Mutation Report for: G117H_human-BCHE

The single residue mutation of butyrylcholinesterase (BChEG117H) hydrolyzes a number of organophosphosphorus (OP) anticholinesterases. Whereas other BChE active site/proximal mutations have been investigated, none are sufficiently active to be prophylactically useful. In a fundamentally different computer simulations driven strategy, we identified a surface peptide loop (residues 278-285) exhibiting dynamic motions during catalysis and modified it via residue insertions. We evaluated these loop mutants using computer simulations, substrate kinetics, resistance to inhibition, and enzyme reactivation assays using both the choline ester and OP substrates. A slight but significant increase in reactivation was noted with paraoxon with one of the mutants, and changes in KM and catalytic efficiency were noted in others. Simulations suggested weaker interactions between OP versus choline substrates and the active site of all engineered versions of the enzyme. The results indicate that an improvement of OP anticholinesterase hydrolysis through surface loop engineering may be a more effective strategy in an enzyme with higher intrinsic OP compound hydrolase activity.

Human butyrylcholinesterase (hBChE) is currently being developed as a detoxication enzyme for the catalytic hydrolysis or stoichiometric binding of organophosphates (OPs). Previously, rationally designed hBChE mutants (G117H and E197Q) were reported in the literature and showed the feasibility of engineering OP hydrolytic functional activity into hBChE. However, the OP hydrolysis rate for G117H is too low for clinical utility. Additional OP-resistant hBChE variants with greater hydrolysis rates are needed as OP nerve-agent countermeasures for therapeutic utility. As described herein, a directed molecular evolution process was used to identify amino acid residues that contribute to OP-resistant functional activity of hBChE variants. In this article, we describe the development and validation of a novel method to identify hBChE variants with OP-resistant functional activity (decreased rate of OP inhibition). The method reported herein used an adenoviral protein expression system combined with a functional screening protocol of OP nerve-agent model compounds that have been shown to have functional properties similar to authentic OP nerve-agent compounds. The hBChE screening method was robust for transfection efficiency, library diversity, and reproducibility of positive signals. The screening approach not only identified the previously reported hBChE G117H variant, but also identified a series of additional hBChE variants, including hBChE G117N, G117R, E197C, and L125V, that exhibited OP-resistant functional activities not reported previously. The mammalian functional screening approach can serve as a cornerstone for further optimization and screening for OP-resistant hBChEs for potential therapeutic applications.

This study is aimed at understanding the hydrolysis mechanism of organophosphate (OP) compounds by G117H-BChE. It is a theoretical study that focuses on the role of the G117H mutation in the dephosphorylation step. Various proposed mechanisms are examined. We show that His117 acts as a general base by activating a water molecule, and thus assisting its nucleophilic attack on the phosphate. The calculated reaction energy profile agrees well with the experimental data. Moreover, analysis of the reaction via its two hypothetical elementary steps, proton transfer and hydroxide attack, supports the role of His117 as a general base. Further support to the proposed mechanism is gained by structural comparison of the active site to RNAse A, which has similar composition of substrate and functional groups. The similarity between these enzymes extends beyond the structure and also becomes evident when comparing functionality of various active sites residues as well as rate-pH dependence obtained in the two cases. Moreover, it is demonstrated that an extended form of Bevilacqua's model (Biochemistry 2003;42:2259-2265) may resolve the apparent contradictions between the proposed mechanism and various experimental observations regarding rate-pH dependence. Finally, that same model is shown to rationalize the hydrolase activity of G117D BChE, an observation which is considered puzzling. It is concluded that G117H-BChE hydrolyzes echothiophate and possibly other OP compounds via a general acid-base mechanism. On the basis of this mechanism, one can now proceed with rational design aimed at improving the enzyme by exploiting both the structural and mechanistic knowledge.

Human butyrylcholinesterase (BChE, EC 3.1.1.8) is an efficient scavenger of nerve agents and organophosphorus (OP) pesticides; one molecule of BChE inactivates one molecule of OP in a suicide reaction that irreversibly inhibits BChE. By contrast the BChE mutant, G117H, inactivates many molecules of OP. The OP makes a covalent bond with the active site serine and then the serine is dephosphorylated by the action of His117. In an effort to understand the mechanism by which is 117 achieves dephosphorylation, 62 new mutants of human BChE were tested for OP hydrolase activity, using a new screening assay. It was found that not only G117H, but also G117D, G117E, and L286H mutants were OP hydrolases. These results support the hypothesis that a hydrogen-bond acceptor acts as a general base t activate a water molecule which in turn dephosphorylates the active site serine The screening assay provides a convenient means for identifying cholinesterase mutants with OP hydrolase activity.

Because of deficiencies in the present treatments for organophosphorus anticholinesterase poisoning, we are attempting to develop a catalytic scavenger that can be administered as prophylactic protection. Currently known enzymes are inadequate for this purpose because they have weak binding and slow turnover, so we are trying to make an appropriate enzyme by protein engineering techniques. One butyrylcholinesterase mutant, G117H, has the desired type of activity but reacts much too slowly. This communication describes an attempt to determine the reason for the slow reaction so that a more efficient enzyme might be designed. The results indicate that the mutation at residue 117 has resulted in a distortion of the transition state of the reaction of organophosphorus compounds with the active site serine. This information will be used to develop other mutants that avoid transition state stabilization sites.

Organophosphorus acid anhydride (OP) "nerve agents" are rapid, stoichiometric, and essentially irreversible inhibitors of serine hydrolases. By placing a His near the oxyanion hole of human butyrylcholinesterase (BChE), we made an esterase (G117H) that catalyzed the hydrolysis of several OP, including sarin and VX [Millard et al. (1995) Biochemistry 34, 15925-15930]. G117H was limited, however, because it was irreversibly inhibited by pinacolyl methylphosphonofluoridate (soman); soman is among the most toxic synthetic poisons known. This limitation of G117H has been overcome by a new BChE (G117H/E197Q) that combines two engineered features: spontaneous dephosphonylation and slow aging (dealkylation). G117H/E197Q was compared with the single mutants BChE G117H and E197Q. Each retained cholinesterase activity with butyrylthiocholine as substrate, although kcat/Km decreased 11-, 11- or 110-fold for purified G117H, E197Q, or G117H/E197Q, respectively, as compared with wild-type BChE. Only G117H/E197Q catalyzed soman hydrolysis; all four soman stereoisomers as well as sarin and VX were substrates. Phosphonylation and dephosphonylation reactions were stereospecific. Double mutant thermodynamic cycles suggested that the effects of the His and Gln substitutions on phosphonylation were additive for PSCR or PRCR soman, but were cooperative for the PSCS stereoisomer. Dephosphonylation limited overall OP hydrolysis with apparent rate constants of 0.006, 0.077, and 0.128 min-1 for the PR/SCR, PSCS, and PRCS soman stereoisomers, respectively, at pH 7.5, 25 degrees C. We conclude that synergistic protein design converted an archetypal "irreversible inhibitor" into a slow substrate for the target enzyme.

The G117H mutant of human butyrylcholinesterase (EC 3.1.1.8) was expressed in Chinese hamster ovary cells. Substitution of Gly 117 with His to make the G117H mutant endowed butyrylcholinesterase with the ability to catalyze the hydrolysis of organophosphate esters. G117H was still able to hydrolyze butyrylthiocholine, benzoylcholine, and o-nitrophenyl butyrate, but in addition it had acquired the ability to hydrolyze the antiglaucoma drug echothiophate and the pesticide paraoxon. Wild-type butyrylcholinesterase was irreversibly inhibited by echothiophate and paraoxon, but G117H regained 100% activity within 2-3 min following reaction with these compounds. On a polyacrylamide gel, the same bands that stained for activity with butyrylthiocholine also stained for activity with echothiophate. G117H is the only enzyme known that hydrolyzes echothiophate. Diethoxyphosphorylated G117H aged with a half-time of 5.5 h, a rate 600 times slower than the rate of hydrolysis. Echothiophate and paraoxon were hydrolyzed with the same kcat of 0.75 min-1. This calculates to a rate acceleration of 100,000-fold for hydrolysis of echothiophate and paraoxon by the G117H mutant compared to the nonenzymatic rate.

Serine esterases and proteases are rapidly and irreversibly inhibited by organophosphorus (OP) nerve agents. To overcome this limitation, we selected several residues that were predicted to be within 3-10 A of both the active site Ser O gamma and the oxyanion hole of human butyrylcholinesterase for mutation to His (G115H, G117H, Q119H, and G121H). In remarkable contrast with wild-type (WT) and all other His mutants tested, G117H underwent spontaneous reactivation following OP inhibition to regain 100% of original esterase activity with maximum k3 values of approximately 6.8 x 10(-5) and 16 x 10(-5) s-1 for GB (sarin) and VX, respectively, in 0.1 M Bis-Tris, 25 degrees C. The free energy of activation for k3 was 19 kcal mol-1, and measurement of pH dependence suggested that reactivation resulted from an acidic group with pKa 6.2. To evaluate further the importance of His in achieving this result, we changed the same Gly to Lys (G117K) and compared its substrate and inhibitor kinetics with those of G117H. Both mutants retained esterase activity with Km values similar to those of WT for neutral ester hydrolysis, but G117K did not reactivate. Complete reactivation proves that G117H is not irreversibly inhibited but instead functions as a catalyst for OP hydrolysis. Dephosphonylation is the rate-limiting step, and G117H effects overall rate constant enhancements of approximately 100- and 2000-fold above the uncatalyzed hydrolysis of GB and VX, respectively, at pH 6.0, 25.0 degrees C. We conclude that an appropriately positioned imidazolium ion in the oxyanion hole catalyzes dephosphonylation and, thereby, confers a novel organophosphorus acid anhydride hydrolase activity upon butyrylcholinesterase.