The conformational properties of the
N-
tert-butylacetyl-
L-
tert-butylglycyl-
L-
N,
N-dimethylasparagyl-
L-alanyl methyl ketone (MK)
1 and its terminal
N-isopropylacetyl analogue
2 were investigated. Whereasthese compounds are weak (mM IC
50 range) inhibitors of the human cytomegalovirus (HCMV) protease, theiractivated carbonyl analogues are >1000-fold more potent (e.g., trifluoromethyl ketone
3, IC
50 = 1.1
M). Acombination of NMR techniques demonstrated that MK
2 exists in solution as a relatively rigid and extendedpeptide structure and that the bulky side chains, notably the P3
tert-butyl group, greatly contribute to maintainingthis solution conformation. Furthermore, transferred nuclear Overhauser effect (TRNOE) studies provided anenzyme-bound conformation of MK
2 that was found to be similar to its free solution structure and comparesvery well to the X-ray crystallographic structure of a related peptidyl inhibitor complexed to the enzyme. Thefact that ligands such as MK
2 exist in solution in the bioactive conformation accounts, in part, for the observedinhibitory activity of activated ketone inhibitors bearing comparable peptidyl sequences. Comparison of theX-ray structures of HCMV protease apoenzyme and that of its complex with a related peptidyl
-ketoamideinhibitor allowed for a detailed analysis of the previously reported conformational change of the enzyme uponcomplexation of inhibitors such as
1 and
3. The above observations indicate that HCMV protease is a novelexample of a serine protease that operates by an induced-fit mechanism for which complexation of peptidylligands results in structural changes which bring the enzyme to a catalytically active (or optimized) form.Kinetic and fluorescence studies are also consistent with an induced-fit mechanism in which a considerableproportion of the intrinsic ligand-binding energy is used to carry out the conformational reorganization of theprotease. Issues related to the rational design of both mechanism- and nonmechanism-based inhibitors of HCMVprotease, notably in light of the peptidyl ligand-induced optimization of its catalytic functioning, are discussed.