p.G117K Gly117Lys (p.G145K Gly145Lys in primary sequence with 28 amino-acids signal peptide) Phosphotriesterase activity OP sensitive
Kinetic parameters
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References:
Title: Protein engineering of a human enzyme that hydrolyzes V and G nerve agents: design, construction and characterization Broomfield CA, Lockridge O, Millard CB Ref: Chemico-Biological Interactions, 119-120:413, 1999 : PubMed
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.
        
Title: Organophosphorus acid anhydride hydrolase activity in human butyrylcholinesterase: synergy results in a somanase Millard CB, Lockridge O, Broomfield CA Ref: Biochemistry, 37:237, 1998 : PubMed
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.
        
Title: Design and expression of organophosphorus acid anhydride hydrolase activity in human butyrylcholinesterase Millard CB, Lockridge O, Broomfield CA Ref: Biochemistry, 34:15925, 1995 : PubMed
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.