Fluorohydrolase preparation

CCMPs have been prepared, which have the ability to catalyze the hydrolysis of organofluorophosphates such as diisopropylphosphorofluoridate and phenylmethylsulfonylfluoride, using several different starting proteins. In addition, semisynthetic fluorohydrolases prepared by the conformational modification of RNase with HMPA were crosslinked with diimidates of chain lengths from Ci to Ce to determine the optimum crosslink for the maximum fluorohydrolase activity. 

Figure 1. Fluorohydrolase activity of CCMPs prepared from RNase as a function of the dimension of the cross-linking span of the diimidate reagent. Activity was measured at pH 7.4 and 30 C using both DFP ( ) and PMSF (o) as substrates. Each activity is the average of two independent assay methods.

Diisopropylphosphoric acid produced the greatest activity (Table 1) while diethylisopropylphosphoric acid gave approximately half the specific activity of DPA and is unstable. Since DEIP is unstable and DPA is not commercially available, we used HMPA as the modifier for preparing and characterizing semisynthetic fluorohydrolases. Furthermore, HMPA has been shown to generate activity with other proteins as well as RNase (Table 2). 

The production of semisynthetic enzymes by conformational modification has been well documented in the literature. 23,39 utility and versatility of this process is exemplified by the numerous starting proteins that have been successfully modified to produce new catalysts. Examples include the preparation of amino acid esterases, fluorohydrolases, and glucose isomerases. In many cases these semisynthetic cata-... 

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