A A

of @1:1), does not coelute with CMVP S132A and only very slightly coelutes with CMVP E122V/A144G (in a molar ratio of CMVP:TFMK of 1:<0.05).

The specificity of these coelution results are consistent with S132 as the active site nucleophile of CMVP and demonstrates that this screening methodology can detect specific binding of inhibitors to CMVP. From the crystal structure, the active site residues of CMVP are S132, H157 and H63 [26-29]. Mutation of S132 is therefore predicted to prevent binding to CMVP of active site directed inhibitors such as TFMK. A stabilized (reversible) hemiacetal protease-inhibitor complex is believed to have formed with CMVP A144L, as schematically illustrated in Fig. 2.21 (bottom). However, when S132 is replaced with a lipophylic amino acid residue such as alanine, the active site is destroyed and the mutated protease is incapable of tightly binding to the inhibitor. E122 is quite far from the active site and is involved in a salt bridge within the protease. Thus, the conformation of protease mutant E122V may be significantly different from the wild-type CMVP due to disruption of the salt bridge. CMVP E122V is enzymatically inactive and this study shows (Fig. 2.21D) that this mutant protease cannot bind significantly to the TFMK inhibitor.

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