Inhibition of lipoxygenase by saturated monohydric alcohols through hydrophobic bondings

ARCHIVES

OF

Inhibition

BIOCHEMISTRY

AND

BIOPHYSICS

of Lipoxygenase

664-669

118,

by Saturated Hydrophobic

HISATERU

MITSUDA,

(1967)

Monohydric

through

Bondings

KYODEN

YASUMOTO,

Department o.f dgricultural Faculty of Agriculture, Kyoto Sakyo-ku, Kyoto, Received

Alcohols

October

AND

AIJIRO

YAMARIOTO

Chemistry University Japan

19, 1966

A series of saturated monohydric alcohols has been examined in the search for evidence pertaining to hydrophobic bondings during the reaction of lipoxygenase from defatted soybean meal. The alcohols produce a reversible inhibition of the enzymic reaction, the degree of which increases with an increase in bhe chain length of the alcohols. Can’t Hoff plots for the inhibition indicate a positive entropy change for the combination of the alcohols with the enzyme. Hydrophobic bond formation between the alcohols and the enzyme is not only accounted for by these facts bllt also anticipated from close relationships established between the inhibitory activity of the alcohols and their physicochemical properties. From Lineweaver-Burk plots it follows that the type of the inhibition is a mixed type. The alcohols are unable to prevent the enzyme from the inactivating action of hydrogen peroxide, which destroys the catalytic site in the enzyme. The possibility that the alcohols combine with the enzyme at a hydrophobic region which serves as binding site for the substrate is discussed.

Lipoxygenase (E.C. 1.13.1.13, linoleate: oxygen oxidoreductase) specifically catalyzes the oxidation of cis-methylene interrupted unsatura.ted fatty acids and their esters to respective hydroperoxides. The most typical substrates are naturally occurring isomers of linoleic, linolenic, and arachidonic acids (1). In addit’ion to these, I-bromooctadecadiene acts as substrate, peroxide formation being of same order as that for linoleic acid (2). It thus appears that properties of the polar group in the subst,rates are not critical for the enzymic reaction. Amino acids analyses have revealed that the enzyme protein is relat,ively abundant with valine, leucine, and isoleucine, which have nonpolar side chains (3), and imply that a hydrophobic region composed by some of these amino acids residues on an affinity

the

surfacse

of

the

protein

marily responsible for their affinity to the enzyme. Ko direct evidence is yet) obtained for his suggestion, and relat,ively little is known about the function of saturated hydrocarbon moieties attaching t,o the diene system in the substrates. In this paper, inhibition of lipoxygenase by snt8urated monohydric alcohols has been st,udied t’o present several lines of evidence for their hydrophobic interaction with the enzyme along with the discussion on the significance of the bonding in this enzyme cat’alysis. MATERIALS

AND

METHODS

Enzyme. Crystalline lipoxygenase was prepared from defatted soybean meals by a modification of the method of Theorell et al. (5) as described previously (6). The final enzyme preparation was homogeneous electrophoretically and Idtracentrifugally. Specific activity determined by spectrophotometric method (7) was 5.0 X lo2 Theorell unit per absorbancy at 280 mp, which is slightly higher than that reported by Theorell et al. (5). The protein concentration was determined spec-

has

for nonpolar moieties of the substrates. Holman (4) has suggested that unsaturated moieties of fatty acids are pri664

LIl’OSY(:ESASE tro1~hot~oniet~rically, based on a molar absorbancy of ~280 = 1.82 X 106, which was calculated from molecular weight of 102,000 and the absorbancy value of 17.8 for l?& lipoxygenase (5). .tsscr~/. The lipoxygenase artivity was conveniently meaa~u-ed I)y using a Clark oxygen electrode alrd was expressed as the initial rate of oxygen uptake at 25” as described in previous paper (6). The enzymic reaction was started 1)~ adding 0.1 ml of enzyme solution of proper dilrltion to 2.90 1111 of substrate solution which was allowed in advance to equilibrate with atmosphere at 25”. The sllbstrate solut,ion was prepared before use according to the direction of Tappel et al. (8) from linoleic acid (2 98r: pure) purchased from Fluka AG, ISlIcks SC:, Switzerland. Inhibitors. Most cornpormds used as illhibitora were of reliable commercial sources and were used withotlt further purification. Conjugated isomer of linoleic acid was prepared from linoleic acid according to the procedllres of White and&uackenbush (9). Estimation of ‘(e$ective curbon chain length.” The ability of the alcohols to form hydrophobic bondillgs to the enzyme protein was assumed to be expressed in terms of their “effective carbon chain length,” (‘,, This follows directly from general rule that the contribution of each CH? group to hydrophobic bonding is additive to a very good approximation but decreases with increasing degree of branching (10). c’,, vallies for n-alcohols were thlls taken as the number of carbon atoms as being involved in the molecldes. c’, values for the branched alcohols were estimated in the following ways with a supposition that only some fraction of the carbon atoms are capable of interacting with the nonpolar residues of the protein to form hydrophobic bonds. Affinity of n-alcohols to a certain protein, such as pKi values or of similar nature reported by other investigators were plotted against their numbers of carbon atoms. To the resldted linear regression line determined by the method of least squares were fitted the affinity constants of branched alcohols, and then corresponding hypothetical members of carbon atoms were read out. These procedures were repeat,ed for affinit,y constants of alcohols to other proteins: lellcine aminopept,idase (II), ribonuclease (12), and cold insoluble fraction of soybean proteins (13), respectively. The e, values thus obtained were averaged for each branched alcohols and used in the abscissa of Fig. 2. RESULTS

AND

I~ISCUSSIO~

Inhibition by a series of saturated monohydric alcohols. While little is known about

the inhibitor

for lipoxygenase except for

INHIBITIOS

ti6.S

nonsubst.rate higher fatty acid and several antioxidants, a significant inhibition was observed for t,he saturat,ed monohydric alcohols whic*h have carbon atoms from 1 to 7. So specific struc%ure requirement was inferred for the inhibitors, but a marked increase of t,he inhibition was observed with increasing carbon chain length for a given concentration of added alcohol. The branched alcohols, however, produced the inhibition lower than the st,raight-chain isomers; twtbutyl al~*ohol was proved the poorest inhibitor in t’he butyl series. There were only insignificant changes in the dielect)ric constant of the alcohol-water solutions for different members of the homologous series. The inhibition is thus not, primarily due to :I change in the dielwtric constant, of t’he reaction medium but essentially due to nonspecific hydrophobic bonding formed between the alcohols and the enzyme protein. Inhibition by most’ alcohols except, for n-heptyl alcohol did not follow a simple mass action law. The experimental data shown in Fig. 1 indicate a wmbinat,ion of 1 mole of inhibitor per binding site of the enzyme at the lower c,oncentrat,ion, but 2 moles at the higher c,oIlc,entration. For convenience of analyzing the experimental data, the concentration of alcohols which reduced the rate of oxygen uptake just by half was taken as a measure for Ki , the dissociation const,ant of enzyme-inhibitor complex. Alt,hough t,he Ki values thus assessedare of apparent nature, they st,ill provide an adequate measure for the inhibitory effect of the alcohols and lead to a comparison of their inhibitory activity mit’h relat#ive impunity for possiblemisunderstanding. Figure 2 shows the dependcncc of the pKi values obtained in this way on the “effect& carbon chain length,” C, , of the alcohols which were estimated as described in Materials and flfethods in order to interpret systematically the inhibitory effect of both n- and branched alcohols. Several physicwhemical consstantsof alcohols, such as solubility in water (KS), boiling point (15), and partition coefficient between water and n-o&y1 alcohol (IG), are known to associate with the mutual hydrophobic interaction and cwnsequcntly to be com-

666

MITSUI>A,

YASUMOTO,

posed additively from each CH2 group contribution (10). Similar additivity is also obtainable for the absolute entropy value. When the pK; values were plotted against these physicochemical constants or t,heir derivatives, significant correlations were obtained as shown in Table I. These result’s seem also to bear sufficient evidences for hydrophobic interaction of the alcohols to the nonpolar binding site of the protein. Reversibility of the inhibition. The reversibility of the inhibition by alcohols was fairly complete with respect t,o the enzyme activity. One of the typical experiments gave t,he following results. Lipoxygenase, being assayed in presence of 1.00 and 0.033 M n-propyl alcohol under t,he conditions given in the legend to Fig. 1, but with 1O-3 M linoleic acid, was inhibited by 52.1 and 4.6 %, respectively, whereas 94.6% of the original activity was recovered with the enzyme incubated with 1.00 M alcohol at 10” for an

isd-Butand -2 -4

1

I

I

I

-3

-2

-I

0

Lo13

(INHIBITOR)

(M)

FIG. 1. Diminution of the rate of linoleate oxidation as a function of alcohol concentration. The plots of log(cu/l--0l) against log(inhibitor) were based on a simple mass action law which gives the equation, pKi = log(a/l--a)--n log(inhibitor), where Q: is the fraction of enzyme activity vanished, and n is the number of moles of alcohol which combine with each binding site of enzyme. The enzymic reaction was started by the addition of 0.10 ml of enzyme solution (4.8 pg protein) to 2.90 ml of 0.1 M borate buffer containing 10d4 M ammonium linoleate and varied concentration of alcohol. The assay medium was maintained at 25” and allowed to equilibrate with atmosphere before the reaction started (oxygen dissolves by 240 PM under these conditions).

AND

YA%IAILIOTO

3-

2-

“EFFECTIVE

-I

CARBON

CHAIN

LENGTH”

_

FIG. 2. A linear relationship between pKi and “effective carbon chain length,” 6, , of inhibitor. Cn’s and pKi’s were estimated as described in the text. The linear regression line determined by the method of least squares yielded, pKi = 0.472C,c 0.743, with correlation coefficient of 0.995. The enzyme activity was determined as described in the legend to Fig. 1.

hour and then assayed in the aqueous substrate solution so that the alcohol concentration diluted 30-fold. It is evident that reversal of the inhibition is obtained by dilution and that the residual inhibition is due to the amount of alcohol remaining in the assay medium. A complete recovery of the enzyme activity after fractionation between 10 and 20% (v/v) ethyl alcohol, which was employed as an effective procedure for the enzyme purification (5, 6), is another distinct evidence for the reversibility of the inhibit,ion by alcohols. Temperature dependence of the inhibition. A second line of evidences for the hydrophobic bonding comes from the positive change in entropy, AS, around room temperature (17). As shown in Fig. 3, Van’t Hoff plots for the inhibition by n-heptyl alcohol are curved over the employed temperature range, which give enthalpy changes, AH, O-17,000 Cal/mole and thus AS of 12-69 e.u. These positive AS may be ascribed to a possible change of water structure around the hydrocarbon portion of the added alcohol and the nonpolar side chain of the enzyme to which the alcohol binds

LII’OSYC:ESASE TABLE RELATIONSHIP

BETWEEN

I

pk’i’s AND SOME PHYSICOCHEMICAL OF ALCOHOLS

Boiling point (T, “C; 760 mm Hg) Solubility in water (C; M; 25”, 760 mm Hg) Partition coefficient (P; f’,,.ater/Cn-octanol ; 25”, 760 mm Ed Absolute entropy (S”; e.u.; 25”, 760 mm Hg)

tx7

ISHIBITIOX

pKi

~0NS’I’AN’I.S

= 0.0240T - 1.631 -0.651 1ogC + 1.171 = 0.944 1ogP + 0.830

=

= 0.05989”--2.08

a Content in the parentheses represents the abbreviation used in the equation, stant, and the conditions under which the constant was obtained. b The pK& used in these calculations were the same as in Fig. 2.

12 12

0.990 -0.903 0.984

9

0.992

7

the unit of the con

results were also obtained with other alcohols. The theory of this mixed type inhibition is developed with t’he assumption that the inhibition not only affects the enzymesubstrate combination but also completely prevents the breakdown of the complex (21). 2.5 It is likely that t’he alcohols occupy the Y, hydrophobic region in lipoxygenase t,o retard the interaction with substrate and that catalytic inactiveness of the enzyme-inhibitor-sub&rate complex is a result of the possible inaccessibilit)y of the unsaturated system of the substrate to t’he catalyt’ic site 2.0 of the enzyme. I I I I Possible n&u/-e of hydf*ophobic site in Zipozygenase. The contribution of each CH:! l/$L?AT3uR4E, ~lo=K-~, group to free energy change, AF, in the FIG. 3. T’an’t Hoff plots for the inhibition of inhibition of lipoxygenase by alcohols is lipoxygenase by n-heptyl alcohol. Assay condi-650 Cal/mole, which was calculated from tions were same as in Fig. 1 except for varied the slope of st)raight line in Fig. 2. Similar temperature. values have been reported for AF increment per each CH, group in protein-nonpolar (18-20). The experimental data in Fig. 3 group interaction (22). For the inhibition of also indicate a trend that AH and AS ap- leucine aminopeptidase by alcohols, t’he AF proach to negative values at higher branch increment is -400 cal/mole (I 1) ; for the of employed temperat’ure. This fact seems denaturation of (hold insoluble fraction of compatible with the theory proposed by soybean proteins, -:378 (13); for the therN6methy and Scheraga (B-20). mal transition of ribonuclease, -398 (23). Type of the inhibition. As the substrate On the other hand, increment of AF of it’self has a large nonpolar portion in its activation per each CH, group for horse molecule, some competition of alcohols with liver estcrase-catalyzed hydrolysis of n-fat,ty substrate for t’he enzyme was first expected. acid salicylates is -550 cal/mole (24) ; and Double reciprocal plots, however, prove the for chymotrypsin cat,alyzed hydrolysis of inhibition by n-heptyl alcohol to be a mixed n-fatty acid tyrosine esters, -650 (25). type of partially competitive and purely These differences in AF increment, per each noncompetitive, as shown in Fig. 4. Similar CH, group would be interpreted as a rc-

0

5 I/ (LINOLEIC

ACID)

IO (104.M-‘1

FIG. 4. Double reciprocal plots for lipoxygenase reaction in the presence and absence of 2.5 X 10-s M n-heptyl alcohol. Assay conditions were same as in Fig. 1 except for varied concentration of substrate. of the nature of hydrophobic site in the respective protein, but no available theory is effect’ive to explain these differences in terms of structure or nature of hydrophobic region. It may be worthwhile, however, to point out the fact that the AF increments calculated for these protein reactions fall within a certain range. The degree of hydrophobic interaction per each CH2 group would be thus of a similar order and irrespective of what kind of protein is concerned. It is quite a reasonable assumption that the area of hydrophobic region in the protein is limited to a certain extent and does not cover the whole surface of the protein molecule. From this assumption it follows that limited dimension of the hydrophobic region in the protein leads to a saturation of affinity of the protein to hydrocarbon portion of inhibitor or substrate at a certain carbon chain length. Actually, V, for the hydrolysis of n-fatty acid tyrosine esters catalyzed by chymotrypsin is reported to saturate at about 7 carbon chain length (2fi); K, for the hydrolysis of n-fatty acid tyrosine esters catalyzed by horse liver esterase, at about 9 (26); the inhibition of leucine aminopeptidase by alcohols and n-fatty acids, at 5 (27). The straight line in Fig. 2 does not flection

give any significant evidence for the saturation with respect> t,o carbon chain length within the employed range. It thus appears that the hydrophobic region in the enzyme is large or sufficient to accommodate the whole hydrocarbon portion of n-heptyl alcohol. The concept of mixed type inhibition (21) has a connotat,ion t’hat the catalytic site is close to but dist,inctly different from the site wit’h which the alcohols bind. Table II furnishes further evidence for making a differentiation between these two sites. Lipoxygenase easily loses its activity by oxidation with hydrogen peroxide, permanganate, iodine, and so on. Conjugated linoleate, a competitive inhibitor described by Holman (l), prevents fairly well the inactivation

of

the

enzyme

by

hydrogen

peroxide. Thus the catalytic site of the enzyme is seemingly concerned with t’his oxidative inactivation, while it is yet obscure what amino acid residue, or other grouping if any, function as bhe cat’alyt’ic site. On the contrary, none of the employed alcohols is effective in preventing the enzyme from the inactivation by hydrogen peroxide. The most likely interpretation of these results is that the conjugated linoleate does not allow hydrogen peroxide to atStack the TABLE

II

EFFECTSOFCONJUGATEDLINOLEATEAND~-BUTYL ALCOHOL ON INACTIVATION OF LIPOXYGENASE BY HYDROGEN PEROXIDE" "zldactiv-

Addition

Hydrogen peroxide Hydrogen peroxide Conjugated linoleate n-Butyl alcohol (2 Hydrogen peroxide linoleate (1V M) Hydrogen peroxide alcohol (2 X 10-l

(1P M) (10e5 M) (1OP M) X 10-l M) (1OP M) + conjugated (10m5

M)

+

n-butyl

0 27 31 96 31 32

M)

a The enzyme activity was determined as described in the text, after incubation with t,he additions of indicated concentration in 0.02 M phosphate buffer of pH 7.5 at 25” for 2 hours. * This value was expressed in y0 of control run.

catalyt,ic site by ovvupying the site, whereas the alcohols do, since they can merely attach to a site other than the catalytic site. This int,erpret’ation is compatible with the conclusion drawn from the vonccpt, of mixed type inhibition by the alcohols. Most investiffntors find it convenient8 to divide the surface of enzyme arbitrarily into a number of regions from the observations made on t,he enzyme activity altered either by exposing it to different’ external media or by c+hcmkal reaction in which simple organic or cnzymics reagents arc used. In this paper the not,ation of “hydrophobic site” is proposed instead of hydrophobic* region, in order to describe the region, cxeluding the catalyt’ic site, in direct contact with the substrate, which is general!y cglled the binding site. The function of the hydrophobic site of lipoxygenase is perhaps to provide the catalytic site with the anchor which permits a specific interaction between the sub&ate and enzyme, and increasesthe rate of csatalysis. This tjentativc conclusion may be substantiated by the recent finding of Hamberg and Samuelsson (28) that t,he lipoxygenasc-catalyzed introduction of oxygen into substrates is of a higher degree of specificity than formerly imagined; the diene systems starting at position R counted from the met8hyl ends, and those starting at position 9 caountedfrom the varboxyl ends, are susceptible to the enzymic reaction. It map be inferred from their finding that a nonpolar portion of a definit)e length attaching to the diene system is required t)o endow the substrate \vit,h a specific affinity to the enzyme. REFEREXCES T., AND BERGSTI~~M, S., in “The Enzymes” (J. B. SlLmner and K. MyrbAek, eds.), first, edition, T.01. II, p. 559. Academic Press, Sew ‘J-ork (1952). 2. BI,AIN, J. A., .IND SHE:AHER, (i., J. Sri. Food ilgr. 16, 373 (1965). 3. HOLMAN, I?. T., l'anz~:rt, F., SCHWEIGERT, B. S., AND AMN’:s, s. R., .4wh. Biochem. 26, 199 (1950). 4. Hormah-, I:. T., Arch. Hiochem. 15, 403 (194i). 5. THEORELL, H., HoIIM.4N, IZ. T., AXI) AKESON, A., Acfn (‘hem. &and. 1, 571 (1947). 1. H~LM~N,

H.

6. Mrwm4, ZNI)

H., Kr.s\so,

Y.4sr:~o~ro,

‘I‘.,

dlgr.

K., Biol.

YAMAIIOTC~,

(‘hrm.

A.,

31,

115

(lS(i7). H.,

7. THWRELL,

A., Pharm. 8. TAPIWI., w.

A. 0..

BEIK:SWX~M,

S.,

.~NI)

Al
-4cfu HP~P. 21, 318 (1946). L., BO~ISR, I’. I)., ASI) LUSDBEILG, --l/d.

Niochem.

&oph!/s.

42,

293

(1953). 9. \vHI'L'E,

H. B., JR., AND ~l;i\cliE2iRI.sH, F. W., Oil C&w/. Sot. 36, 653 (1959). KETELAAR, J. A. A., in “Chemical Const,it IItion,” p. 356. Elsevier, London (1958).

J. .l,,r.

10.

11. HIM,, IL. L., AND SMITH, E. L., J. Biol. Chem. 224, 209 (1957). 12. SCHRIEII, E. E., 1NGWSI,L, It. T., AND SCHEILAGA, H. i\., J. l’h!/.s. (‘hem. 69, 298 (1965). 13. FUKUSHIMA, I>., J. Hiochun~. (rl’ok!go) 75, 822 (1965). 14. KINOSHITA, Ii., Hfr/(. (‘hem. Sot. Jupan 31, 1081 (1958). 15. &ISSNER, H. I’., (‘henl. Eng. Plog1’. 45, 149 (1949). 16. IWASA, J., FUJIYA, T. ANO HASSCH, C., J. .Iled. Chem. 8, 150 (1905). 17. KAIZMANN: W., .ldoar~. I’rofein Chem. 14, 1 (1959). 18. X~ME,~HY, G., AND SCHEIL~GA, H. A., J. C’hcm. Phys. 36, 3382 (1962). 19. S~ME~THY, G., ANIJ SCHERAGA, Ii. A., J. Chem. Ph!/s. 36, 3101 (1962). 20. N~YEYHY., G., ANI) SCHERAGA, H. A., J. Phyx. (‘hem. 66, 1773 (1962). 21. I~IWWENWALD, .J. S., AND S~AENGWYN-DAVIES, G. I)., in “A Symposirun on the Mechanisms of Enzyme Act,ion” (W. D. McElroy and B. Glass, eds.), p. 154. Johns Hopkins Press, Baltimore (1954). 22. LUMRY, Il.. in “The Enzymes” (1’. I). Bayer, H. Lardy, and K. RIyrbilck, cds.), second edition, 1-01. 1. p. 157. Academic Press, Yew Tork (1959). E. E., .4x1) SCHEILAGA, Ii. A., Rio23. SCHRIER, rhim. Hiophys. /t&a 64, 406 (1962). 24. HOFBTEE, B. H. J., J. Uiol. Chum. 199, 365 (1952). 25. HOPSTEE, B. H. J., Iliochim. Bioph,ys. dcta 24, 211 (1957). 26. HOFYTEY:, B. H. J., J. Hid. Chem. 20’7, 219 (1954). 27. SMI'L‘H, E. L., Lunlnu, II., ANI) I'OLGLBSE, W. J., J. Ph,ys. Colloid Chem. 55, 125 (1951). 28. HAMBERG, M., AND SAMUELSSON, B., Biochwz. Biophys. RPS. Co~nrr~un. 21, 531 (1965).