Characterization of the Human Cytomegalovirus Protease As an Induced-Fit Serine Protease and the Implications to the Design of Mechanism-Based Inhibitors

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• 作者：Steven R. LaPlante ; Pierre R. Bonneau ; Norman Aubry ; Dale R. Cameron ; Robert Dé ; ziel ; Chantal Grand-Maî ; tre ; Cé ; line Plouffe ; Liang Tong ; and Stephen H. Kawai
• 刊名：Journal of the American Chemical Society
• 出版年：1999
• 出版时间：April 7, 1999
• 年：1999
• 卷：121
• 期：13
• 页码：2974 - 2986
• 全文大小：289K
• 年卷期：v.121,no.13(April 7, 1999)
• ISSN：1520-5126

文摘

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 IC₅₀ range) inhibitors of the human cytomegalovirus (HCMV) protease, theiractivated carbonyl analogues are >1000-fold more potent (e.g., trifluoromethyl ketone 3, IC₅₀ = 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.