to covalent adduct formation, is proposed for activated inhibitors such as 1 and 2.
Bonneau
P R
PR
Department of Biochemistry, Bio-Méga Research Division, Boehringer Ingelheim (Canada) Ltd., Laval, Québec H7S 2G5, Canada.
Grand-Maître
C
C
Greenwood
D J
DJ
Lagacé
L
L
LaPlante
S R
SR
Massariol
M J
MJ
Ogilvie
W W
WW
O'Meara
J A
JA
Kawai
S H
SH
D003587:Cytomegalovirus / Q000737:chemistry* / Q000201:enzymology; D010450:Endopeptidases / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D007659:Ketones / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D009682:Magnetic Resonance Spectroscopy; D011480:Protease Inhibitors / Q000737:chemistry* / Q000378:metabolism / Q000494:pharmacology; D011487:Protein Conformation; D012697:Serine Endopeptidases
D007659:Ketones; D011480:Protease Inhibitors; D010450:Endopeptidases; D012697:Serine Endopeptidases; C071910:assemblin