Stereochemical nature of the products of linoleic acid oxidation catalyzed by lipoxygenases from potato and soybean

Vol. 170, No. 2, 1990 BIOCHEMICAL AND BIOPHYSKAL RESEARCH COMMUNICATIONS

July 31, 1990 Pages 491-496

STEREOCHEMICAL NATURE OF THE PRODUCTS OF LINOLEIC ACID OXIDATION CATALYZED BY

LIPOXYGENASES FROM POTATO AND SOYBEAN

Vladimir Nikolaev, Pallu Reddanna, Jay Whelan, George Hildenbrandt and C. Charma Reddy*

Environmental Resources Research Institute and Department of Veterinary Science The Pennsylvania State University

University Park, Pennsylvania 16802

Received June 4, 1990

SUMMARY: When linoleic acid was incubated with the purified potato lipoxygenase under 0, atmosphere, a mixture of 9 and 13-hydroperoxyoctadecadienoic acids was formed. Stereochemical analysis of the respective methyl-hydroxyoctadecadienoic acids revealed that the 9-isomer was in S-configuration whereas 13-hydroxyoctadecadienoic acid was a mixture of S (39%) and R (61%). Exactly the opposite was the case with the soybean lipoxygenase products, where the 13-isomer was found to be in S-configuration and 9-hydroxyoctadecadienoic acid - a mixture of S (73%) and R (27%). A general scheme is proposed for the stereochemical nature of oxidation products of enzymes which are predominantly either [ +2] or [-21 lipoxygenases. @I990 Academic Press, Inc.

Lipoxygenases (EC 1.13.11.12) catalyze the first committed step in the biosynthesis of leukotrienes, lipoxins and many other physiologically active oxygenated fatty acids. These enzymes can oxygenate a number of polyenoic fatty acids containing one or more 1,4-cis,cts-pentadiene structures. Until recently it was assumed that lipoxygenases exhibit stringent positional specificity with respect to the removal of a hydrogen atom from a double allylic carbon atom. Also, it was thought that most lipoxygenases insert 0, stereospecifically forming only one of the possible optical isomers. N-onregiospecificity, however, has been recently demonstrated with a number of lipoxygenase preparations from both plant and animal sources (l-3). Furthermore, the steric analysis of the reaction products revealed the occurrence of a mixture of optical isomers. Interestingly, the formation of different optical isomers seems to coincide with the nonregiospecificity of lipoxygenases (3).

We have recently demonstrated that an electrophoretically pure lipoxygenase preparation from potato tubers produces all six possible HPETEs from arachidonic acid (4,s). Our preliminary data on the steric analysis of the oxygenated products of

*To whom correspondence should be addressed.

ABBREVIATIONS: H(P)ETE, hydroxy(hydroperoxy)eicosatetraenoic acid; H(P)ODE, hydroxy(hydroperoxy)octadecadienoic acid; HPLC, high-pressure liquid chromatography; SP, straight phase; CP, chiral phase.

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Vol. 170, No. 2, 1990 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

arachidonic acid revealed that 5, 8, and ll-HETEs were in S-configuration whereas 9-, 12-, and 15HETEs were non-racemic mixtures of R and S (V. Nikolaev and C. C. Reddy, unpublished results). Given the strict antarafacial relationship between the removal of a hydrogen atom and the insertion of O,, the occurrence of a mixture of optical isomers in these reactions might reveal some important mechanistic clues to the understanding of the lipoxygenase-catalyzed reactions. In the present study, we have analyzed the optical isomerism of the potato lipoxygenase-catalyzed linoleic acid oxidation products and compared it to that of the soybean lipoxygenase products. Based on the stereochemical nature of all these products, a common mechanism is suggested for the lipoxygenases showing either [ + 21 or [-21 rearrangement of the fatty acid radical.

MATERIALS AND METHODS

Materials: 9,12-&,cz&octadecadienoic acid (linoleic acid) was a product of Sigma Chemical Co. (St. Louis, MO). Soybean lipoxygenase-1 was obtained from U.S. Biochemical Corp. (Cleveland, OH).

Isolation and Assav of Linoxyeenases: The details of the purification procedure for the lipoxygenase from potato tubers are described elsewhere (4). Enzyme activities were determined polar0 raphically at 3BC in 150 mM sodium phosphate buffer (pH 6.8 for potato and pH 9.0 f or soybean lipoxygenase). The reaction was initiated by the addition of 0.18 mM linoleic acid. One unit of lipoxygenase activity is defined as one p mole of 0, consumed per min.

Prenaration of Primarv Oxveenation Products of Linoleic Acid: To obtain HPODEs, 25 U of purified potato or soybean lipoxygenase was added to 50 ml of oxygen-saturated 150 mM potassium phosphate buffer (pH 6.8 or 9.0, respectively). The reaction was started by the addition of 9 pmoles linoleic acid in 0.25 ml of ethanol. The mixture was incubated under 0, for 30 set at 22oC, with vigorous stirring. The reaction was terminated by adjusting the pH to 2.0 with 6 N HCl and extracted immediately with two volumes of hexane:ether (l:l,v/v). The organic extract was dried over anhydrous sodium sulfate and evaporated to dryness under vacuum. To assess the contribution of nonenzymic oxidation as well as hydroperoxide contamination of the commercial linoleic acid, parallel analyses were conducted on reaction mixtures incubated with heat- denatured enzymes and on unincubated linoleic acid. The HPODEs were reduced to their respective alcohols (HODEs) with NaBHa (50 mg/lOOOAU) at 0°C for 1 min. Following acidification with HCl and dilution with water, hydroxy fatty acids were extracted as described above, and separated by SP-HPLC.

The SP-HPLC analysis of the Dynamax 60A Silica 25xlOcm

roducts was carried out on a semi-preparative (partic e size 8 pm) column (Rainin Instruments Co., f

Woburn, MA). The samples were eluted isocratically at 5 ml/min with a solvent system of hexane/2-propanol/acetic acid (984:15:1, v/v/v). The effluent was monitored at 234 nm with an ultraviolet spectromonitor-165 (Beckman Instruments Co., Palo Alto, CA). The peaks were collected individually and the UV spectra of each peak was recorded on a Beckman DU-7 Spectrophotometer. Quantification of the HPLC products was estimated from the area of the individual peaks measured by Hitachi D-2000 Chromato- Integrator.

S&&&&s& bv HPLC of HODE Methylesters: Pure HODEs were methylated with excess of etheral diazomethane at 0°C for 15 min. Methylesters of HQDEs were purified again on SP-HPLC and their enantiomers were resolved on a dinitrobenzoyl Bellefonte, PA P

henyl glycine chiral phase HPLC column (25Ox4.6mm) (Supelco Inc., , using a solvent system of hexane:2-propanol:acetic acid (996:3:1, v/v/v)

at a flow rate of 2 ml/min. The R and S assignments were established by cross co- chromatography between the soybean and potato products. It is well known that potato

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lipoxygenase produces 9-HPODE in S configuration (6), while soybean lipoxygenase gives rise to 13(S)-HPODE (7).

RESULTS AND DISCUSSION

The purified potato lipoxygenase converted linoleic acid mainly to 9-HPODE, while soybean lipoxygenase produced predominantly 13-HPODE as shown on the HPLC analysis of their respective HODEs (Fig.1). The percentage compositions of all positional, geometrical, and optical isomers of the different HODEs are summarized in Table 1. ‘These findings, together with the results reported by others (6,8,9), indicate that the potato enzyme is primarily a [-21 lipoxygenase, in that it exerts strict control in the rearrangement of the fatty acid radical towards the carboxylic group (major pathway) and to a much lesser extent [ + 21, i.e. towards the methyl group (minor pathway). On the other hand, soybean lipoxygenase showed mainly [ + 21 rearrangement of the fatty acid radical (major pathway) and to a certain extent [-21 rearrangement (minor pathway), which is in agreement with previous reports (7). Evidently, some still unknown intrinsic properties of the respective lipoxygenases determine the [ + 21 or [-21 rearrangement as the major pathway.

It ‘was recently suggested that autooxidation and/or nonenzymic oxygenation of the pentadienyl radical generated by the soybean enzyme may be responsible for the formation of the [-21 rearrangement products, namely 9-HODE and (EE)-diene isomers, in the soybean reaction (10). However, the products formed in our control experiments when linoleic acid was incubated with heat-denatured enzyme constituted in most cases

A B CD

Potato Lipoxygenase

Soybean Lipoxygenase

Control

I 0 10 20

Elution Time (min)

Figure 1. Straight-phase HPLC profile of the reduced yducts of lin;ol$ haciz oxidation catalyzed b potato and soybean ipoxygenases, denatured enzyme. d 1 profiles represent equal amounts of extract from reactions differing only in the enzyme used. A: 13-(9Z,llE)-HODE, B: 13- (9E,llE)-HODE; c: 9-(lOE,12Z)-HODE; and D: 9-(lOE,l2E)-HODE.

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Table 1. Compositions of positional, geometrical and optical isomers of H(P)ODEs formed from linoleic acid by soybean-l or by potato lipoxygenasea

Enzyme Conversion Positional Geometrical Optical ratios isomersb isomer& isome&

[%I HODEs 13-HODEs 9-HODEs 13-HODE 13-HODE 9-HODE 9-HODE PI (W WI OW

13:9 (ZE):(EE) (EZ):(EE) S:R S:R S:R S:R

Control 0.3 51:49 77:23 82:18 49:51 50:50 49:51 50:50

Potato LO 75 % of non-enzymic productsd

Soybean LO 70

% of non-enzymic productsc

9:91 40:60 96:4 39:61 48:52 loo:o 92:8

(2) (0) (4) (1) (0) (1) (5) (3) (1) (1) (0) (1) (7)

95:5 98:2 67~33 loo:o 93:7 73:27 53:47

(0) (4) (0) (3) (5) (3) (0) (2) (22) (3) (9) (2) (3)

a Oxygenations and isomer determinations carried out in triplicate (SD<0.8% for the control and SD<5% for the experimental groups). The zero figures reflect values less than 0.5.

b Analyzed as HODEs by straight-phase HPLC. c Analyzed as Methyl-HOD& by chiral-phase HPLC .

d Percent of the respective product that could be accounted for by the control reaction.

less than 5% of the enzymatically formed hydroperoxides. In fact, the same amount of oxidation products was obtained from the unincubated linoleic acid substrate indicating that they came from the commercial preparation or storage of the substrate in ethanol and were not formed during the incubation. The distribution of the geometrical isomers in our control experiments agrees well with the earlier reports on the oxidation of Iinoleic acid in organic solvents (11). Therefore, nonenzymic oxidation of linoleic acid during the reaction is negligible and would not contribute in any significant way to the differences in the distribution of isomers.

Occurrence of (EE) forms of the HODEs was also found in other studies with soybean lipoxygenase (10,12,13). This phenomenon may be attributed to autooxidative isomerization of (EZ)-diene hydroperoxides by decomposition of peroxyradicals and reversible oxygen addition (14,15). The shorter reaction times, much greater conversion ratios, and lower concentrations of linoleic acid employed in our study seem to prevent any significant nonenzyrnic isomerization of the product hydroperoxides. Thus, the enzyme may be responsible for the transformation of the (EZ) products into (EE)- hydroperoxides observed in our experiments. Recently, it was shown that during oxygenation soybean lipoxygenase catalyzes even the thermodynamically unfavorable E to 2 rearrangement (13). The authors suggested that this conversion takes place at the

494


stage of the intermediate pentadienyl radical where much of the barrier to rotation of the double bond has been relieved.

Enzyme involvement in this geometrical isomerization is further supported by the stereochemical profile of the hydroperoxides. Both geometrical isomers of the major pathway products (13-HPODE and 9-HPODE for soybean and potato lipoxygenases, respectively) were predominantly in S-configuration (Table 1). During the geometrical isomerization, the enzyme appears to be largely in control of the stereochemistry, yielding gr’eater than 90% retention of the S configuration (Fig. 2) .

The steric analysis of the minor pathway products indicated enzyme participation and control over oxygen introduction during their generation. This resulted in a

predominantly R configuration (61%) for potato lipoxygenase-catalyzed 13-HPODE (EZ), while soybean-produced 9-HPODE (EZ) which was 73%S. This decreased stereoselectivity has lead to the suggestion that the lipoxygenases abstract hydrogen atoms stereospecifically when the major pathway products are formed and less selectively in the formation of the minor pathway products (16). Also, it has been suggested that the lipoxygenases act on the substrate molecule in two orientations and that the antarafacial relation does not hold during formation of the minor products (17). It has even been suggested that the linoleic acid radical is released from the ferrous enzyme because of low oxygen concentrations as proposed by Ludwig et al. (18). In this case, one would expect a stereorandom oxygenation giving rise to a racemic mixture of hydroperoxides which is inconsistent with our findings.

S

S

S R

r!d

Soybean Lipoxygenase

S

Potato

:dD’,’ 13&E) 9(EZ) HODE HODE

EE)

Elution Time

Figure 2. Chiral-phase HPLC profile of Methyl-HODEs obtained from the HPODEs generated by potato and soybean lipoxygenases from linoleic acid.

495


The results of our stereochemical analyses led us to the conclusion that when exerting their major pathway of rearrangement ([-21 or [ + 2]), at least at their pH optima, the lipoxygenases produce dioxygenation product(s) with an S-configuration. On the contrary, the minor pathway rearrangement leads to the formation of a nonracemic mixture of S and R compounds. This is supported by the data available for the lipoxygenases from corn germ (1,17), reticulocytes, wheat, pea seeds II (19) and tomato (20), as well as by a recent in viva study performed with reticulocyte lipoxygenase (21). Some lipoxygenases display no clear preference towards either of the two major rearrangements. This is the case with soybean-2 (22,23), pea seed I and quasi- lipoxygenase of hemoglobin (19), which produce virtually all possible isomers in equal amounts. Thus, further investigations are needed to check the validity of the proposed general scheme for other lipoxygenases and fatty acids.

1.

2.

3.

4.

5.

!: 8:

9.

::* 12:

13. 14.

1.5. 16.

17.

18.

19.

20.

2

23.

REFERENCES

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Stereochemical nature of the products of linoleic acid oxidation catalyzed by lipoxygenases from potato and soybean