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in CMVP by comparing the non-covalent binding affinities of the inhibitor to CMVP mutants A144L, S132A and E122V/A144G. CMVP A144L represents wild type CMVP (with respect to enzymatic activity) and is the reference protease for the binding studies. Serine residue 132 is the active site residue predicted to be responsible for nucleophilic attack of the DFMK class of inhibitors and is likely to be essential for the non-covalent binding of these inhibitors to CMVP. Mutation of S132 to an alanine residue in CMVP S132A inactivates the protease and is likely to prevent binding of the DFMK and TFMK class of inhibitors to the protease. Mutation of E122 to a valine residue in CMVP E122V/A144L has also been shown to destroy the enzymatic activity of CMVP [25] but the effect on inhibitor binding is not known.

The ESI mass spectrum for inhibitor TFMK (MW 545 Da), prior to passage through a GPC spin column (Fig. 2.21A), exhibits the characteristic molecular ions (M+H)1+, (M+H2O+H)1+, (M+H2O+Na)1+ and (M+H2O+K)1+ at m/z 546.3, 564.3, 586.3 and 602.1, respectively, as well as one fragment ion [M-C(CH3)3+2H]1+ at m/z 490.2. In a control study, the ESI mass spectrum for the spin column eluate of pure TFMK (not illustrated) shows the absence of TFMK in the low mass region from m/z 415 to m/z 620. The ESI mass spectra of the spin column eluates of TFMK incubated with the CMVPs A144L, S132A and E122V/A144G (each originally prepared at a molar ratio of CMVP:TFMK of 1:40) are illustrated in Fig. 2.21B, C, D, respectively. All the spectra are normalized for the abundance of pure TFMK (in Fig. 2.21A) that corresponds to a @1:1 molar ratio of CMVP:TFMK. (The ion distributions for the proteases occur over the m/z range of 750 to 1200 and are not depicted.) In a control study, the ESI mass spectrum of pure CMVP A144L (not illustrated) shows the presence of background peaks at m/z 538 and m/z 598, which correspond to solvent complexes of acetic acid (indicated with stars in Fig. 2.21B, C, D). From these data, it is evident that TFMK coelutes with CMVP A144L (in a CMVP:TFMK molar ratio eb5! (A) TFMK (MW 545)

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