Butyrylcholinesterase GII7H/EI97Q is a somanase

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

Research output: Contribution to journalArticle

Abstract

We reported previously on the design and expression of a human butyrylcholinesterase (BuChE GII7N) that reactivates spontaneously to catalyze hydrolysis of the nerve agents, sarin and VX [JferAtwri734,(IM5)]. Unfortunately, GII7H is irreversibly inhibited by the most toxic nerve agent, soman (CD). To overcome this limitation, we designed, expressed and partially purified a double mutant (Gl I7H/EI97Q) based upon the hypothesis that removal of the acid at EI97 would slow the so-called "aging" reaction of GD sufficiently to allow the new HI 17 to catalyze reactivation. For comparison, we also made the single mutant EI97Q. All three enzymes retained esterase activity with Km values for butyrylthiocholine (BuSCh) of 0.05±O.OI mM (GII7H),0.078±0.005 mM (E197Q) and 028±0.02 mM (GII7H/EI97Q) at pH 7.5,25°C ;[wildtype (WT) Km=0.025±0.006 mM]. Like WT, substrate activation was observed with Km values of 0.8,23 and 120 mM for GII7H, EI97Q and G117H/E197Q, respectively, compared with 0.9 for WT. All were slowly inhibited by GD. For example, apparent It, values at pH 7.5, 25°C for the CD P(-)C(-) isomer were: > 10s M-1sec-1 (WT); 7,600 ±400 M-1sec-1(GII7H);3,600±300 M W(EI97Q); and6IO± 40 HV(GII7H/EI97Q). Following inhibition and subsequent removal of excess GD by gel filtration, Gl I7H/EI97Q underwent spontaneous reactivation, with approximate k; values of 11.4 × 10-5 and 210 × 10-5 sec-1 for the P(-)C(-) and P()C(+) GD stereoisomen, respectively, at pH 7.5, 25°C. No recovery of activity was observed for WT, GII7H or E197Q inhibited by GD. Measurement of free inhibitor using a separate, indirect assay confirmed that GII7H/EI97Q reactivation resulted in destruction of GD. We conclude that G117H/E197Q is a cholinesterase with novel somanase activity.

Original languageEnglish (US)
JournalFASEB Journal
Volume10
Issue number6
StatePublished - Dec 1 1996

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diisopropyl-fluorophosphatase
Butyrylcholinesterase
cholinesterase
Butyrylthiocholine
Sarin
Soman
Poisons
Cholinesterases
Esterases
Isomers
Hydrolysis
Assays
mutants
Aging of materials
Gels
Chemical activation
Recovery
esterases
Acids
isomers

ASJC Scopus subject areas

  • Biotechnology
  • Biochemistry
  • Molecular Biology
  • Genetics

Cite this

Hillard, C. B., Lockridge, O., & Broomfield, C. A. (1996). Butyrylcholinesterase GII7H/EI97Q is a somanase. FASEB Journal, 10(6).

Butyrylcholinesterase GII7H/EI97Q is a somanase. / Hillard, C. B.; Lockridge, Oksana; Broomfield, C. A.

In: FASEB Journal, Vol. 10, No. 6, 01.12.1996.

Research output: Contribution to journalArticle

Hillard, CB, Lockridge, O & Broomfield, CA 1996, 'Butyrylcholinesterase GII7H/EI97Q is a somanase', FASEB Journal, vol. 10, no. 6.
Hillard, C. B. ; Lockridge, Oksana ; Broomfield, C. A. / Butyrylcholinesterase GII7H/EI97Q is a somanase. In: FASEB Journal. 1996 ; Vol. 10, No. 6.
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title = "Butyrylcholinesterase GII7H/EI97Q is a somanase",
abstract = "We reported previously on the design and expression of a human butyrylcholinesterase (BuChE GII7N) that reactivates spontaneously to catalyze hydrolysis of the nerve agents, sarin and VX [JferAtwri734,(IM5)]. Unfortunately, GII7H is irreversibly inhibited by the most toxic nerve agent, soman (CD). To overcome this limitation, we designed, expressed and partially purified a double mutant (Gl I7H/EI97Q) based upon the hypothesis that removal of the acid at EI97 would slow the so-called {"}aging{"} reaction of GD sufficiently to allow the new HI 17 to catalyze reactivation. For comparison, we also made the single mutant EI97Q. All three enzymes retained esterase activity with Km values for butyrylthiocholine (BuSCh) of 0.05±O.OI mM (GII7H),0.078±0.005 mM (E197Q) and 028±0.02 mM (GII7H/EI97Q) at pH 7.5,25°C ;[wildtype (WT) Km=0.025±0.006 mM]. Like WT, substrate activation was observed with Km values of 0.8,23 and 120 mM for GII7H, EI97Q and G117H/E197Q, respectively, compared with 0.9 for WT. All were slowly inhibited by GD. For example, apparent It, values at pH 7.5, 25°C for the CD P(-)C(-) isomer were: > 10s M-1sec-1 (WT); 7,600 ±400 M-1sec-1(GII7H);3,600±300 M W(EI97Q); and6IO± 40 HV(GII7H/EI97Q). Following inhibition and subsequent removal of excess GD by gel filtration, Gl I7H/EI97Q underwent spontaneous reactivation, with approximate k; values of 11.4 × 10-5 and 210 × 10-5 sec-1 for the P(-)C(-) and P()C(+) GD stereoisomen, respectively, at pH 7.5, 25°C. No recovery of activity was observed for WT, GII7H or E197Q inhibited by GD. Measurement of free inhibitor using a separate, indirect assay confirmed that GII7H/EI97Q reactivation resulted in destruction of GD. We conclude that G117H/E197Q is a cholinesterase with novel somanase activity.",
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AU - Hillard, C. B.

AU - Lockridge, Oksana

AU - Broomfield, C. A.

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N2 - We reported previously on the design and expression of a human butyrylcholinesterase (BuChE GII7N) that reactivates spontaneously to catalyze hydrolysis of the nerve agents, sarin and VX [JferAtwri734,(IM5)]. Unfortunately, GII7H is irreversibly inhibited by the most toxic nerve agent, soman (CD). To overcome this limitation, we designed, expressed and partially purified a double mutant (Gl I7H/EI97Q) based upon the hypothesis that removal of the acid at EI97 would slow the so-called "aging" reaction of GD sufficiently to allow the new HI 17 to catalyze reactivation. For comparison, we also made the single mutant EI97Q. All three enzymes retained esterase activity with Km values for butyrylthiocholine (BuSCh) of 0.05±O.OI mM (GII7H),0.078±0.005 mM (E197Q) and 028±0.02 mM (GII7H/EI97Q) at pH 7.5,25°C ;[wildtype (WT) Km=0.025±0.006 mM]. Like WT, substrate activation was observed with Km values of 0.8,23 and 120 mM for GII7H, EI97Q and G117H/E197Q, respectively, compared with 0.9 for WT. All were slowly inhibited by GD. For example, apparent It, values at pH 7.5, 25°C for the CD P(-)C(-) isomer were: > 10s M-1sec-1 (WT); 7,600 ±400 M-1sec-1(GII7H);3,600±300 M W(EI97Q); and6IO± 40 HV(GII7H/EI97Q). Following inhibition and subsequent removal of excess GD by gel filtration, Gl I7H/EI97Q underwent spontaneous reactivation, with approximate k; values of 11.4 × 10-5 and 210 × 10-5 sec-1 for the P(-)C(-) and P()C(+) GD stereoisomen, respectively, at pH 7.5, 25°C. No recovery of activity was observed for WT, GII7H or E197Q inhibited by GD. Measurement of free inhibitor using a separate, indirect assay confirmed that GII7H/EI97Q reactivation resulted in destruction of GD. We conclude that G117H/E197Q is a cholinesterase with novel somanase activity.

AB - We reported previously on the design and expression of a human butyrylcholinesterase (BuChE GII7N) that reactivates spontaneously to catalyze hydrolysis of the nerve agents, sarin and VX [JferAtwri734,(IM5)]. Unfortunately, GII7H is irreversibly inhibited by the most toxic nerve agent, soman (CD). To overcome this limitation, we designed, expressed and partially purified a double mutant (Gl I7H/EI97Q) based upon the hypothesis that removal of the acid at EI97 would slow the so-called "aging" reaction of GD sufficiently to allow the new HI 17 to catalyze reactivation. For comparison, we also made the single mutant EI97Q. All three enzymes retained esterase activity with Km values for butyrylthiocholine (BuSCh) of 0.05±O.OI mM (GII7H),0.078±0.005 mM (E197Q) and 028±0.02 mM (GII7H/EI97Q) at pH 7.5,25°C ;[wildtype (WT) Km=0.025±0.006 mM]. Like WT, substrate activation was observed with Km values of 0.8,23 and 120 mM for GII7H, EI97Q and G117H/E197Q, respectively, compared with 0.9 for WT. All were slowly inhibited by GD. For example, apparent It, values at pH 7.5, 25°C for the CD P(-)C(-) isomer were: > 10s M-1sec-1 (WT); 7,600 ±400 M-1sec-1(GII7H);3,600±300 M W(EI97Q); and6IO± 40 HV(GII7H/EI97Q). Following inhibition and subsequent removal of excess GD by gel filtration, Gl I7H/EI97Q underwent spontaneous reactivation, with approximate k; values of 11.4 × 10-5 and 210 × 10-5 sec-1 for the P(-)C(-) and P()C(+) GD stereoisomen, respectively, at pH 7.5, 25°C. No recovery of activity was observed for WT, GII7H or E197Q inhibited by GD. Measurement of free inhibitor using a separate, indirect assay confirmed that GII7H/EI97Q reactivation resulted in destruction of GD. We conclude that G117H/E197Q is a cholinesterase with novel somanase activity.

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