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Inhibition of flyhead acetylcholinesterase by dimethoxon analogs
PESTICIDE
BIOCHEMISTRY
Inhibition
AND
of Flyhead
Acetylcholinesterase
C. T. of Entomology,
Department
4, 249-253
PHYSIOLOQY
HUANG
lVorth
W. C.
AND
Carolina
State
by Dimethoxon
Analogs]
DAUTERMAN
University,
Raleigh,
North
CaroKna
27607
AND
F. L. USDA
Forest
Service,
HASTINGS
Southeastern Forest Experiment Research Triangle Park, North Received
June
1, 1973;
accepted
Station, Carolina
Forestry 27709
February
Sciences
Laboratory,
21, 1974
The inhibition of purified housefly-head acetylcholinesterase by 16 dimethoxon analogs was studied and the phosphorylation and affinity constants determined. Except for compound XV, all the analogs were better inhibitors of flyhead acetylcholinesterase than of bovine erythrocyte acetylcholinesterase. The large bimolecular reaction constants were the result of higher affinity and phosphorylation rates. Mono-substitution on the carbamoyl nitrogen primarily affected reactivity (kz), whereas di-substitution affected affinity (K,). Modification of the 0,0-dialkyl moiety had little effect on affinity although reactivity decreased proportionately with chain length. There is some indication that the interaction of these inhibitors with flyhead AChE is due to a considerable electrostatic contribution. The difference between target enzymes (housefly versus erythrocyte AChE) accounts in part for the selectivity of dimethoate and its analogs.
individual rate constants are kr, k-1, Its, and kS. Main (2) derived equations demonstrating that the inhibition reaction followed first-order kinetics based on the assumptions that k3 = 0, k-1 > kq, and [PX] >> [El. An experimental procedure based on these conditions was developed to solve the following linear form of the inhibition equation (3) :
INTRODUCTION
The inhibition of acetylcholinesterases by organophosphates is considered to involve formation of a reversible complex, EPX, followed by conversion of EPX to the phosphorylated enzyme, EP. (1):
kt kz E+PXGEPX+EP--tE k-1 L
kz Products
iAt/2.3A
+ products, ahertr E is enzyme, PX is an organophosphate consisting of the alkylphosphoryl group P and the leaving group X. The 1 Paper No. 4064 of the Journal Series of the North Carolina State University Agricultural Experiment Station, Raleigh, North Carolina. Work supported in part by PHS Research Grant ES-00044 from the Institute of Environmental Health Sciences. 249 Copyright -111 rights
@ 1974 by Academic Press. Inc. of reprodwtion in any form reserved.
log v = i/k, + l/ki,
where i is the inhibitor concentration, 2.3Alog v/At is the first-order rate constant for a particular value of i, K, = k-l/k1 is the affinity constant, k? is the phosphorvlation constant and ki = k?/K, is the bimolecular reaction constant. Using Main’s kinetics, a number of studies have been made on the effect of
250
HUANG,
DAUTERMAN
structure of carbamates and organophosphates as cholinesterase inhibitors (P-11). Hellenbrand (10) found that housefly acetylcholinesterase appeared to be more sensitive to carbamate inhibition than erythrocyte cholinesterase. Kinetic studies showed that flyhead acetylcholinesterase contained two cation binding sites, one for the substrate and the other for the inhibitor (12) while vertebrate cholinesterases appear to have only one (1). With regard to the esteratic site, flyhead acetylcholinesterase consists of four subunits and one esteratic site (Huang and Dauterman, unpublished work) while vertebrate cholinesterases consist of four subunits and two esteratic sites (13, 14). Since certain differences between flyhead acetylcholinesterase and vertebrate cholinesterases appear to exist, the question arises whether these differences may result in different rates of inhibition by organophosphates. The present study was undertaken to investigate the inhibition of purified flyhead acetylcholinesterase by a series of dimethoxon analogs and to compare these findings with those of a comparable study with bovine erythrocyte acetylcholinesterase (9). MATERIALS
AND
METHODS
Substrates Acetylcholine bromide was obtained from Eastman Organic Chemicals, Rochester, New York and acetylthiocholine iodide was purchased from Sigma Chemical Company, St. Louis, Missouri.
AND
HASTINGS
Enzyme Acetylcholinesterase (EC 3.1.1.7) (AChE) was purified from housefly heads, lMusca domestica (L.) (CSMA strain) according to the method of Huang and Dauterman (15). The purified enzyme used in this study was 1300-fold purified and had a specific activity of 370 units/mg (1 unit hydrolyzed 1 pmole acetylthiocholine per minute at pH 8.0, 25°C). The working solution contains 5.2 units/ml and was prepared in 0.02 M phosphate buffer, pH 7.0 containing 1.0 M sodium chloride. Inhibition
Procedure
The procedure of Main and Iverson (3) was slightly modified. Enzyme (0.2 ml) was placed into one sidearm of the inhibition reaction vessel and 0.2 ml of inhibitor into the other. The inhibition reactions were carried out at 25”C, pH 7.0 for a desired period of time from O-16 sec. The residual enzyme activity was determined at 25°C and pH 7.0 using a Radiometer pH-Stat (Copenhagen, Denmark) in 2.0 mM acetylcholine bromide. The rate of the inhibition reaction of the enzyme with various concentrations of the inhibitor (i) was determined by plotting the log of velocity (v) against time (t). The slopes were calculated by regression analysis to give 2.3 A log v/At or (p) and their standard errors. The K,, kz, and ki values and their standard errors were calculated by regression analysis according to the equation i/p = i/k2 + l/ki where l/k2 is the slope, l/ki is the y intercept and K, = kz/ki.
Inhibitors Dimethoxon and O,O-dimethyl S- (Nisopropylcarbamoylmethyl) phosphorothiolate were kindly supplied by American Cyanamid Company, Princeton, New Jersey and Montecatini Edison, Milano, respectively. The remaining dimethoxon analogs were synthesized and purified as described by Hastings and Dauterman (9).
RESULTS
AND
DISCUSSION
The K, and corresponding kz and ki values of the 16 dimethoxon analogs investigated are given in Table 1. Although compound I was studied by Hastings and Dauterman (9), the data from this compound could not be obtained due to insufficient material.
INHIBITION
OF
FLYHEAD
TABLE Aginity
Constants,
Phosphorylation Against
1
and Bimolecular Reaction Con&ants for Acetylcholinestera.se at WY!, pH 7.0
Constants, HouseJEy-Head
Compound
kz
XIr” II XIII XIV XV XVI XVII
Analogs
Number of coneentrations
-
2
(CHaO)zR (CzHrO)zR (n-CaHrO)aR (iseCaHIO)eR (n-GHs0)2R (C?HnO)zP(O)S CH-CHr-C(O)NHCHx (GHsO)*P(O)S(;‘H-C(O)NHCHa
y K. and ks could
Dimethoxon
ki (M-l min-1) ( x 10”)
(min-1) R==(CzHaO)zP(O) SCH&(O) I RNHz II RNHCHI III RNHCzHh I\’ RNHn-CaH? V RNHiso--CsHI VI RNHn-C,Hs VII RN(CH+? VIII RN(C&.)? IX RN (n-‘&H,) X RN&-&HI)* XI0 RN(n-C,Hs)r F+P~)SCHI-C(0)
251
ACETYLCHOLINESTERASE
not be determined
2.0 1.77 2.0 1.09 1.97 1.14 0.28 0.0287 .376
1x9
2z.5 41.16 98.5
25.2 24.7 13.8 31.1 3.6 20.5 64.4 78.1 -
6%:‘: 63.2 34.95 40.1 9.38 -
lZ3 13.4 19.2 7.5 21 2.1 7.3 19.6 12.3
12z9 23.22 49.1 49.55 33.9 55.6 124.0 1393 24.9 2980
4.46 2.0 2.75 4.2 2.3 7.12
10.5 17.9 24.5 12.0 25.9 11.1
160.6 248.5 37.0 3.13 23.84 26.87
8.4 13.8 17.0 5.4 14.4 6.3
36.0 123.9 13.43 ,745 10.3 3.77
1.2
22.4
140.76
15.4
115.7
since no appreciable
slope
Similar K, values for the N-alkyl compounds (II-VI) were obtained, indicating that the lengthening of the monoalkyl substituent on the amide nitrogen had only a small effect on their binding. However, the K, values of the N,N-dialkyl compounds (VIII-X) were much lower, and here the values decreased as the length of the dialkyl substituent increased from methyl to n-propyl. These results suggest that the second alkyl group in the N,Ndialkyl compounds was involved in binding, resulting in increased affinity. Since K, (k-l/kJ is an equilibrium constant, the calculation of the free energy of binding (AG = RT In l/K,) of the dimethoxon analogs is justified. An average increase in binding energy for each additional methylene group in the N,N-dialkyl series from methyl to n-propyl was about 545 Cal/mole. The increase in binding energy is apparently the result of hydrophobic bonding of each additional methylene group, although the calculated increase in energy was only about 50% of theoretical (16). Similar observations have bren reported in other studies (4-9). The phosphorylation
was measured
in the i/p versus
iY9 12.6 5.7 7.2 11.4 1.7 14.2 45.7 69.4
0.00750.34 0.12 o:;“o I 0.10 0.07 0.01 0.01
- 1.57 - 0.98 - 0.0s - 0.95
2.6 4.9 8.0 7.4 12.5 5.3
0.14 0.00750.21 0.21 0.08 1.04
3.0 1.5 - 2.11 -10.7 - 3.3 -10.4
0.06
-
7.7
1.5 2.71 0.96 as2
0.46
i plot.
constants (kp) of the N,N-dialkyl compounds were smaller than their corresponding N-alkyl analogs. The variation in the k2 values of the two groups of analogs was not affected significantly by an increase in the length of their leaving groups. Therefore, it is concluded that the high bimolecular reaction constants (ki) of the N,N-dialkyl compounds is mainly due to their good affinities. The K, value of the di-n-butyl analog was not determined since no appreciable slope in the i/p versus i was obtained over a lo-fold range of inhibitor concentration. The K, values of the O,O-dialkyl compounds (XII-XV) were relatively larger than compound II, but the variation bctween them was small. The results indicated that the acyl-binding site of the enzyme was fully occupied by one or both methylene groups of the alkyl phosphoryl portion of the inhibitor. Apparently there was no additional hydrophobic bonding bctween the enzyme and the inhibitor with an increase in the methylene substituents. Similar findings were reported in studies with O,O-dialkyl malaoxon, paraoxon and
252
HUANG,
TABLE
DAUTERMAN
2
Comparison of Standard Free Energies of Binding of Compounds ZZ, XVI and XVIZ with Flyhead and Bovine Erythrocyte Acetylcholinesterase ___-Inhibitor
Enzyme
Flyhead AChE Ik&e
0. Data
eryth’
from
K. (mM)
AC” (Cd mole-‘)
AAG” (Cal mole-‘)
x::
2.0
z;:g
-756
XVII x::
::i2 17.2 16.8
-3982 -2421 -2435
-
XVII
21.9
-2263
reference
14
(9).
dimethoxon analogs and bovine erythrocyte AChE (5, 9). The phosphorylation constants of the dialkylphosphoryl compounds (II, XIIXV) ranged from a high of 248 min-’ for compound II to a low of about 3 mill-’ for compound XIV. The kp values were in decreasing order: diethyl > dimethpl > dirz-propyl > di-n-butyl > di-iso-propyl. By contrast, with bovine erythrocyte AChE the k, values increased with an increase in chain length (9). Hastings and Dauterman postulated that the longer dialkylphosphoryl substituents influenced reactivity by some form of attachment to the enzyme. The nature of the binding was unknown but it probably was not hydrophobic. A similar explanation might apply to the housefly enzyme but instead of enhancing reactivity, the larger substituents decreased k?. The complementary fit of an alkylphosphoryl group to the esteratic site of bovine crythrocyte AChE appears to be important for phosphorylation when binding is involved at the anionic site (9, 17). The distance between these two sites in housefly-head AChE and bovine erythrocyte AChE has been reported to be 4.;i5.9 A and 2.5-4.5 A, respectively (18). Compound XVI has an additional methylcne group between the phosphorus atom and the carbonyl, the distance between these two atoms being increased to 3.36.0 A. The affinity decreased 3.6-fold and the phosphorylation rate constant was 9 times lower than compound II, its closest
AND
HASTINGS
analog. The decrease in affinity and k2 might be explained in terms of a decreased inductive effect which would render the phosphorus atom less electrophilic, or that steric hindrance of the extra methylene prevented optimum orientation and reactivity of the enzyme-inhibitor complex. On the other hand, the bond distance between the phosphorus and carbonyl atoms in compound XVII was the same as other dimethoxon analogs (2.7-4.7) although il had an additional methyl group attached to the single methylene between the sulfur and carbonyl carbon. There was very lit,tle change in the bimolecular reaction const,ant as compared to compound II, although binding increased 40% and phosphorylation rate (lc,) decreased 43%. Comparison of the standard free-energy of binding of compounds II and XVII with flyhead AChE resulted in only 300 cal mole-r difference, indicating that the extra methyl group did not comribute significantly to hydrophobic binding (Table 2). However, a large difference in AG was observed between compounds II and XVI (-756 cal mole-‘). By contrast, variation in the structure of compound II to form XVI and XVII had no effect on binding for the bovine AChE. This would suggest that factors controlling K, are different for the two enzymes. The possibility that there is a considerable electrostatic contribution for the binding to flyhead AChE of compounds II and XVII is in keeping with the findings concerning the possible distribution of the cation binding sites on these two enzymes. The fact t,hat all dimethoxon analogs (with the exception of compound X) had higher affinities for housefly AChE than crythrocyte AChE supports this contention. It is interesting to compare the K,, kz and k; values of housefly AChE (Table 1) and bovine erythrocytc AChE (9). Except for the N,N-di-iso-propyl compound all dimethoxon analogs had higher affinities for housefly AChE than for erythrocytc:
INHIBITION
OF
FLYHEAD
AChE (2.k2.9-fold). Comparing the kt values, compounds XIII and XV had higher phosphorylation rates with erythrocyte AChE than with flyhead AChE while the remaining compounds all phosphorylated flyhead AChE much faster. The bimolecular reaction constant of compound XIII was approximately equal with both enzymes, whereas t,hc Ici for compound XV and crythrocyte AChE was 2.7-fold faster; the remaining compounds had higher k; values with flyhead AChE. The data indicate that a difference does exist in the binding and phosphorylation rates between housefly AChE and bovine erythrocyte AChE wi’h analogs of dimethoxon. The evidence presented by Chen and Dauterman (19) indicates no correlation between carboxyamidase degradation of dimethoatc analogs and their toxicity. Therefore, the role of amidase activity in selectivity appears to be of minor importanc(l. The present data show a difference in the inhibition of flyhead AChE and erythrocyte AChE and would suggest that the difference in the target enzymes may account in part for the selectivity of dimethoate and it’s analogs. REFERENCES
1. I. B. Wilson, “The Enzymes,” P. D. Boyer, H. Lardy, and K. Myrblck, Eds.), 2nd ed. vol. 4, p. 501; Academic Press, New York, 1960. 2. A. R. Main, Affinity and phosphorylation constants for the inhibition of esterases by organophosphates, Science 144, 992 (1964). 3. A. R. Main and F. Iverson, Measurement of t,he affinity and phosphorylation constants governing irreversible inhibition of cholinesterases by di-isopropyl phosphorofluoridate, Hiochem. J. 100, 825 (1966). 4. A. R. Main and F. L. Hastings, A comparison of acetylation, phosphorylation and binding in related substrates and inhibitors of serum cholinesterase, Biochem. J. 101, 584 (1966). -5. Y. C. Chiu, A. 12. Main, and W. C. Dauterman, Affinity and phosphorylation constants of a series of O,&dialkyl malaoxons and paraoxons
253
ACETYLCHOLINESTERASE
with
acetylcholinesterase,
18, 2171
Biochem.
PhaTnzacol.
(1969).
6. P. Bracha and R. D. O’Brien, Trialkyl phosphate and phosphorothiolate anticholinest,erases. I. Amiton analogs, Biochemistry 7, 1545 (1968). 7. P. Bracha and R. D. O’Brien, Trialkyl phosphate and phosporothiolate anticholinesterases. II. Effect of chain length on potency, Biochemistry 7, 1555 (1968). 8. P. Bracha and R. D. O’Brien, Hydrophobic bonding of trialkyl phosphates and phosphorothiolate to acetylcholinesterases, Riochemistry 9, 741 (1970). 9. F. L. Hastings and W. C. Dauterman, Phosphorylation and affinity constants for the inhibition of acetylcholinesterase by dimethoxon analogs, Pestic. Biochem. and Physiol. 1, 248 (1971). 10. K. Hellenbrand, Inhibition of housefly acetylcholinesterase by carbamates, J. Agr. Food Chem. 1.5, 825 (1967). 11. J. H. Davies, W. R. Campbell, and C. W. Kearns, Inhibition of fly head acetylcholinesterase by bis-[ (nz-hydroxyphenylj-trimethylammonium iodide] esters of polymethylenedicarbamic acids, Biochem. J. 117, 221 (1970). 12. IX. Hellenbrand and R. M. Krupka, Kinetic studies on the mechanism of insect acetylcholinesterase, Biochemistry 9, 4665 (1970). 1% W. Leuzinger, M. Goldberg, and E. Cauvin, Molecular properties of acetylcholinesterase, J. Mol. Biol. 40, 217 (1969). 14. A. It. Main, E. Tarkan, J. L. Aull, and W. G. Soucie, Purification of horse serum cholinesterase by preparative polyacrylamide gel electrophoresis, J. Biol. Chem. 247, 566 (1972). 15. C. T. Huang and W. C. Dauterman, Purification of flyhead cholinesterase, J. Ins& Biocham. 3, 325 (1973). 16. C. Tanford, “Physical chemistry of macromolecules,” p. 130, Wiley, New York, 1961. 17. Y. C. Chiu and W. C. Dauterman, The affinity and phosphorylation constants of a series of branched-chain homologs of diethyl malaoxon and acetoxon with acetylcholinesterase, Biochm. Pharmacol. 18, 1665 (1969). 18. W. B. Neely, The use of molecular orbital calculations as an aid to correlate the structure and activity of cholinesterase inhibitors, iVIol. Pharmacol. 1, 137 (1965). 19. P. R. S. Chen and W. C. Dauterman, Studies on the toxicity of dimethoate analogs and their hydrolysis by sheep liver amidase, Pestic. Riochem. and Physiol. 1, 340 (1971).
BIOCHEMISTRY
Inhibition
AND
of Flyhead
Acetylcholinesterase
C. T. of Entomology,
Department
4, 249-253
PHYSIOLOQY
HUANG
lVorth
W. C.
AND
Carolina
State
by Dimethoxon
Analogs]
DAUTERMAN
University,
Raleigh,
North
CaroKna
27607
AND
F. L. USDA
Forest
Service,
HASTINGS
Southeastern Forest Experiment Research Triangle Park, North Received
June
1, 1973;
accepted
Station, Carolina
Forestry 27709
February
Sciences
Laboratory,
21, 1974
The inhibition of purified housefly-head acetylcholinesterase by 16 dimethoxon analogs was studied and the phosphorylation and affinity constants determined. Except for compound XV, all the analogs were better inhibitors of flyhead acetylcholinesterase than of bovine erythrocyte acetylcholinesterase. The large bimolecular reaction constants were the result of higher affinity and phosphorylation rates. Mono-substitution on the carbamoyl nitrogen primarily affected reactivity (kz), whereas di-substitution affected affinity (K,). Modification of the 0,0-dialkyl moiety had little effect on affinity although reactivity decreased proportionately with chain length. There is some indication that the interaction of these inhibitors with flyhead AChE is due to a considerable electrostatic contribution. The difference between target enzymes (housefly versus erythrocyte AChE) accounts in part for the selectivity of dimethoate and its analogs.
individual rate constants are kr, k-1, Its, and kS. Main (2) derived equations demonstrating that the inhibition reaction followed first-order kinetics based on the assumptions that k3 = 0, k-1 > kq, and [PX] >> [El. An experimental procedure based on these conditions was developed to solve the following linear form of the inhibition equation (3) :
INTRODUCTION
The inhibition of acetylcholinesterases by organophosphates is considered to involve formation of a reversible complex, EPX, followed by conversion of EPX to the phosphorylated enzyme, EP. (1):
kt kz E+PXGEPX+EP--tE k-1 L
kz Products
iAt/2.3A
+ products, ahertr E is enzyme, PX is an organophosphate consisting of the alkylphosphoryl group P and the leaving group X. The 1 Paper No. 4064 of the Journal Series of the North Carolina State University Agricultural Experiment Station, Raleigh, North Carolina. Work supported in part by PHS Research Grant ES-00044 from the Institute of Environmental Health Sciences. 249 Copyright -111 rights
@ 1974 by Academic Press. Inc. of reprodwtion in any form reserved.
log v = i/k, + l/ki,
where i is the inhibitor concentration, 2.3Alog v/At is the first-order rate constant for a particular value of i, K, = k-l/k1 is the affinity constant, k? is the phosphorvlation constant and ki = k?/K, is the bimolecular reaction constant. Using Main’s kinetics, a number of studies have been made on the effect of
250
HUANG,
DAUTERMAN
structure of carbamates and organophosphates as cholinesterase inhibitors (P-11). Hellenbrand (10) found that housefly acetylcholinesterase appeared to be more sensitive to carbamate inhibition than erythrocyte cholinesterase. Kinetic studies showed that flyhead acetylcholinesterase contained two cation binding sites, one for the substrate and the other for the inhibitor (12) while vertebrate cholinesterases appear to have only one (1). With regard to the esteratic site, flyhead acetylcholinesterase consists of four subunits and one esteratic site (Huang and Dauterman, unpublished work) while vertebrate cholinesterases consist of four subunits and two esteratic sites (13, 14). Since certain differences between flyhead acetylcholinesterase and vertebrate cholinesterases appear to exist, the question arises whether these differences may result in different rates of inhibition by organophosphates. The present study was undertaken to investigate the inhibition of purified flyhead acetylcholinesterase by a series of dimethoxon analogs and to compare these findings with those of a comparable study with bovine erythrocyte acetylcholinesterase (9). MATERIALS
AND
METHODS
Substrates Acetylcholine bromide was obtained from Eastman Organic Chemicals, Rochester, New York and acetylthiocholine iodide was purchased from Sigma Chemical Company, St. Louis, Missouri.
AND
HASTINGS
Enzyme Acetylcholinesterase (EC 3.1.1.7) (AChE) was purified from housefly heads, lMusca domestica (L.) (CSMA strain) according to the method of Huang and Dauterman (15). The purified enzyme used in this study was 1300-fold purified and had a specific activity of 370 units/mg (1 unit hydrolyzed 1 pmole acetylthiocholine per minute at pH 8.0, 25°C). The working solution contains 5.2 units/ml and was prepared in 0.02 M phosphate buffer, pH 7.0 containing 1.0 M sodium chloride. Inhibition
Procedure
The procedure of Main and Iverson (3) was slightly modified. Enzyme (0.2 ml) was placed into one sidearm of the inhibition reaction vessel and 0.2 ml of inhibitor into the other. The inhibition reactions were carried out at 25”C, pH 7.0 for a desired period of time from O-16 sec. The residual enzyme activity was determined at 25°C and pH 7.0 using a Radiometer pH-Stat (Copenhagen, Denmark) in 2.0 mM acetylcholine bromide. The rate of the inhibition reaction of the enzyme with various concentrations of the inhibitor (i) was determined by plotting the log of velocity (v) against time (t). The slopes were calculated by regression analysis to give 2.3 A log v/At or (p) and their standard errors. The K,, kz, and ki values and their standard errors were calculated by regression analysis according to the equation i/p = i/k2 + l/ki where l/k2 is the slope, l/ki is the y intercept and K, = kz/ki.
Inhibitors Dimethoxon and O,O-dimethyl S- (Nisopropylcarbamoylmethyl) phosphorothiolate were kindly supplied by American Cyanamid Company, Princeton, New Jersey and Montecatini Edison, Milano, respectively. The remaining dimethoxon analogs were synthesized and purified as described by Hastings and Dauterman (9).
RESULTS
AND
DISCUSSION
The K, and corresponding kz and ki values of the 16 dimethoxon analogs investigated are given in Table 1. Although compound I was studied by Hastings and Dauterman (9), the data from this compound could not be obtained due to insufficient material.
INHIBITION
OF
FLYHEAD
TABLE Aginity
Constants,
Phosphorylation Against
1
and Bimolecular Reaction Con&ants for Acetylcholinestera.se at WY!, pH 7.0
Constants, HouseJEy-Head
Compound
kz
XIr” II XIII XIV XV XVI XVII
Analogs
Number of coneentrations
-
2
(CHaO)zR (CzHrO)zR (n-CaHrO)aR (iseCaHIO)eR (n-GHs0)2R (C?HnO)zP(O)S CH-CHr-C(O)NHCHx (GHsO)*P(O)S(;‘H-C(O)NHCHa
y K. and ks could
Dimethoxon
ki (M-l min-1) ( x 10”)
(min-1) R==(CzHaO)zP(O) SCH&(O) I RNHz II RNHCHI III RNHCzHh I\’ RNHn-CaH? V RNHiso--CsHI VI RNHn-C,Hs VII RN(CH+? VIII RN(C&.)? IX RN (n-‘&H,) X RN&-&HI)* XI0 RN(n-C,Hs)r F+P~)SCHI-C(0)
251
ACETYLCHOLINESTERASE
not be determined
2.0 1.77 2.0 1.09 1.97 1.14 0.28 0.0287 .376
1x9
2z.5 41.16 98.5
25.2 24.7 13.8 31.1 3.6 20.5 64.4 78.1 -
6%:‘: 63.2 34.95 40.1 9.38 -
lZ3 13.4 19.2 7.5 21 2.1 7.3 19.6 12.3
12z9 23.22 49.1 49.55 33.9 55.6 124.0 1393 24.9 2980
4.46 2.0 2.75 4.2 2.3 7.12
10.5 17.9 24.5 12.0 25.9 11.1
160.6 248.5 37.0 3.13 23.84 26.87
8.4 13.8 17.0 5.4 14.4 6.3
36.0 123.9 13.43 ,745 10.3 3.77
1.2
22.4
140.76
15.4
115.7
since no appreciable
slope
Similar K, values for the N-alkyl compounds (II-VI) were obtained, indicating that the lengthening of the monoalkyl substituent on the amide nitrogen had only a small effect on their binding. However, the K, values of the N,N-dialkyl compounds (VIII-X) were much lower, and here the values decreased as the length of the dialkyl substituent increased from methyl to n-propyl. These results suggest that the second alkyl group in the N,Ndialkyl compounds was involved in binding, resulting in increased affinity. Since K, (k-l/kJ is an equilibrium constant, the calculation of the free energy of binding (AG = RT In l/K,) of the dimethoxon analogs is justified. An average increase in binding energy for each additional methylene group in the N,N-dialkyl series from methyl to n-propyl was about 545 Cal/mole. The increase in binding energy is apparently the result of hydrophobic bonding of each additional methylene group, although the calculated increase in energy was only about 50% of theoretical (16). Similar observations have bren reported in other studies (4-9). The phosphorylation
was measured
in the i/p versus
iY9 12.6 5.7 7.2 11.4 1.7 14.2 45.7 69.4
0.00750.34 0.12 o:;“o I 0.10 0.07 0.01 0.01
- 1.57 - 0.98 - 0.0s - 0.95
2.6 4.9 8.0 7.4 12.5 5.3
0.14 0.00750.21 0.21 0.08 1.04
3.0 1.5 - 2.11 -10.7 - 3.3 -10.4
0.06
-
7.7
1.5 2.71 0.96 as2
0.46
i plot.
constants (kp) of the N,N-dialkyl compounds were smaller than their corresponding N-alkyl analogs. The variation in the k2 values of the two groups of analogs was not affected significantly by an increase in the length of their leaving groups. Therefore, it is concluded that the high bimolecular reaction constants (ki) of the N,N-dialkyl compounds is mainly due to their good affinities. The K, value of the di-n-butyl analog was not determined since no appreciable slope in the i/p versus i was obtained over a lo-fold range of inhibitor concentration. The K, values of the O,O-dialkyl compounds (XII-XV) were relatively larger than compound II, but the variation bctween them was small. The results indicated that the acyl-binding site of the enzyme was fully occupied by one or both methylene groups of the alkyl phosphoryl portion of the inhibitor. Apparently there was no additional hydrophobic bonding bctween the enzyme and the inhibitor with an increase in the methylene substituents. Similar findings were reported in studies with O,O-dialkyl malaoxon, paraoxon and
252
HUANG,
TABLE
DAUTERMAN
2
Comparison of Standard Free Energies of Binding of Compounds ZZ, XVI and XVIZ with Flyhead and Bovine Erythrocyte Acetylcholinesterase ___-Inhibitor
Enzyme
Flyhead AChE Ik&e
0. Data
eryth’
from
K. (mM)
AC” (Cd mole-‘)
AAG” (Cal mole-‘)
x::
2.0
z;:g
-756
XVII x::
::i2 17.2 16.8
-3982 -2421 -2435
-
XVII
21.9
-2263
reference
14
(9).
dimethoxon analogs and bovine erythrocyte AChE (5, 9). The phosphorylation constants of the dialkylphosphoryl compounds (II, XIIXV) ranged from a high of 248 min-’ for compound II to a low of about 3 mill-’ for compound XIV. The kp values were in decreasing order: diethyl > dimethpl > dirz-propyl > di-n-butyl > di-iso-propyl. By contrast, with bovine erythrocyte AChE the k, values increased with an increase in chain length (9). Hastings and Dauterman postulated that the longer dialkylphosphoryl substituents influenced reactivity by some form of attachment to the enzyme. The nature of the binding was unknown but it probably was not hydrophobic. A similar explanation might apply to the housefly enzyme but instead of enhancing reactivity, the larger substituents decreased k?. The complementary fit of an alkylphosphoryl group to the esteratic site of bovine crythrocyte AChE appears to be important for phosphorylation when binding is involved at the anionic site (9, 17). The distance between these two sites in housefly-head AChE and bovine erythrocyte AChE has been reported to be 4.;i5.9 A and 2.5-4.5 A, respectively (18). Compound XVI has an additional methylcne group between the phosphorus atom and the carbonyl, the distance between these two atoms being increased to 3.36.0 A. The affinity decreased 3.6-fold and the phosphorylation rate constant was 9 times lower than compound II, its closest
AND
HASTINGS
analog. The decrease in affinity and k2 might be explained in terms of a decreased inductive effect which would render the phosphorus atom less electrophilic, or that steric hindrance of the extra methylene prevented optimum orientation and reactivity of the enzyme-inhibitor complex. On the other hand, the bond distance between the phosphorus and carbonyl atoms in compound XVII was the same as other dimethoxon analogs (2.7-4.7) although il had an additional methyl group attached to the single methylene between the sulfur and carbonyl carbon. There was very lit,tle change in the bimolecular reaction const,ant as compared to compound II, although binding increased 40% and phosphorylation rate (lc,) decreased 43%. Comparison of the standard free-energy of binding of compounds II and XVII with flyhead AChE resulted in only 300 cal mole-r difference, indicating that the extra methyl group did not comribute significantly to hydrophobic binding (Table 2). However, a large difference in AG was observed between compounds II and XVI (-756 cal mole-‘). By contrast, variation in the structure of compound II to form XVI and XVII had no effect on binding for the bovine AChE. This would suggest that factors controlling K, are different for the two enzymes. The possibility that there is a considerable electrostatic contribution for the binding to flyhead AChE of compounds II and XVII is in keeping with the findings concerning the possible distribution of the cation binding sites on these two enzymes. The fact t,hat all dimethoxon analogs (with the exception of compound X) had higher affinities for housefly AChE than crythrocyte AChE supports this contention. It is interesting to compare the K,, kz and k; values of housefly AChE (Table 1) and bovine erythrocytc AChE (9). Except for the N,N-di-iso-propyl compound all dimethoxon analogs had higher affinities for housefly AChE than for erythrocytc:
INHIBITION
OF
FLYHEAD
AChE (2.k2.9-fold). Comparing the kt values, compounds XIII and XV had higher phosphorylation rates with erythrocyte AChE than with flyhead AChE while the remaining compounds all phosphorylated flyhead AChE much faster. The bimolecular reaction constant of compound XIII was approximately equal with both enzymes, whereas t,hc Ici for compound XV and crythrocyte AChE was 2.7-fold faster; the remaining compounds had higher k; values with flyhead AChE. The data indicate that a difference does exist in the binding and phosphorylation rates between housefly AChE and bovine erythrocyte AChE wi’h analogs of dimethoxon. The evidence presented by Chen and Dauterman (19) indicates no correlation between carboxyamidase degradation of dimethoatc analogs and their toxicity. Therefore, the role of amidase activity in selectivity appears to be of minor importanc(l. The present data show a difference in the inhibition of flyhead AChE and erythrocyte AChE and would suggest that the difference in the target enzymes may account in part for the selectivity of dimethoate and it’s analogs. REFERENCES
1. I. B. Wilson, “The Enzymes,” P. D. Boyer, H. Lardy, and K. Myrblck, Eds.), 2nd ed. vol. 4, p. 501; Academic Press, New York, 1960. 2. A. R. Main, Affinity and phosphorylation constants for the inhibition of esterases by organophosphates, Science 144, 992 (1964). 3. A. R. Main and F. Iverson, Measurement of t,he affinity and phosphorylation constants governing irreversible inhibition of cholinesterases by di-isopropyl phosphorofluoridate, Hiochem. J. 100, 825 (1966). 4. A. R. Main and F. L. Hastings, A comparison of acetylation, phosphorylation and binding in related substrates and inhibitors of serum cholinesterase, Biochem. J. 101, 584 (1966). -5. Y. C. Chiu, A. 12. Main, and W. C. Dauterman, Affinity and phosphorylation constants of a series of O,&dialkyl malaoxons and paraoxons
253
ACETYLCHOLINESTERASE
with
acetylcholinesterase,
18, 2171
Biochem.
PhaTnzacol.
(1969).
6. P. Bracha and R. D. O’Brien, Trialkyl phosphate and phosphorothiolate anticholinest,erases. I. Amiton analogs, Biochemistry 7, 1545 (1968). 7. P. Bracha and R. D. O’Brien, Trialkyl phosphate and phosporothiolate anticholinesterases. II. Effect of chain length on potency, Biochemistry 7, 1555 (1968). 8. P. Bracha and R. D. O’Brien, Hydrophobic bonding of trialkyl phosphates and phosphorothiolate to acetylcholinesterases, Riochemistry 9, 741 (1970). 9. F. L. Hastings and W. C. Dauterman, Phosphorylation and affinity constants for the inhibition of acetylcholinesterase by dimethoxon analogs, Pestic. Biochem. and Physiol. 1, 248 (1971). 10. K. Hellenbrand, Inhibition of housefly acetylcholinesterase by carbamates, J. Agr. Food Chem. 1.5, 825 (1967). 11. J. H. Davies, W. R. Campbell, and C. W. Kearns, Inhibition of fly head acetylcholinesterase by bis-[ (nz-hydroxyphenylj-trimethylammonium iodide] esters of polymethylenedicarbamic acids, Biochem. J. 117, 221 (1970). 12. IX. Hellenbrand and R. M. Krupka, Kinetic studies on the mechanism of insect acetylcholinesterase, Biochemistry 9, 4665 (1970). 1% W. Leuzinger, M. Goldberg, and E. Cauvin, Molecular properties of acetylcholinesterase, J. Mol. Biol. 40, 217 (1969). 14. A. It. Main, E. Tarkan, J. L. Aull, and W. G. Soucie, Purification of horse serum cholinesterase by preparative polyacrylamide gel electrophoresis, J. Biol. Chem. 247, 566 (1972). 15. C. T. Huang and W. C. Dauterman, Purification of flyhead cholinesterase, J. Ins& Biocham. 3, 325 (1973). 16. C. Tanford, “Physical chemistry of macromolecules,” p. 130, Wiley, New York, 1961. 17. Y. C. Chiu and W. C. Dauterman, The affinity and phosphorylation constants of a series of branched-chain homologs of diethyl malaoxon and acetoxon with acetylcholinesterase, Biochm. Pharmacol. 18, 1665 (1969). 18. W. B. Neely, The use of molecular orbital calculations as an aid to correlate the structure and activity of cholinesterase inhibitors, iVIol. Pharmacol. 1, 137 (1965). 19. P. R. S. Chen and W. C. Dauterman, Studies on the toxicity of dimethoate analogs and their hydrolysis by sheep liver amidase, Pestic. Riochem. and Physiol. 1, 340 (1971).