Fluorohydrolase

The A-esterases now classified as diisopropyl fluorophosphatases (diiso-propyl-fluorophosphate fluorohydrolase, DFPase, somanase, EC 3.1.8.2) were previously listed under EC 3.8.2.1. These enzymes, which hydrolyze P-F and P-CN bonds such as those of nerve gases, should be described as organophosphorus acid anhydrolases rather than phosphatases [56]. Diisopropyl-fluoro-phosphatases exist in different forms with contrasting substrate specificities. One form is able to hydrolyze paraoxon at a low rate, while others have no paraoxonase activity. The different forms differ in their molecular weights and in their requirements for bivalent cations for activity [56]. 

The first two examples of CCMP described herein, have been studied and reported elsewhere in the literature. Only the highlights of these studies are given. The last example, fluorohydrolases, includes our latest results and are given in more detail. 

In the balance of this paper, we describe the application of our method of chemical modification to the generation of fluorohydrolases The most common substrate to measure fluorohydrolase activity is diiso-propylphosphorofluoridate (DFP). Although DFP is not found in nature, Mazur reported the enzymatic hydrolysis of this highly toxic organo-fluorophosphate by an enzyme isolated from hog kidney.Since then, diisopropyl phosphorofluoridate fluorohydrolase (DFPase, E.C. 3.8.2.1) has been extensively studied and well characterized. In addition, enzymes with organofluorophosphate hydrolyzing activity have been shown to be widely distributed phylogenetically and sources are known from bacteria, protozoa, invertebrates, and vertebrates. 

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. 

Conformationally modified RNase, derivatized with dimethyl pimelimi-date, had the highest fluorohydrolase activity toward DFP however, the highest activity toward PMSF was obtained for dimethyl suberimidate crosslinked protein. The best overall activity for both substrates was obtained with the pimelimidate (Cs) crosslinked modified RNase. A plot of activity towards PMSF versus pH, after chromatography on G-15 Sephadex, shows that the optimum pH is approximately 7.5 (Figure 2). The values of... 

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.

Table 2. Fluorohydrolase fied with HMPA. activity of various proteins modi-...

Figure 2. Fluorohydrolase activity as a function of pH for dimethyl pimelimidate crosslinked CCMP. Activity was measured using 5 mM PMSF as the substrate at 30°C. Each activity is the average of two separate measurements obtained by HPLC and fluoride electrode methods described in the text. 

In previous articles, we described a procedure for the induction of fluorohydrolase activity in bovine albumin, casein, egg albumin, hexokin-ase and ribonuclease gy perturbing these proteins at pH 3 and adding a modifier, significant activity towards DFP and PMSF was obtained. The initial activity was measured before protein crosslinking indicating that the active conformation was retained for several minutes in the presence of modifier at neutral pH. If the perturbed protein was chromatographed on G-15 to remove modifier, no activity could be detected. 

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). 

To compare the catalytic efficiency of catalysts, it is helpful to compare the enhancement ratios (E.R.). E.R. is calculated by dividing the kcat by the kuncat (the rate constant for the uncatalyzed reaction). Enormous rate enhancements are achieved by enzymes. For example, hydrolytic enzymes often exhibit rate enhancements of 10 -10 2 compared with the spontaneous water-catalyzed or the acid/base-catalyzed reaction at about neutral pH. e For purposes of comparison, the kcat, kuncat, and E.R. values for two hydrolytic abzymes, as well as CCMP fluorohydrolase chromatographed on G-15 Sephadex gel is presented in Table 3. The fluorohydrolase, chromatographed on G-15 Sephadex, has an E.R. four times that of the two abzymes. In addition, the enhancement ratios of natural DFPases from various sources as well as CCMP fluorohydrolase are presented in Table 4. In all cases, the initial E.R. (before any purification) is higher for semisynthetic fluorohydrolases than for any of the natural unpurified DFPases. 

Table 3. Comparison of semisynthetic enhancement ratios of fluorohydrolases. abzymes and...

Table 4. Comparison of enhancement ratios of natural DFPases and CCMP fluorohydrolases. 

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-... 

Semisynthetic fluorohydrolase has activity comparable to the naturally occurring DFPases and does not require a divalent cation as does the "Mazur-type" enzyme. It operates over a pH range from 6.5 to 8.0 with the optimum at approximately 7.5. A comparison of natural and semisynthetic fluorohydrolase activity is shown in Table 4. An examination of the enhancement ratio (2.2 x 10 ) shows that the CCMP fluorohydrolase is a remarkably good catalyst. If the crosslinking reaction is further optimized to increase the yield of the dimer along with further purification steps, a stable highly efficient semisynthetic fluorohydrolase should be achieved. 

The authors wish to thank the Army Research Office (Contract No. DAAL03-86-C-0021) for their financial support of the fluorohydrolase project. We also express our thanks to the Department of Energy - Office of Basic Energy Science (Contract No. DE-AC02-81erl2003) for support of the earlier amino acid esterase studies. Additional support was given by Owens-Illinios, Inc. and Anatrace, Inc. 

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