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Ions of the rare earths as possible reactivators of acetylcholinesterase inhibited by some organophosphorus compounds
BIOCHIMICA ET BIOPHYSICA ACTA
427
BBA 26640
IONS OF T H E RARE E A R T H S AS POSSIBLE REACTIVATORS OF ACETYLCHOLINESTERASE I N H I B I T E D BY SOME ORGANOPHOSPHORUS COMPOUNDS
D. J. H O W E L L S AY~ D. B. COULT
Chemistry Division Chemical Defence Establishment, Porton Down, Salisbury, Wiltshire (Great Britain) (Received March 3oth, 1971)
SUMMARY
I. The reactivation of 'aged' phosphonylated acetylcholinesterase may form the basis for a therapy for poisoning by certain organophosphorus compounds. Since it is known that rare earth metal ions catalyse the hydrolysis of p-nitrophenyl hydrogen methylphosphonate--a simple model compound for the 'aged' enzyme--it was considered of interest to investigate the possibility that rare earth metal ions might reactivate the 'aged' inhibited enzyme itself. 2. Studies of the effects of rare earth metal ions on acetylcholinesterase indicated that they were reversible competitive inhibitors of the enzyme. 3. Using two criteria of reaction, namely, the reappearance of enzyme activity and the release of ~3~Plphosphonate, it was shown that the rare earth metal ions did not catalyse the dephosphonylation of organophosphorus-inhibited acetylcholinesterase. 4. The reasons for the lack of reaction are discussed briefly and it is concluded that studies with rare earth metal ions are unlikely to lead to a new therapy for poisoning by organophosphates.
Poisoning by certain organophosphorus compounds, notably Soman (1,2,2trimethylpropyl methylphosphonofluoridate), does not respond to treatment with atropine and oxime I as does poisoning by most organophosphates2, 3. It has become clear that this lack of response is associated with the change of the organophosphorusinhibited acetylcholinesterase I (EC 3.1.1.7) to the 'aged' dealkylated enzyme II 4-11.
I
IX
m"
Abbreviations: P2A, pyridine-2-aldoxime; P2S, pyridine-2-aldoxime methyl methanesulphonate.
Biochim. Biophys. Acta, 244 (1971) 427-431
428
D.J.
H O W E L L S , D. B. COULT
A possible therapy for poisoning by these organophosphorus compounds could therefore be based on the reactivation of the 'aged' dealkylated enzyme. Thus, when it was demonstrated that rare earth metal ions catalyse the hydrolysis of p-nitrophenyl hydrogen methylphosphonate III12,~% it became of interest to know whether the rare earth metal ions could catalyse the dephosphonylation of II and, if so, whether such dephosphonylation was accompanied by the reappearance of enzyme activity. As the rare earth metal ions were being assessed as possible reactivators of inhibited acetylcholinesterase it was essential to know what effects, if any, these materials had upon the uninhibited enzyme. The acetylcholinesterase (bovine erythrocyte ; Sigma (London) Chemical) activity was determined titrimetrically (Radiometer Copenhagen automatic titrimeter) using O.Ol M NaOH. The assays were carried out under N~ in a total volume of IO ml NaC1 solution (o.15 M) containing acetylcholine (o.8 raM) at pH 7.4 and at 25 °. During investigations of the effects of the rare earth metal ions on the enzyme activity, various concentrations of rare earth salts (KochLight Laboratories, or K and K Laboratories, Plainview, N.Y., U.S.A.) with and without pyridine-2-aldoxime (P2A) (synthesised at the Chemical Defence Establishment) were mixed with the enzyme (approx. 0.5 I.U.) in saline solution; the pH was adjusted to 7.4 and the reaction started by addition of substrate. The rare earth metal ions (except promethium) were found to be reversible inhibitors of acetylcholinesterase: rare earth metal ion (5 raM) completely inhibited the enzyme under the normal assay conditions but over 95% of the original activity could be recovered by exhaustive dialysis. The inhibition, which in the case of lanthanum was apparently competitive with regard to substrate (Fig. i), could be removed by EDTA (o.oi M) and neither
c
0.5
g~ L
m
I
o o2o4&&
I
,.o
I
I
,.4
&
I
2O
Enzyme reaction v e l o c i t y x103 ( l / m i n ) Substrate concentration
Fig. i . I n h i b i t i o n of a c e t y l c h o l i n e s t e r a s e b y l a n t h a n u m . T h e a s s a y s w e r e p e r f o r m e d a t 25 ° a n d p i t 7.4 i n o.15 M NaC1 s o l u t i o n u s i n g a n a u t o m a t i c t i t r i m e t e r . L a n t h a n u m w a s p r e s e n t a t a c o n c e n t r a t i o n of o (o), o . i (&) a n d i m M (D). F u r t h e r e x p e r i m e n t a l d e t a i l s a r e g i v e n i n t h e t e x t .
Biochim. Biophys. Acta, 244 ( i 9 7 I) 4 2 7 - 4 3 i
POSSIBLE REACTIVATORS OF ACETYLCHOLINESTERASE
429
this concentration of E D T A nor the order of addition of the rare earth metal ion and the E D T A influenced the subsequently measured enzyme activity. However, addition of 1.25 mlV[ P2A which also complexes with the rare earth metal ions (I raM) did not reduce their inhibition of the esterase but appeared to increase it b y approximately 5-fold. Further investigations revealed that the uninhibited enzyme (2.5-3 I.U./ml) could be incubated with rare earth metal ions (I mM) with and without P2A (1.25 raM) at 37 ° and at various p H values (pH 7-8.4; o.I M 1,4-diazobicycloE2,2,21-octane buffer la) for at least 6 h with no detectable irreversible loss of activity and that it was only after prolonged incubation (24 h) under these conditions that any irrecoverable loss of activity (20% of control samples) occurred. The possibility of any reaction between the rare earth metal ions and the phosphonylated aeetylcholinesterase (forms I and II) was examined using two criteria of reaction, namely, the reappearance of esterase activity and the release of radioactive E32PI phosphonate. All the rare earth metal ions (except promethium) were used in the experiments with unlabelled inhibited enzyme but only yttrium was selected for use in the experiments with radioactive materials (see ref. 13). Undealkylated inhibited enzyme and 'aged' dealkylated enzyme were prepared in aqueous solution by treating acetylcholinesterase with Sarin (isopropyl methylphosphonofluoridate) and Soman respectively, and separating the phosphonylated protein from excess inhibitor by column chromatography 11. Both unlabelled and 32P-labelled inhibitors were used. The resulting solutions of inhibited esterase were equivalent to approx. 5 I.U./ml and were used immediately for incubation with rare earth metal ions (I mM) as described above. Samples of the incubation mixtures were taken after various time intervals from 0-24 h and were immediately made o.oi M with respect to EDTA. Duplicate determinations were made on each sample of (I) enzyme activity, (2) unbound radioactivity by the ultrafiltration techniquelL and (3) bound label after exhaustive dialysis. Solutions were assayed for radioactivity either directly using a Geiger-Mtiller counter for liquids (see ref. I I ) or after addition of a dioxane-based scintillator mixture 1'~using a Tritomat 6o12 Liquid Scintillation Counter (Isotope Developments). The results of the present investigations demonstrated that the rare earth metal ions cannot reactivate acetylcholinesterase inhibited by organophosphates either before or after dealkylation of the inhibited enzyme. The experiments carried out with radioactively-labelled inhibited acetylcholinesterase supported the findings of the studies with the unlabelled inhibited enzyme and also demonstrated that no dephosphonylation reaction occurred in the presence of yttrium except in the case of Satininhibited enzyme (Table I). A release of labelled phosphonate occurred in this case only after prolonged incubation (24 h) but there was no reappearance of esterase activity. No explanation for this apparently anomolous observation can be proffered at the present time. However, in parallel experiments using I mM pyridine-2-aldoxime methyl methanesulphonate (P2S) (synthesised at the Chemical Defence Establishment) instead of a rare earth metal ion, the enzyme inhibited b y Sarin was rapidly reactivated with the concomitant release of asp and, as expected, no reactivation and no dephosphonylation of the esterase inhibited by Soman occurred. The presence of P2A and yttrium during reactivation of Sarin-inhibited enzyme b y P2S was found to reduce the activity recovered after i h by about 200/0. The inability of the rare earth metal ions to reactivate undealkylated inhibited enzyme was not totally unexpected as they do not catalyse the hydrolysis of the neuBiochim. Biophys. Acta, 244 (I97 I) 427-431
430
D . j . HOWELLS, D. B. COULT
TABLE I RESULTS PHORUS
OF
INCUBATION
COMPOUNDS
OF
V¢ITtt
ACETYLCHOLINESTERASE
POSSIBLE
INHIBITED
BY
a2p-LABELLED
ORGANOPHOS-
REACTIVATORS
Inhibited acetylcholinesterase (approx. 5 I.U./ml) was incubated with either P2S (i mM) or Yttrium-P2A mixture (i mM and 1.25 mM respectively) at pH 8. 4 and at 37 °. Bound a~p ("a"; counts/Ioo sec per mt of incubation mixture), unbound ~2p ("b"; units as for "a") and enzyme activitv ("c"; I.U./o.2 ml of incubation mixture) were determined as described in the text. Blank values without addition of possible reactivators are given. Inhibitor
Reactivator
Measured value
Sarin
P2S
a b c a b
Yttriunl/P2A
Soman
Incubation time (h) : o I 24
95 1.8 44 ° o
45 32o 3.9 42o o
45 315 4 -I 45 255
Blank
41o o o 35 ° o
C
O
O
O
O
P2S
a b
285 o
285 o
280 o
245 o
C
0
0
0
0
Yttrium/P2A
a b
45 ° o
460 o
44 ° o
590 o
C
0
0
0
0
t r a l e s t e r e t h y l p - n i t r o p h e n y l m e t h y l p h o s p h o n a t e to a n y m a r k e d degree12, la. H o w ever, as t h e r a r e e a r t h m e t a l ions are r e a c t i v e t o w a r d s t h e m o n o b a s i c a c i d e s t e r I I I , t h e r e a c t i o n of t h e s e m a t e r i a l s w i t h t h e p h o s p h o r u s c e n t r e of t h e d e a l k y l a t e d i n h i b i t e d e n z y m e was c o n s i d e r e d likely. T h e r e a s o n s for t h e l a c k of r e a c t i o n w i t h S o m a n - i n h i b i t e d e n z y m e are o b s c u r e at t h e p r e s e n t t i m e b u t in g e n e r a l t e r m s c e r t a i n possibilities m a y be c o n s i d e r e d . I n t h e first p l a c e c o m p o u n d I I I is a v e r y s i m p l e m o l e c u l e c o m p a r e d w i t h t h e p h o s p h o n y l a t e d e n z y m e ; as a r e s u l t t h e p h y s i c o c h e m i c a l e n v i r o n m e n t of t h e p h o s p h o n y l c e n t r e in t h e l a t t e r is m u c h m o r e c o m p l e x t h a n in I I I , a n d it is l i k e l y t h a t c e r t a i n factors, s u c h as t h e steric a n d p o l a r p r o p e r t i e s of this c e n t r e in t h e p r o t e i n , m a y p r e v e n t a t t a c k b y t h e r a r e e a r t h m e t a l ions. S e c o n d l y , t h e r a r e e a r t h m e t a l ions w e r e s h o w n to be i n h i b i t o r s of t h e e s t e r a s e in t h e p r e s e n c e a n d a b s e n c e of P 2 A a n d , as this i n h i b i t i o n is a p p a r e n t l y c o m p e t i t i v e w i t h r e g a r d to s u b s t r a t e , it is p r o b a b l e t h a t t h e s e m a t e r i a l s are b o u n d at or n e a r t h e a c t i v e site of t h e e n z y m e : also t h e r a r e e a r t h m e t a l i o n - P 2 A m i x t u r e a p p a r e n t l y r e d u c e d t h e r e a c t i v a t i o n of S a t i n i n h i b i t e d e n z y m e b y P2S. I t is t h e r e f o r e possible t h a t t h e r a r e e a r t h m e t a l ions are p r e f e r e n t i a l l y b o u n d to a p a r t i c u l a r site on t h e p r o t e i n a n d h e n c e are n o t o n l y u n a b l e to a t t a c k t h e p h o s p h o n y l c e n t r e b u t also i n t e r f e r e w i t h t h e access of o t h e r m o l e c u l e s to t h e p h o s p h o n y l c e n t r e . T h e p r e s e n t s t u d i e s s u g g e s t t h a t I I I is p r o b a b l y a p o o r m o d e l c o m p o u n d for t h e d e a l k y l a t e d i n h i b i t e d e n z y m e ; it m i g h t be of i n t e r e s t t h e r e fore t o s t u d y t h e effects of r a r e e a r t h m e t a l ions o n t h e h y d r o l y s i s of s i m p l e a c i d p h o s p h o n a t e esters h a v i n g an a l k y l g r o u p or a c h a i n of 1,2 or m o r e a m i n o a c i d r e s i d u e s in p l a c e of t h e p - n i t r o p h e n y l l e a v i n g g r o u p . REFERENCES I A. T. LOOMIS AND B. S A L A F S K Y , Toxicol. A p p l . Pharmacol., 5 (I963) 685. 2 I. V~L COLEMAN, P. E. LITTLE AND G. A. GRANT, Can. J. Biochem. Physiol., 38 (196o) lO35. 3 F. HOBBIGER,in G. B. KOELLE, Handbuch der Experimentellen Pharmakologie, Vol. 15, Springer-Verlag, Berlin, 1963. Biochim. Biophys. Acts, 244 (1971) 427-431
POSSIBLE REACTIVATORS OF ACETYLCHOLINESTERASE
431
4 A. F. CHILDS, D. R. DAVIES, A. L. GREEN AND J. P. RUTLAND,Br. J. Pharmacol. Chemother., io (1955) 462. 5 ]3. M. ASKEW, Br. J. Pharmacol. Chemother., I I (1956) 417 . 6 F. HOBBIGER, Br. J. Pharmacol. Chemother., 12 (1957) 438. 7 F. BERENDS, C. H. POSTHUMUS, I. V. D. SLUYS AND F. A. DIEIRKAUF, Biochim. Biophys. Acla, 34 (1959) 576 . 8 F. 13ERENDS, Doctoral Dissertation, U n i v e r s i t y of Leiden, 1964. 9 J. H. FLEISCHER AND L. W. HARRIS, Biochem. Pharmacol., 14 (1965) 641. IO W. t{. BERRY AND D. t{. DAVIES, Biochem. J., IOO (1966) 572. I I D. 13. COULT,D. J. MARSH AND G. READ, Biochem. J., 98 (1966) 869. 12 R. j . WITHEY, Can. J. Chem., 47 (1969) 4383 . 13 F. McC. 13LEWETT AND P. WATTS,J. Chem. Soc., in the press. 14 1~. M. KEEN, Br. J. Pharmacol. Chemother., 26 (1966) 7o4 . 15 F. E. 13UTLER, Anal. Chem., 33 (1961) 409.
Biochim. Biophys. Acta, 244 (1971) 427-431
427
BBA 26640
IONS OF T H E RARE E A R T H S AS POSSIBLE REACTIVATORS OF ACETYLCHOLINESTERASE I N H I B I T E D BY SOME ORGANOPHOSPHORUS COMPOUNDS
D. J. H O W E L L S AY~ D. B. COULT
Chemistry Division Chemical Defence Establishment, Porton Down, Salisbury, Wiltshire (Great Britain) (Received March 3oth, 1971)
SUMMARY
I. The reactivation of 'aged' phosphonylated acetylcholinesterase may form the basis for a therapy for poisoning by certain organophosphorus compounds. Since it is known that rare earth metal ions catalyse the hydrolysis of p-nitrophenyl hydrogen methylphosphonate--a simple model compound for the 'aged' enzyme--it was considered of interest to investigate the possibility that rare earth metal ions might reactivate the 'aged' inhibited enzyme itself. 2. Studies of the effects of rare earth metal ions on acetylcholinesterase indicated that they were reversible competitive inhibitors of the enzyme. 3. Using two criteria of reaction, namely, the reappearance of enzyme activity and the release of ~3~Plphosphonate, it was shown that the rare earth metal ions did not catalyse the dephosphonylation of organophosphorus-inhibited acetylcholinesterase. 4. The reasons for the lack of reaction are discussed briefly and it is concluded that studies with rare earth metal ions are unlikely to lead to a new therapy for poisoning by organophosphates.
Poisoning by certain organophosphorus compounds, notably Soman (1,2,2trimethylpropyl methylphosphonofluoridate), does not respond to treatment with atropine and oxime I as does poisoning by most organophosphates2, 3. It has become clear that this lack of response is associated with the change of the organophosphorusinhibited acetylcholinesterase I (EC 3.1.1.7) to the 'aged' dealkylated enzyme II 4-11.
I
IX
m"
Abbreviations: P2A, pyridine-2-aldoxime; P2S, pyridine-2-aldoxime methyl methanesulphonate.
Biochim. Biophys. Acta, 244 (1971) 427-431
428
D.J.
H O W E L L S , D. B. COULT
A possible therapy for poisoning by these organophosphorus compounds could therefore be based on the reactivation of the 'aged' dealkylated enzyme. Thus, when it was demonstrated that rare earth metal ions catalyse the hydrolysis of p-nitrophenyl hydrogen methylphosphonate III12,~% it became of interest to know whether the rare earth metal ions could catalyse the dephosphonylation of II and, if so, whether such dephosphonylation was accompanied by the reappearance of enzyme activity. As the rare earth metal ions were being assessed as possible reactivators of inhibited acetylcholinesterase it was essential to know what effects, if any, these materials had upon the uninhibited enzyme. The acetylcholinesterase (bovine erythrocyte ; Sigma (London) Chemical) activity was determined titrimetrically (Radiometer Copenhagen automatic titrimeter) using O.Ol M NaOH. The assays were carried out under N~ in a total volume of IO ml NaC1 solution (o.15 M) containing acetylcholine (o.8 raM) at pH 7.4 and at 25 °. During investigations of the effects of the rare earth metal ions on the enzyme activity, various concentrations of rare earth salts (KochLight Laboratories, or K and K Laboratories, Plainview, N.Y., U.S.A.) with and without pyridine-2-aldoxime (P2A) (synthesised at the Chemical Defence Establishment) were mixed with the enzyme (approx. 0.5 I.U.) in saline solution; the pH was adjusted to 7.4 and the reaction started by addition of substrate. The rare earth metal ions (except promethium) were found to be reversible inhibitors of acetylcholinesterase: rare earth metal ion (5 raM) completely inhibited the enzyme under the normal assay conditions but over 95% of the original activity could be recovered by exhaustive dialysis. The inhibition, which in the case of lanthanum was apparently competitive with regard to substrate (Fig. i), could be removed by EDTA (o.oi M) and neither
c
0.5
g~ L
m
I
o o2o4&&
I
,.o
I
I
,.4
&
I
2O
Enzyme reaction v e l o c i t y x103 ( l / m i n ) Substrate concentration
Fig. i . I n h i b i t i o n of a c e t y l c h o l i n e s t e r a s e b y l a n t h a n u m . T h e a s s a y s w e r e p e r f o r m e d a t 25 ° a n d p i t 7.4 i n o.15 M NaC1 s o l u t i o n u s i n g a n a u t o m a t i c t i t r i m e t e r . L a n t h a n u m w a s p r e s e n t a t a c o n c e n t r a t i o n of o (o), o . i (&) a n d i m M (D). F u r t h e r e x p e r i m e n t a l d e t a i l s a r e g i v e n i n t h e t e x t .
Biochim. Biophys. Acta, 244 ( i 9 7 I) 4 2 7 - 4 3 i
POSSIBLE REACTIVATORS OF ACETYLCHOLINESTERASE
429
this concentration of E D T A nor the order of addition of the rare earth metal ion and the E D T A influenced the subsequently measured enzyme activity. However, addition of 1.25 mlV[ P2A which also complexes with the rare earth metal ions (I raM) did not reduce their inhibition of the esterase but appeared to increase it b y approximately 5-fold. Further investigations revealed that the uninhibited enzyme (2.5-3 I.U./ml) could be incubated with rare earth metal ions (I mM) with and without P2A (1.25 raM) at 37 ° and at various p H values (pH 7-8.4; o.I M 1,4-diazobicycloE2,2,21-octane buffer la) for at least 6 h with no detectable irreversible loss of activity and that it was only after prolonged incubation (24 h) under these conditions that any irrecoverable loss of activity (20% of control samples) occurred. The possibility of any reaction between the rare earth metal ions and the phosphonylated aeetylcholinesterase (forms I and II) was examined using two criteria of reaction, namely, the reappearance of esterase activity and the release of radioactive E32PI phosphonate. All the rare earth metal ions (except promethium) were used in the experiments with unlabelled inhibited enzyme but only yttrium was selected for use in the experiments with radioactive materials (see ref. 13). Undealkylated inhibited enzyme and 'aged' dealkylated enzyme were prepared in aqueous solution by treating acetylcholinesterase with Sarin (isopropyl methylphosphonofluoridate) and Soman respectively, and separating the phosphonylated protein from excess inhibitor by column chromatography 11. Both unlabelled and 32P-labelled inhibitors were used. The resulting solutions of inhibited esterase were equivalent to approx. 5 I.U./ml and were used immediately for incubation with rare earth metal ions (I mM) as described above. Samples of the incubation mixtures were taken after various time intervals from 0-24 h and were immediately made o.oi M with respect to EDTA. Duplicate determinations were made on each sample of (I) enzyme activity, (2) unbound radioactivity by the ultrafiltration techniquelL and (3) bound label after exhaustive dialysis. Solutions were assayed for radioactivity either directly using a Geiger-Mtiller counter for liquids (see ref. I I ) or after addition of a dioxane-based scintillator mixture 1'~using a Tritomat 6o12 Liquid Scintillation Counter (Isotope Developments). The results of the present investigations demonstrated that the rare earth metal ions cannot reactivate acetylcholinesterase inhibited by organophosphates either before or after dealkylation of the inhibited enzyme. The experiments carried out with radioactively-labelled inhibited acetylcholinesterase supported the findings of the studies with the unlabelled inhibited enzyme and also demonstrated that no dephosphonylation reaction occurred in the presence of yttrium except in the case of Satininhibited enzyme (Table I). A release of labelled phosphonate occurred in this case only after prolonged incubation (24 h) but there was no reappearance of esterase activity. No explanation for this apparently anomolous observation can be proffered at the present time. However, in parallel experiments using I mM pyridine-2-aldoxime methyl methanesulphonate (P2S) (synthesised at the Chemical Defence Establishment) instead of a rare earth metal ion, the enzyme inhibited b y Sarin was rapidly reactivated with the concomitant release of asp and, as expected, no reactivation and no dephosphonylation of the esterase inhibited by Soman occurred. The presence of P2A and yttrium during reactivation of Sarin-inhibited enzyme b y P2S was found to reduce the activity recovered after i h by about 200/0. The inability of the rare earth metal ions to reactivate undealkylated inhibited enzyme was not totally unexpected as they do not catalyse the hydrolysis of the neuBiochim. Biophys. Acta, 244 (I97 I) 427-431
430
D . j . HOWELLS, D. B. COULT
TABLE I RESULTS PHORUS
OF
INCUBATION
COMPOUNDS
OF
V¢ITtt
ACETYLCHOLINESTERASE
POSSIBLE
INHIBITED
BY
a2p-LABELLED
ORGANOPHOS-
REACTIVATORS
Inhibited acetylcholinesterase (approx. 5 I.U./ml) was incubated with either P2S (i mM) or Yttrium-P2A mixture (i mM and 1.25 mM respectively) at pH 8. 4 and at 37 °. Bound a~p ("a"; counts/Ioo sec per mt of incubation mixture), unbound ~2p ("b"; units as for "a") and enzyme activitv ("c"; I.U./o.2 ml of incubation mixture) were determined as described in the text. Blank values without addition of possible reactivators are given. Inhibitor
Reactivator
Measured value
Sarin
P2S
a b c a b
Yttriunl/P2A
Soman
Incubation time (h) : o I 24
95 1.8 44 ° o
45 32o 3.9 42o o
45 315 4 -I 45 255
Blank
41o o o 35 ° o
C
O
O
O
O
P2S
a b
285 o
285 o
280 o
245 o
C
0
0
0
0
Yttrium/P2A
a b
45 ° o
460 o
44 ° o
590 o
C
0
0
0
0
t r a l e s t e r e t h y l p - n i t r o p h e n y l m e t h y l p h o s p h o n a t e to a n y m a r k e d degree12, la. H o w ever, as t h e r a r e e a r t h m e t a l ions are r e a c t i v e t o w a r d s t h e m o n o b a s i c a c i d e s t e r I I I , t h e r e a c t i o n of t h e s e m a t e r i a l s w i t h t h e p h o s p h o r u s c e n t r e of t h e d e a l k y l a t e d i n h i b i t e d e n z y m e was c o n s i d e r e d likely. T h e r e a s o n s for t h e l a c k of r e a c t i o n w i t h S o m a n - i n h i b i t e d e n z y m e are o b s c u r e at t h e p r e s e n t t i m e b u t in g e n e r a l t e r m s c e r t a i n possibilities m a y be c o n s i d e r e d . I n t h e first p l a c e c o m p o u n d I I I is a v e r y s i m p l e m o l e c u l e c o m p a r e d w i t h t h e p h o s p h o n y l a t e d e n z y m e ; as a r e s u l t t h e p h y s i c o c h e m i c a l e n v i r o n m e n t of t h e p h o s p h o n y l c e n t r e in t h e l a t t e r is m u c h m o r e c o m p l e x t h a n in I I I , a n d it is l i k e l y t h a t c e r t a i n factors, s u c h as t h e steric a n d p o l a r p r o p e r t i e s of this c e n t r e in t h e p r o t e i n , m a y p r e v e n t a t t a c k b y t h e r a r e e a r t h m e t a l ions. S e c o n d l y , t h e r a r e e a r t h m e t a l ions w e r e s h o w n to be i n h i b i t o r s of t h e e s t e r a s e in t h e p r e s e n c e a n d a b s e n c e of P 2 A a n d , as this i n h i b i t i o n is a p p a r e n t l y c o m p e t i t i v e w i t h r e g a r d to s u b s t r a t e , it is p r o b a b l e t h a t t h e s e m a t e r i a l s are b o u n d at or n e a r t h e a c t i v e site of t h e e n z y m e : also t h e r a r e e a r t h m e t a l i o n - P 2 A m i x t u r e a p p a r e n t l y r e d u c e d t h e r e a c t i v a t i o n of S a t i n i n h i b i t e d e n z y m e b y P2S. I t is t h e r e f o r e possible t h a t t h e r a r e e a r t h m e t a l ions are p r e f e r e n t i a l l y b o u n d to a p a r t i c u l a r site on t h e p r o t e i n a n d h e n c e are n o t o n l y u n a b l e to a t t a c k t h e p h o s p h o n y l c e n t r e b u t also i n t e r f e r e w i t h t h e access of o t h e r m o l e c u l e s to t h e p h o s p h o n y l c e n t r e . T h e p r e s e n t s t u d i e s s u g g e s t t h a t I I I is p r o b a b l y a p o o r m o d e l c o m p o u n d for t h e d e a l k y l a t e d i n h i b i t e d e n z y m e ; it m i g h t be of i n t e r e s t t h e r e fore t o s t u d y t h e effects of r a r e e a r t h m e t a l ions o n t h e h y d r o l y s i s of s i m p l e a c i d p h o s p h o n a t e esters h a v i n g an a l k y l g r o u p or a c h a i n of 1,2 or m o r e a m i n o a c i d r e s i d u e s in p l a c e of t h e p - n i t r o p h e n y l l e a v i n g g r o u p . REFERENCES I A. T. LOOMIS AND B. S A L A F S K Y , Toxicol. A p p l . Pharmacol., 5 (I963) 685. 2 I. V~L COLEMAN, P. E. LITTLE AND G. A. GRANT, Can. J. Biochem. Physiol., 38 (196o) lO35. 3 F. HOBBIGER,in G. B. KOELLE, Handbuch der Experimentellen Pharmakologie, Vol. 15, Springer-Verlag, Berlin, 1963. Biochim. Biophys. Acts, 244 (1971) 427-431
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4 A. F. CHILDS, D. R. DAVIES, A. L. GREEN AND J. P. RUTLAND,Br. J. Pharmacol. Chemother., io (1955) 462. 5 ]3. M. ASKEW, Br. J. Pharmacol. Chemother., I I (1956) 417 . 6 F. HOBBIGER, Br. J. Pharmacol. Chemother., 12 (1957) 438. 7 F. BERENDS, C. H. POSTHUMUS, I. V. D. SLUYS AND F. A. DIEIRKAUF, Biochim. Biophys. Acla, 34 (1959) 576 . 8 F. 13ERENDS, Doctoral Dissertation, U n i v e r s i t y of Leiden, 1964. 9 J. H. FLEISCHER AND L. W. HARRIS, Biochem. Pharmacol., 14 (1965) 641. IO W. t{. BERRY AND D. t{. DAVIES, Biochem. J., IOO (1966) 572. I I D. 13. COULT,D. J. MARSH AND G. READ, Biochem. J., 98 (1966) 869. 12 R. j . WITHEY, Can. J. Chem., 47 (1969) 4383 . 13 F. McC. 13LEWETT AND P. WATTS,J. Chem. Soc., in the press. 14 1~. M. KEEN, Br. J. Pharmacol. Chemother., 26 (1966) 7o4 . 15 F. E. 13UTLER, Anal. Chem., 33 (1961) 409.
Biochim. Biophys. Acta, 244 (1971) 427-431