Evidence of a Conformational Change in the Human Cytomegalovirus Protease upon Binding of Peptidyl-Activated Carbonyl Inhibitors
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- 作者：Pierre R. Bonneau ; Chantal Grand-Maî ; tre ; Daniel J. Greenwood ; Lisette Lagacé ; Steven R. LaPlante ; Marie-José ; e Massariol ; William W. Ogilvie ; Jeff A. O'Meara ; and Stephen H. Kawai
- 刊名：Biochemistry
- 出版年：1997
- 出版时间：October 14, 1997
- 年：1997
- 卷：36
- 期：41
- 页码：12644 - 12652
- 全文大小：387K
- 年卷期：v.36,no.41(October 14, 1997)
- ISSN：1520-4995
文摘
A series ofN-tert-butylacetyl-L-tert-butylglycyl-L-N
,N-dimethylasparagyl-L-alanyl-derivedinhibitors (trifluoromethyl ketone 1, pentafluoroethylketone, 2, methyl ketone 3, and 
-ketoamide4,with respective KI values of 1.1, 0.1, 2100, and0.2 
M) of the human cytomegalovirus protease wereused to study the effect of binding of peptidyl inhibitors on theintrinsic fluorescence and CD propertiesof the enzyme. In the presence of saturating concentrations ofcompounds 1, 2, and 4, an identicalblueshift in the fluorescence maximum of the enzyme upon specifictryptophan excitation was observed relativeto that of the free protease. In the case of the methyl ketone3, whose inhibition of the enzyme does notinvolve formation of a covalent adduct as evidenced by 13CNMR studies of carbonyl-labeled inhibitors,the blue shift in the emission was also observed. For bothcompounds 1 and 2 which exhibit slow-binding kinetics, the observed rate constants for the slow onset ofinhibition of substrate hydrolysis correlatewell with the kobs values of the time-dependentchange in the emission spectra. Studies employing adouble mutant of HCMV protease Ala143Gln/Trp42Phe identified Trp-42 asthe principal fluorescencereporter. Taken together with information provided by our recentelucidation of the crystallographicstructure of the enzyme [Tong, L., Qian, C., Massariol, M.-J.,Bonneau, P. R., Cordingley, M. G., &Lagacé, L. (1996) Nature 383, 272], theseobservations are consistent with the inhibition of HCMVproteaseby peptidyl ketones involving a conformational change of the protease.A mechanism involving a konlimited by dehydration of the hydrated species, followed by rapidligand binding and a conformationalchange prior to covalent adduct formation, is proposed for activatedinhibitors such as 1 and 2.
,N-dimethylasparagyl-L-alanyl-derivedinhibitors (trifluoromethyl ketone 1, pentafluoroethylketone, 2, methyl ketone 3, and -ketoamide4,with respective KI values of 1.1, 0.1, 2100, and0.2 M) of the human cytomegalovirus protease wereused to study the effect of binding of peptidyl inhibitors on theintrinsic fluorescence and CD propertiesof the enzyme. In the presence of saturating concentrations ofcompounds 1, 2, and 4, an identicalblueshift in the fluorescence maximum of the enzyme upon specifictryptophan excitation was observed relativeto that of the free protease. In the case of the methyl ketone3, whose inhibition of the enzyme does notinvolve formation of a covalent adduct as evidenced by 13CNMR studies of carbonyl-labeled inhibitors,the blue shift in the emission was also observed. For bothcompounds 1 and 2 which exhibit slow-binding kinetics, the observed rate constants for the slow onset ofinhibition of substrate hydrolysis correlatewell with the kobs values of the time-dependentchange in the emission spectra. Studies employing adouble mutant of HCMV protease Ala143Gln/Trp42Phe identified Trp-42 asthe principal fluorescencereporter. Taken together with information provided by our recentelucidation of the crystallographicstructure of the enzyme [Tong, L., Qian, C., Massariol, M.-J.,Bonneau, P. R., Cordingley, M. G., &Lagacé, L. (1996) Nature 383, 272], theseobservations are consistent with the inhibition of HCMVproteaseby peptidyl ketones involving a conformational change of the protease.A mechanism involving a konlimited by dehydration of the hydrated species, followed by rapidligand binding and a conformationalchange prior to covalent adduct formation, is proposed for activatedinhibitors such as 1 and 2.