Physical aspects of the inhibition of enzymes by hydrocarbons: The inhibition of α-chymotrypsin by chlorinated aromatics and alkanes

ENVIRONMENTAL

RESEARCH

36, 193-205 (1985)

Physical Aspects of the Inhibition of Enzymes by Hydrocarbons: The Inhibition of wchymotrypsin by Chlorinated Aromatics and Alkanes ROBERT GuelphBiochemistry,

H. LAW, ALAN

MELLORS,

AND F. Ross HALLETT”

Warerloo Centre

for Graduate Work in Chemistry, and *GuelphWaterloo Program for Graduate Physics, University of Guelph, Guelph. Onturio

Department of Chemistry and Work in Physics, Depurtmenf of NIG 2W1, Canuda

Accepted March 6, 1984 The inhibition of a-chymotrypsin by a series of chlorinated hydrocarbons, including polychlorinated biphenyls, has been studied. The solubility of the hydrocarbons was determined by autocorrelation analysis of light scattering. Kinetic analysis indicated that inhibition of the enzyme occurs when l-2 molecules of inhibitor bind per molecule of enzyme. Chlorinated aromatics including polychlorinated biphenyls were more potent than monochloroalkanes in inhibition of the enzyme. Considerable inhibition was seen when some compounds were present as micelles. Molar volume correlations suggest that chlorinated hydrocarbons exert effects on soluble enzymes similar to their effects on membrane-bound enzymes, and that a membrane lipid phase is not essential for this type of inhibition. 0 1985 Academic

Press. Inc.

INTRODUCTION

Physical toxicity in biological systems is shown by many substances and is primarily determined by the physical properties of those substances, and not by their chemical properties (McGowan, 1954). Physically toxic effects can be caused by compounds such as the inert gases that do not undergo ionic, covalent, or hydrogen bonding under physiological conditions (White, 1974). Physical toxicity often correlates well with hydrophobicity, and there is little indication of chemical or stereochemical specificity. On the other hand, chemical toxicity is dependent on specific functional groups and there is a high degree of stereospecificity, as for example in the binding of agonists or antagonists to receptors. Physical toxicity has been demonstrated for many living organisms, for isolated cells and tissues, and for membrane-bound enzymes (Sharom and Mellors, 1980). Physical toxicity, one aspect of which is general anesthesia, has been correlated with many molecular factors, especially volume (McGowan, 1952), surface area (Miller and Hildebrand, 1968), and partition coefficients between water and organic liquids (Richet, 1893). It is not clear whether membrane lipids are involved, or if hydrophobic protein areas in essential enzymes satisfy all the requirements of the critical biophase for physical toxicity. The hydrophobicity and lack of specific structural requirements for physically toxic substances have suggested that the cell membrane is a probable site of action. The membrane is generally accepted as the site of anesthetic or narcotic action, though there is some question as to whether it is the bulk bilayer or the boundary layer of lipid around membrane-bound proteins that is the site of drug action (Franks and Lieb, 1978). A 193 0013-9351/85 $3.00 Copyright 0 1985 by Academic Press, Inc. All rights of reproduction in any form reserved.

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third possibility is that the anesthetic acts at the hydrophobic regions within proteins (Johnson et al., 1954), and this study describes a test of this theory by searching for physical toxicity in a soluble, lipid-free, enzyme system. cx-Chymotrypsin was chosen as a model in this study because it is a water-soluble, lipidfree, well-characterized enzyme, in which the only hydrophobic areas available for interaction with physically toxic compounds would be amino acid residues in the enzyme itself. A series of chlorine-substituted alkanes and aromatics was used to inhibit o-chymotrypsin. Light-scattering techniques were used to determine the concentrations at which micellar aggregates of hydrocarbons appeared. MATERIALS

AND METHODS

Materials

a-Chymotrypsin (a-CT, EC 3.4.21.1) (Type II, three times crystallized from bovine pancreas), benzoyl-L-tyrosine ethyl ester (BTEE), and N-acetyl-L-tyrosine ethyl ester (ATEE) were obtained from Sigma Chemical Company (St. Louis, MO.). The chlorinated biphenyl inhibitors, 4-chlorobiphenyl (4-PCB), 2,2’-dichlorobiphenyl (2,2’-PCB), and 2,2’,4,5tetrachlorobiphenyl (2,2’,4,5,-PCB) were obtained from Ultra Scientific Ltd.; chloroheptane, chloroctane, and chlorodecane were purchased from Aldrich Chemical Company (Milwaukee, Wise.); other chlorinated hydrocarbons were kindly donated by Dr. N. Bunce and were from BDH Chemical Co. (Toronto, Ontario) or Aldrich Chemical Company. Enzyme Assays

The activity of the (w-CT was checked by hydrolysis of benzoyl-L-tyrosine ethyl ester (Hummel, 1959). One unit is equivalent to 1 kmole hydrolysedlmin at pH 7.8 and 25°C and a sp act of 39.5 units/mg enzyme was determined. Inhibition studies were performed using ATEE hydrolysis monitored by a pH-stat consisting of an automatic titrator, recorder, and a syringe burette (Radiometer Ltd., Copenhagen). The substrate was 2 mM ATEE solution in 0.1 M KCl. The a-CT solution was prepared by dissolving 5 mg of enzyme protein in 50 ml of cold dilute HCl (pH 3.0). The ATEE solution (20 ml) was equilibrated at 25.o”C, adjusted to pH 8.0 by the addition of 0.05 M NaOH, and then maintained at this pH by the automatic addition of 0.005 M NaOH from the syringe burette. A 50 u.1aliquot of the enzyme solution was added to the substrate in the reaction vessel, giving a final enzyme concentration of 250 rig/ml. Inhibitors were added in 0.1 ml dimethylsulfoxide (DMSO) to the 20-ml substrate solution, and preincubated with stirring at 37°C for 2 hr prior to addition of enzyme. Controls contained substrate preincubated with DMSO only. The amount of base added to maintain a pH of 8.0 in a reaction mixture containing 20 ml of 2 mM ATEE in 0.1 M KCl, 100 t.~.lof DMSO, and 5 mg of a-CT protein, at 25”C, over a period of 8 min was used as the measure of enzyme activity after subtraction of the base addition necessary to maintain a constant pH in the absence of enzyme. From a plot of percentage of control activity versus log[inhibitor], the concentration of inhibitor that gave 50% inhibition (I,,) was estimated.

ENZYME

Culculation

of Churacteristic

INHIBITION

BY

HYDROCARBONS

195

Volume

The characteristic volume (V,v) of each inhibitor compound was calculated by the method described previously (McGowan et al., 1979). The characteristic atomic volumes of the atoms in the inhibitor molecule were added together, and a value was subtracted for each covalent bond between atoms, the same value being used for all bond types whether single, double, or triple. This type of calculation can be illustrated by determining the characteristic volume of the compound chlorobenzene. The characteristic atomic volumes for the component atoms are: carbon, 1.635 x lo-“; hydrogen, 0.871 X 10e5; and chlorine, 2.095 x 10es m3 mole-‘. Chlorobenzene is composed of six carbon atoms, five hydrogen atoms, one chlorine atom, and twelve bonds between these atoms. No differentiation is made between single or double bonds and each is assigned a contribution of -0.656 x lop5 m3 mole-’ to the characteristic volume of the molecule. The calculated value of V,yfor chlorobenzene is therefore: 6(1.635) + X0.871) + (2.095) + 12(-0.656) = 8.388 x lo-” m3 mole-‘. It has been shown that the physical properties of a compound, such as its partition coefficient or its physical toxicity, is related to the characteristic volume multiplied by an experimentally determined constant, k, in the form -log x = kV, + E. In this study we examined the relationship between inhibition of the enzyme (r-CT and the characteristic volume of several chlorinated hydrocarbons by plotting the -log of the inhibition constant against the V, of each compound. The constant used was 36,000 which is the experimentally determined constant for relating a compound’s characteristic volume with its partition coefficient for nonassociating solvents. The interaction term E is solely determined by the single chlorine atom on each molecule, and should be a constant, although the absolute value has not been determined for this system. Quasi-elastic Light Scattering Quasi-elastic light scattering was used to estimate the solubility characteristics of the hydrophobic inhibitors used in this study. A 15 mW helium-neon laser (Jodon Eng. Assoc. Inc.) producing light of wavelength 6328 A was used to illuminate the sample. The scattering chamber has been described in detail elsewhere (Racey et al., 1981). The detection system used was a PAR Instruments Inc. quantum photometer (Model 1140A with amplifier/discriminator model 114OC), set at a scattering angle of 90”, and linked to a DANA counter (Model 8010) and a Langley-Ford Autocorrelator (Model 1096). All solutions used were filtered through a 0.2~km polycarbonate filter (Gelman Inc.) to remove dust particles. The inhibitors tested were added to 10 ml of 0.1 M KC1 in 50-p,l aliquots of DMSO and stirred for 2 hr at 37°C in a sealed container. Samples were then transferred to optical cuvettes for spectroscopy. We used this system to indicate the concentration of inhibitor at which micelles or particles begin to appear in solution. Analysis of the experimental scattering functions from the autocorrelator to obtain particle sizes was performed using the method of cumulants (Koppel, 1972). For these studies, the first cumulant only was used to provide the average

196

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loo50-

AND HALLETT

L-:I

b

-2

-1

100 -

s = p ; i? t

50/If

i,

-4

-3

-2

C

I

f

-4

-3 Log Concsntntlon

-2 (M)

FIG. 1. The inhibition of a-chymotrypsin by three chlorinated hydrocarbons (open symbols) compared with particle formation in 0. I M KC1 as measured by light scattering (closed symbols). The bars show the SEM for triplicate determinations of cu-chymotrypsin, as a percentage of controls. (a) Ichloropropane; (b) I-chlorodecane; (c) 3-chlorotoluene.

correlation time and from this, (using the Stokes-Einstein relation) the average hydrodynamic diameter of the micelles. Several reviews of quasi-elastic light scattering and the analysis procedures have appeared in the literature (Chu, 1974; Schurr, 1977). RESULTS AND DISCUSSION

A number of preliminary experiments were carried out to ensure that only the inhibitor concentration or the enzyme concentration were rate limiting, and that the inhibitor solvent, 0.5% v/v DMSO, was without effect on the catalysis. To ensure that the salt concentration was not limiting, KC1 was replaced in the assay with various concentrations of NaCl or Na,SO, without effect on the rate of hydrolysis of ATEE. Over the limited range examined, the size and concentration of the ions had no significant effect on enzyme activity, contrary to what would be observed if “salting out” of the hydrophobic substrate was the primary cause of the activation of chymotrypsin by salts. Kinetic evidence (Gaudin and Viswanatha, 1972) shows that the effect of salts on chymotrypsin is to increase the

ENZYME

INHIBITION

TABLE INHIBITION

CONSTANTS (I,,), CHARACTERISTIC CHLORINATEDCOMPOUNDS

Compound 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14.

Methylene chloride 1-chloropropane I-chlorobutane 2-chlorobutane Chlorocyclohexane I-Chloroheptane 1-Chloroctane I-Chlorodecane Chlorobenzene 3-chlorotoluene I-chloronaphthalene 4-PCB 2,2’-PCB 2,2’,4,5-PCB

1

VOLUMES (V,J, AND OTHER PROPERTIESFOR SOME WHKH INHIBIT IX-CHYMOTRYPSIN

V, (10e5 m3/mole) 4.94 6.53 7.92 7.92 9.67 12.2 13.6 16.4 8.39 9.81 12.1 14.5 15.7 18.1

197

BY HYDROCARBONS

I 5” 0.44 0.28 0.12 0.32 3.5 x 5.8 x 5.0 x 4.6 x 2.4 x 3.1 x 2.3 x 7.5 x 1.9 x 3.0 x

10-2 lO-2 10-2 1O-2 1O-3 1O-3 1O-4 10-4 10-d 10-4

s 1.6 1.5 1.1 2.0 1.6 2.3 1.5 2.0 1.0 0.8 0.9 1.1 1.1 0.6

Concentration at which particles are observed (M) >l.O >0.75 bO.4 >0.6 4 x 3 x 6 x I x 1.5 x 7.5 x 1x
10-X 10-3 1O-3 10-4 10-Z 10-3 10-4 10-4 10-5 10-5

Note. Values of s were obtained from the slopes of the plots in Fig. 2, at inhibitor concentration I = I,,, that is at the zero intercept on the abscissa. The lower limit of particle size used to determine if particles are present was 0.5 km.

V,,, of ATEE hydrolysis with no change in the K, for ATEE. At the low concentrations of chymotrypsin used in this work no dimerization of the enzyme or precipitation of the enzyme was observed. Based on the equilibrium dimerization constant of 2.1 x lo3 M-I at pH 7.8 (Nichol et al., 1972) no significant dimerization would be expected under these conditions. Figure la-c shows the effect of three chlorinated hydrocarbons on CL-CT activity, compared with the light-scattering data on the appearance of particles in solutions of the inhibitors in 0.1 M KCl. Table 1 summarizes similar results for 14 chlorinated hydrocarbons; the compounds fall into three groups: monochlorinated alkanes, monchlorinated aromatics, and polychlorinated biphenyls. Figure la shows the activity of U-CT as a function of the concentration of I-chloropropane, which is typical of compounds l-4 in Table 1, in that the I,, (concentration of inhibitor for 50% inhibition of u-CT) is less than the solubility limit for the compound as detected by light-scattering. Figure lb shows similar data for l-chlorodecane, typical of compounds 5-8, for which the I,, exceeds the solubility limit as indicated by the formation of small particles. The lack of solubility of the Ichlorodecane and other larger chlorinated alkanes does not prevent a concentration-dependent inhibition of the enzyme at apparent concentrations much higher than the solubility limit. The monochlorinated aromatics show more inhibition of a-CT than similar sized I-chloroalkanes (Table 1, compounds 9-11). For chlorobenzene and 3-chlorotoluene (Fig. lc) the I,, is less than the sollbility limit. The chlorinated biphenyls (compounds 12-14) are very insoluble and yet show substantial potency in the inhibition of a-CT.

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We have analyzed the inhibition of chymotrypsin by hydrocarbons using the approach of Eyring and coworkers (Woodbury et al., 1975) which they applied to the inhibition of bacterial luminescence by anesthetics. The advantage of this treatment is that it allows the estimation of s, the number of anesthetic molecules required to inactivate one protein molecule, from a dose-response curve of the effect D measured as a function of anesthetic concentration, [I]. The relationship is given by: ln(D/l

- D) = In K, + sln[I]

where K, is the association constant of the anesthetic-protein complex. A number of assumptions are made in deriving this relationship; one is that the protein molecule exists in an equilibrium between an active form and an inactive form; that the binding of anesthetic molecules shifts the equilibrium towards the inactive form; another is that the effect D is directly proportional to the fraction of protein that has been converted from active to inactive forms and that D is continuously variable. The relationship has been used to analyze the inhibition of enzymes by anesthetics. In enzyme inhibition studies the relationship is used in the form: ln[A,I(A,

- I)] = In[&/l

- K)] + sln[I]

where A, is the enzyme activity in the absence of inhibitor; A,, that in the presence of inhibitor; and K is the equilibrium constant for the interconversion of active and inactive enzyme molecules. Thus a plot of In[A,/A, - l)] versus ln[I], or versus ln[I/15,] to normalize the abscissa, should yield a straight line of slope S. For example, in the inhibition of glutamate dehydrogenase by the three anesthetics methoxyflurane, halothane, and diethylether the three plots are straight lines with slopes of 2.4, 1.2, and 1.9, respectively (Hulands et al., 1975). These analyses have been used to support the protein conformation change theory of anesthetic action in that the linearity of the plots suggests that one or two anesthetic molecules are sufficient to change a protein conformation from the active to the inactive form. Possible reasons for the non-integer values of s are discussed elsewhere (Woodbury et al., 1975). When the inhibition curves for the effects of 14 chlorinated hydrocarbons on chymotrypsin were plotted in the form ln[A,I(A, - l)] versus In[I/I,,J the chlorinated alkane inhibitors gave straight line plots with values of s from 1 to 2.3, as shown in Table 1 and Fig. 2a,b. These values are similar to those reported by Eyring and coworkers for the inhibition of bacterial luminescence by anesthetics (Woodbury et al., 1975). For the chlorinated aromatics, Fig. 2c,d several of the plots show two regions of linearity with higher values for slope s at lower concentrations of the inhibitors. The sharp inflection in each plot does not correspond to the solubility limit as measured by light scattering, and a more complex cause is likely. At higher concentrations of the inhibitors, where particle formation occurs, it might be expected that more inhibitor molecules would be necessary to inactivate each enzyme molecule but the lower values of s at higher inhibitor concentrations suggest that at these concentrations fewer molecules of inhibitor are required per molecule for inactivation.

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BY HYDROCARBONS

a

b

2, 1

1

0

0

-1

-1

-2

-2

-3

-3

-2 z 5

d

c 1

13 12 A14

1

1

0

0

0

-1

-1

d -2 -3

-3 -2

-3

-2

-1

0

0

l!!!,L

1 2

-3

-2

-1

0

1

In (AdAi-1)

FIG. 2. Plots of ln(A,/Ai - 1) versus ln(I/I,,) for 12 chlorinated hydrocarbons which show inhibition of u-chymotrypsin. The numbered plots refer to 12 of the 14 compounds listed in Table 1: two compounds were omitted for clarity. The slope of the plots, at zero on the abscissa, is the value s and is related to the number of molecules of inhibitor required per molecule of enzyme for inhibition.

The simplest explanations of deviation from a single line for these plots include (a) that the enzyme is not present in only two states; (b) that there are two or more sites for the binding of the inhibitor, and that the highest-affinity site has less effect on activity than the lower affinity site(s). Evidence for two hydrophobic sites in chymotrypsin has been obtained using fluorescent probes (McClure and Edelman, 1967). The question asked in this study is whether physical toxicity can be observed in systems composed only of aqueous protein, or whether a lipid phase is essential for this behavior. Many studies have shown a correlation between the physical toxicity of hydrophobic compounds and partition coefficients, notably those of Hansch and associates (Hansch and Dunn, 1972), and Collander (1950). The partition coefticient is directly related to the free energy required to transfer a molecule from one phase to another within a system. McGowan has shown that the equation, log x = kV,, describes the partition of a noninteracting solute between water and some organic liquid; where x is the partition coefficient, k is some constant value (largely determined by the aqueous phase), and V, is the characteristic molar volume of the solute (McGowan et al., 1979). This relationship arises because the free energy of transfer of a hydrophobic molecule from the organic phase to the aqueous phase is dependent on the extent of disruption of the water structure, which is proportional to the molecular volume of the solute.

200

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HALLETT

It has been found that k is equal to 36,000 for nonassociating solvents such as diethylether. When the solute molecule contains some group which interacts with the water phase, an interaction term Eb may be included in the equation to give log x = kV, - Eb. Eb values are the same for a series of homologous compounds and typical values for the partition of steroids between ether and water are 1.9 for hydroxyl and carbonyl groups and -0.15 for halide atoms. The partition equation can also be used to describe physical toxicity because the concentration of the solute required in the biophase to give some biological effect C’s, will be related to the partition coefficient x, where x = C,lC, and C, is the concentration of solute in the aqueous phase. The equation is used in the form: C,

=

A

+

B

x

1o-‘kVx-Eb)

where A and B are constants for the system; B gives the toxic concentration of the compound in the biophase and A/B is the ratio of the volumes of the nonaqueous phase to the aqueous phase in the system. The Eb terms are slightly different for the biophaselwater system, for example, Eb is 1.2 for aliphatic alcohols, esters, or ethers, and Eb is -0.35 for the halogens. An example of this equation in use is seen in Sharom and Mellors (1980), where the effect of chlorinated compounds, including polychlorinated biphenyls (PCB), on the plasma membrane enzyme 5’nucleotidase was shown. The -log C,, in this case the I,, (concentration of inhibitor required to cause half-maximal activity) was plotted against 36,000 V, - Eb. A curve of two parts was obtained, the initial part of which is a straight line of slope 45” which cuts the log C, axis (x intercept) at log B, the toxic concentration in the biophase. At higher values for V, the slope falls off and approaches a horizontal line where - log C, equals -log A. This fall-off point is dependent on the volume fraction of the biophase in the system as A/B equals the ratio of the nonaqueous phase to the aqueous phase. Figure 3. shows the -log I,, values for the inhibition of u-CT plotted against 36,000 V,, and compared with a similar plot for the inhibition of 5’-nucleotidase (data of Sharom and Mellors, 1980). The value 36,000 was used because it has been shown to give a linear relationship between the molecular volumes of compounds and their partition coefficients between water and many organic solvents, such as diethylether (McGowan, 1954), and this value is close to the average figure for physically toxic effects on a number of enzyme systems (McGowan and Mellors, manuscript in preparation). No interaction term was included because these have only been calculated for partition between water and bulk phases such as organic solvents, and little is known about the interaction terms for specific enzymes. It is clear from Fig. 3 that there are differences and similarities between the soluble a-CT and the membrane-bound 5’-nucleotidase in their inhibition by hydrocarbons. The major difference is that (U-CT, a soluble enzyme, is not as sensitive to inhibition by these organic solvents as are membrane-bound enzymes, such as 5’-nucleotidase. Mouse spleen lymphocyte 5’-nucleotidase is ten times more sensitive to PCB than a-CT is to chlorinated aromatics. The long-chain chlorinated alkanes are less active as inhibitors than their molecular volumes suggest, and much less active than chlorinated aromatics of similar molecular volumes, This could indicate some specific interaction between (YCT and the chlorinated aromatics but this may not necessarily be so. As shown

ENZYME

INHIBITION

201

BY HYDROCARBONS

6

4-

6 0

9 6.

0:’ 21

4 .*I2

:

10 %:

l $3 A4

33 3

A9 2-

10 2

B

AS A6 I-

a7

: d A2

A4

A' ”

1

2

3 36,000

4

6

6

7

Vx

FIG. 3. A plot of inhibitory potency (-log I,,) versus characteristic molecular volume (VJ for chlorinated hydrocarbons in the inhibition of a-chymotrypsin, compared with the inhibition by hydrocarbons of mouse lymphocyte plasma membrane S’nucleotidase (from Sharom and Mellors. 1980). For o-chymotrypsin inhibition (open symbols) the compounds are numbered as in Table 1. For 5’nucleotidase inhibition (closed symbols) the compounds are (1) I-butanol; (2) I-hexanol; (3) I-octanol; (4) I-nonanol; (5) 1-decanol; (6) I-dodecanol; (7) I-tetradecanol: (8) transretinol; (9) I-chlorooctane; (10) I-chlorodecane; (11) 4-PCB; (12) 2,2’-PCB; (13) 2,4,5-PCB; (14) 2,2’,4,5’-PCB; (15) 2,2’,3,4,5’PCB; (16) 2,2’,3,4,4’-PCB; (17) 2,2’,4.4’,5.6’-PCB: (18) 2,2’,3,4,4’.5.6’-PCB; (19) 2,2’,3,4.4’,5.6’-PCB.

in Fig. lb, the long-chain chlorinated alkanes do not significantly inhibit o-CT esterase activity until such high concentrations are reached that a separate organic phase appears in the reaction mixture. For these compounds the true aqueous concentration available to inhibit a-CT is significantly less than the Iso value indicated, as the latter was calculated from the total amount of inhibitor added to the system. The chlorinated aromatics show most inhibition of a-CT and their inhibitory potency shows some resemblance to a physical toxicity curve in that it is proportional to molecular volume up to a limit. A distinct cut-off of inhibitory activity at a specific molar volume would be consistent with association of inhibitor with a “pocket” of fixed dimensions. No such cut-off was seen in this study; though the PCBs are much larger than chlorobenzene, they show similar inhibitory potency. Most of the chlorinated hydrocarbons gave s values for the inhibition of W-CT of between 1 and 2. These are quite similar to the values obtained by Johnson et al. (1954) for the inhibition of bacterial luciferase by anesthetics and which they believe are diagnostic for protein conformational change. It is instructive to compare the s values of around 1, which we obtained for cu-CT inhibition and which Johnson et al. (1954) obtained for bacterial luminescence inhibition, with the s values that we have estimated using the data of Sharom and Mellors (1980) for lymphocyte plasma membrane 5’-nucleotidase inhibition. The membrane enzyme values for s are, for butanol, 4.5; hexanol, 6.8; octanol, 7.2; 4PCB, 5.4; 2,4,5-PCB, 5.6; and 2,2’,4,5-PCB, 6.2. This suggests that in the intact lymphocyte many more molecules of inhibitor are needed per molecule of membrane-bound enzyme for inhibition, compared to the bacterial luminescence system or the chymotrypsin assay. Some earlier studies of inhibition of a-CT by hydrocarbons concluded that the

202

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inhibition was strictly competitive (Miles et al., 1963; Hymes et al., 1969). The inhibition was shown to follow a linear free energy relationship, correlating well with molecular area (Miles et al., 1963) and partition coefficient (Knowles, 1965). This led to the “extraction theory” of enzyme-substrate complex formation in which it is proposed that a hydrophobic site on the enzyme extracts the substrate or inhibitor from its aqueous environment (Miles et al., 1963). Numerous studies have shown that the process is more specific than simple extractions. (Applewhite et al., 1958; Berezin et al., 1970, 1971). It is not always easy to distinguish competitive inhibition from mixed-type inhibition; for example, the effect of anesthetics on bacterial luciferase was first thought to be competitive in that it is overcome by increasing substrate concentrations (Woodbury et al., 1975). The inhibition of xanthine oxidase by benzyl alcohol appears to be competitive at low inhibitor concentrations whereas at higher concentrations the inhibition is mixed (Fridovich, 1966). Evidence that this inhibition of e+CT is consistent with the physical toxicity model rather than competitive inhibition is found in the work of Backes and Canady (1981). They showed the relationships between the free energies of binding and molecular size, for a series of unsubstituted aromatic hydrocarbons binding to three different enzymes. Linear relationships with similar slopes were obtained for a-CT, yeast alcohol dehydrogenase, and human complement C-l esterase. No such similarity is required for competitive interactions but a similar slope is a prediction of the physical toxicity model. It is noteworthy that a similar slope is found for the relationship between size and the free energy of transfer between an organic phase and water. The slope calculated by Backes and Canady is 2.9 kJlmolelcarbon atom of the inhibitor molecule (A@. This value is approximately equivalent to the value of the constant k, 36,000, used in molecular volume plots. When P is the partition coefficient and R and T have their usual thermodynamic meanings then, since RTInP

= AE,

therefore log P = 2900 + (2.303 x 8.31 x 298) = 0.510. Since log P = kV,

and

V, is 1.41 X 1O-5 per C atom,

therefore k x 1.41 x 1O-5 = 0.510 and k = 0.510/1.41

x 1O-5

k = 36,200. There are close parallels between the data of Backes and Canady and physical toxicity plots, though originally the inhibition of c+CT was interpreted as being

ENZYME

INHIBITION

BY HYDROCARBONS

203

caused by competition for a particular hydrophobic site (Miles et al., 1963). These similarities suggest that a physical mechanism of inhibition by hydrocarbons is common to all three different enzymes. In the case of (-U-CT specifically, it might be expected that there would be some interaction between hydrophobic inhibitors and a readily accessible hydrophobic region, the hydrophobic pocket in the enzyme active site. Berezin et al. (1970) suggested that the large enthalpy change observed for (YCT-inhibitor interaction (- 19 kJ/mole) must be due to a conformational change in the enzyme. Direct evidence that hydrophobic inhibitors cause conformational changes in a-CT comes from the work of Smith and Hansch (1973). They looked at cx-CT inhibition during long-term incubations with anionic and cationic hydrophobic inhibitors. They showed that in their system the inhibition was a two-step process, an immediate partial reduction in enzyme activity, followed by a slow continued first-order deactivation, all as a result of reversible hydrophobic associations. Optical rotatory dispersion data indicated that the slow component of the inhibition altered the Cotton effect in the 285-300 nm region, believed to be due to a change in the environment of tryptophan residues in the enzyme. Inhibitor-induced conformational changes may be analogous to the well-studied thermal denaturation of proteins, which also leads to similar changes in the Cotton effect. This view of the process of inhibition of a-CT by hydrocarbons is very similar to the protein conformation change theory of anesthetic action propounded by various groups over the past four decades (Johnson et al., 194.5; Featherstone, 1963; Balasubramanian and Wetlaufer, 1966). In summary, the inhibition discussed here is similar to the process observed by Canady and associates and originally called by them “competitive inhibition.” The process corresponds to the reversible short-term inhibition seen in the first stage of o-CT inhibition by hydrocarbons described by Smith and Hansch (1973). It does not correspond to the second stage of a-CT inhibition seen in the latter study, which is attributed to precipitation of protein. Certain aspects of the inhibition are consistent with partitioning (physical toxicity) while the greater potency of chlorinated aromatic hydrocarbons compared to chlorinated alkanes suggests some specific interactions of unknown origin. Our results support the view that the critical target of anesthetic and physicallytoxic substances may be the hydrophobic regions of proteins, and that the lipid phase of the cell is not essential for impaired protein function. It is likely that these target proteins are much more sensitive to changes in structure and function than the IX-CT of this study, but they need not be intimately associated with a lipid environment. ACKNOWLEDGMENTS This work was supported by Natural Sciences and Engineering Research Council (NSERC) Strategic Grants. We thank Dr. T. Viswanatha for extensive discussions and suggestions, and Dr. N. J. Bunce for gifts of hydrocarbons.

REFERENCES Applewhite, T. H., Martin, R. B., and Niemann, C. (1958). The a-chymotrypsin catalyzed hydrolysis of methylhippurate in aqueous solutions at 25” and pH 7.9, its inhibition by indole and its dependence upon added nonaqueous solvents. J. Amer. Chem. Sot. 80, 1457-1464.

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MELLORS,

AND HALLETT

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