Recent reports have demonstrated the potential of monocyclic
-lactam derivatives as inhibitors ofhuman cytomegalovirus (HCMV) protease. Investigation of the mechanism of inhibition by NMR and massspectrometry has revealed the presence of an acylenzyme intermediate suggesting that
-lactams are hydrolyzedby the enzyme and cause inhibition by competing with substrate. The potential of a fluorogenic
-lactamderivative for convenient kinetic characterization of this mechanism has been evaluated using 4
S-(4-methylumbelliferone)-3
R-methylazetidin-2-one-1-carboxylic acid (4-methylpyridyl) amide
(1). Upon acylationof the enzyme, the fluorescent umbelliferone moiety is released, allowing for continuous monitoring of thehydrolytic process. Examination of a series of progress curves by num
erical analysis has provided valuableinformation on acylation and deacylation rates which relate to the IC
50 values observed for
-lactams. Moreimportantly the potential of compound
1 as an active site titrating agent for HCMV protease has been exploited,and a simple protocol for rapid determination of active enzyme is described. The data are consistent with theHCMV protease dimer being composed of two functional active sites. This titrating agent represents an importanttool that should significantly facilitate the characterization of this novel enzyme.
