Design and Expression of Organophosphorus Acid Anhydride Hydrolase Activity in Human Butyrylcholinesterase

C. B. Millard, Oksana Lockridge, C. A. Broomfield

Research output: Contribution to journalArticle

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Abstract

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 Å of both the active site Ser O γ 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 k 3 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 °C. The free energy of activation for Lt was 19 kcal mol-1, and measurement of pH dependence suggested that reactivation resulted from an acidic group with pK a 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 K m 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 °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.

Original languageEnglish (US)
Pages (from-to)15925-15933
Number of pages9
JournalBiochemistry
Volume34
Issue number49
DOIs
StatePublished - Dec 1995

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Acid Anhydride Hydrolases
Butyrylcholinesterase
Aryldialkylphosphatase
Human Activities
Hydrolysis
Esterases
Sarin
Regain
Serine Proteases
Free energy
Rate constants
Catalytic Domain
Esters
Chemical activation
Ions
Mutation
Catalysts
Kinetics
Substrates

ASJC Scopus subject areas

  • Biochemistry

Cite this

Design and Expression of Organophosphorus Acid Anhydride Hydrolase Activity in Human Butyrylcholinesterase. / Millard, C. B.; Lockridge, Oksana; Broomfield, C. A.

In: Biochemistry, Vol. 34, No. 49, 12.1995, p. 15925-15933.

Research output: Contribution to journalArticle

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abstract = "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 {\AA} of both the active site Ser O γ 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 k 3 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 °C. The free energy of activation for Lt was 19 kcal mol-1, and measurement of pH dependence suggested that reactivation resulted from an acidic group with pK a 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 K m 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 °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.",
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AB - 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 Å of both the active site Ser O γ 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 k 3 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 °C. The free energy of activation for Lt was 19 kcal mol-1, and measurement of pH dependence suggested that reactivation resulted from an acidic group with pK a 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 K m 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 °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.

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