“enzymatic” lipid peroxidation: reactions of mammalian lipoxygenases

Free Radical Biology & Medicine, Vol. 10, pp. 149-159, 1991 0891-5849/91 $3.00 + .00 Printed in the USA. All rights reserved. Copyright © 1991 Pergamon Press plc

- ~ Review Article

" E N Z Y M A T I C " L I P I D P E R O X I D A T I O N : R E A C T I O N S O F M A M M A L I A N

L I P O X Y G E N A S E S

SHOZO YAMAlVlOTO

Department of Biochemistry, Tokushima University, School of Medicine, Kurarnoto-cho, Tokushima 770, Japan

(Received 21 June 1990; Revised and Accepted 9 October 1990)

Abstract--Lipoxygenase is a dioxygenase which incorporates one molecule of oxygen at a certain position of unsaturated fatty acids such as arachidonic and linolenic acids. The enzymatic oxygenation of unsaturated fatty acids is stereospecific concomitant with a steroselective abstraction of hydrogen atom. Fatty acid cyclooxygenase is an atypical lipoxygenase incorporating two molecules of oxygen, and initiates the biosynthesis of prostaglandins and thromboxanes. Arachidonate 5-1ipoxygenase is responsible for the leukotriene synthesis. No such bioactive compound has been found as a metabolite of the 12- and 15-1ipoxygenase pathways, and their physiological roles are still unclarified. These enzymes have been purified, and their molecular and catalytic properties have been investigated. Their cDNA clones have been isolated, and their nucleotide sequences have been determined deducing the primary structures of the enzymes.

Keywords--Lipoxygenase, Cyclooxygenase, Lipid peroxidation, Prostaglandin, Thromboxane, Leukotriene, Lipoxin, Arachidonic acid, Free radicals

INTRODUCTION

The so-called "lipid peroxidation" is a nonenzymatic and nonspecific oxygenation (more precisely dioxygenation) of polyunsaturated fatty acids of both free and esterified forms. As will be discussed in this review article, the reactions catalyzed by lipoxygenase enzymes are regiospecific and stereoselective oxygenation of unsaturated fatty acids.

LIPID PEROXIDATION CATALYZED BY LIPOXYGENASE

ENZYMES

A soybean enzyme termed lipoxidase (now referred to as lipoxygenase) was discovered much earlier, and its catalytic properties have been investigated in relation to

Shozo Yarnamoto, M.D., graduated from Osaka University School of Medicine in 1960. He received a Phi) from Kyoto University Fac- ulty of Medicine. A research associate, lecturer, and associate professor at Kyoto University in 1964-1978, he collaborated with Osamu Hayaishi in an investigation of flavoprotein monooxygenases and the enzymes of arachidonate cascade. As a postdoctoral fellow at Harvard University, Department of Chemistry, he collaborated with Konrad Bloch in an investigation of the enzymes of oxidative cyclization of squalene. He has been Professor of Biochemistry, Tokushima Univer- sity, School of Medicine, since 1979. His current research interests are enzymology and molecular biology of lipoxygenases, lipoxygenase inhibitors, and immunoassays of oxyeicosanoids.

the mechanism of the nonenzymatic lipid peroxidation. As described by Tappel in 1966,1 it was believed at that time that "there was no lipoxidase in animal tissues.

In 1967, the mechanism of prostaglandin biosynthesis by seminal vesicle was studied with reference to the reaction of soybean lipoxygenase, 2 and the lipoxygenase nature of the prostaglandin-synthesizing enzyme (now referred to as fatty acid cyclooxygenase) was shown by the isotope tracer experiments. 3 About 15 years ago, two papers demonstrated the occurrence of a lipoxygenase in human 4 and bovine 5 platelets. Along with the discovery of thromboxane produced from arachidonic acid, production of 12(S)-hydroxy-5Z, 8Z, 10E, 14Z-eicosatetraenoic acid (12(S)-I-IETE) was shown to occur with human platelets. 4 The 12-hydroxy acid was presumed to be derived from a 12(S)-hydroperoxy acid, 4 and, in fact, a study with bovine platelets demonstrated the formation of 12-hydroperoxy-5Z,8Z,10E, 14Z-eicosatetraenoic acid (12-HPETE). 5 In contrast, rabbit leukocytes converted arachidonic acid to 5(S)-hydroxy-6E,SZ, 11 Z, 14Z-eicosatetraenoic acid (5-HETE). 6 This finding implicated the occurrence of a 5-1ipoxyge- nase enzyme which led later to the discovery of leukotrienes. 7 In addition, rabbit leukocytes also produced 15-HETE, and the 15-1ipoxygenase was partially purified to a preparation producing 15(S)-hydroperoxy- 5Z,8Z, 11Z, 13E-eicosatetraenoic acid.S

149

150 S. YAMAMOTO

~ 'v~'x~COOH OH HO0" 12 ' ~ / ~

12-HPETE ~__~__%/%/00H 15-HPETE

i ~ ~ " ~ ~ x j ~ j GOOH PGG 2 5-HPETE

Fig. 1. Mammalian lipoxygenases.

These lipoxygenase enzymes are dioxygenases with polyunsaturated fatty acids as substrate. One molecule of oxygen is incorporated at certain positions of the unsaturated fatty acids. Fatty acid cyclooxygenase is an atypical lipoxygenase incorporating two molecules of oxygen.

REGIOSPECIFIC OXYGENATION

The oxygenation site of the lipoxygenase substrate is different from enzyme to enzyme. Each lipoxygenase is referred to as arachidonate x-lipoxygenase where x is the number of the oxygenated carbon of arachidonic acid as substrate counted starting from the carboxylic carbon. Reference 9 is a guideline for the nomenclature of prostaglandin enzymology.

For example, as shown in Fig. 1, arachidonate 15-1i- poxygenase oxygenates the carbon-15 of arachidonic acid which has 20 carbon atoms. When the same enzyme reacts with linoleic acid of 18 carbon atoms, the oxygenation site is the carbon-13. It should be noted that the carbon-15 of arachidonic acid and the carbon-

Forty Acid Cyclooxygenose

Proetoglandln Hyclroperoxldase

'0"0 ~ O O H

Arochldonlc Acid

o- iR..p T.o O-*E

~ OOH

bOH PGG 2

PGH2

Fig. 2. Reactions of fatty acid cyclooxygenase and prostaglandin hydroperoxidase.

, I 4

1,4-pentodlene

HO-O" ~H

• H ,

.H

Fig. 3. Oxygenase reaction catalyzed by lipoxygenases.

13 of linoleic acid are the (to-6) carbons of both fatty acids. 5-Lipoxygenase oxygenates the carbon-5 of arachidonic acid and produces 5-HPETE. Figure 2 illus- trates the reaction of fatty acid cyclooxygenase. It is presumed that the carbon-11 of arachidonic acid is oxygenated, followed by another oxygenation at the carbon-15 concomitant with cyclization. 3 The l l- peroxyarachidonic acid is presumed as an enzyme- bound intermediate, and the final product is prostaglandin G 2 with 9,11-endoperoxide and 15-hydroperoxide. However,the 11-hydroperoxy acid exogenously added to the enzyme was not metabolized to prostaglandin G 2. ~o

STEREOSPECIFIC OXYGENATION

The oxygenated carbon atom is now an asymmetric carbon (Fig. 3). In the nonenzymatic lipid peroxidation, the oxygenated product is a mixture of hydroperoxides with D R- and Ls-configurations. In sharp contrast, the enzymatic oxygenation is almost stereospecific, and a predominant product is a peroxy acid of either DR- or Ls-configuration. With arachidonic acid as substrate, 5(S)-HPETE is a predominant product by 5-1ipoxygen- ase, 12(S)-HPETE by 12-1ipoxygenase, and 15(5")- HPETE by 15-1ipoxygenase. In the cyclooxygenase reaction, the presumed 11-peroxy intermediate is of R- configuration, and the final product (prostaglandin G2) has a 15(S)-hydroperoxy group. Sea urchin eggs contain

OCed

5S-HPETE OOH OOH 15S-HPETE 12 S-I'~ETEcooH j

8R-HPETE OOH OOH 12R'HPETE I IR-HPETE

Fig. 4. Stereospecific lipoxygenase reactions.

Mammalian lipoxygenases

Table 1. Stereoselective Hydrogen Liberation in Lipoxygenase-Catalyzed Reactions

151

Eliminated Enzyme Source Substrate Product hydrogen References

Cyclooxygenase Seminal Bishomo-?-linolenic acid PC_K31 13-proS(L) 3 vesicle

5-Lipoxygenase Leukocyte 5-Oxygenase 5,6-LTA synthase

12-Lipoxygenase Platelet 12-Oxygenase 14,15-LTA synthase

15-Lipoxygenase Soybean

Arachidonic acid 5-HPETE 7-proS(D) 15 5-HPETE 5,6-LTA a 10-proR(D) 53,56--58

Arachidonic acid 12-HPETE 10-proS(L) 16,17 15-HPETE 14,15-LTA 4 10-proS(L) 17

Bishomo-'y-linolenic acid 15-Hydroperoxy- 13-proS(L) 2 eicosatrienoic acid

l i p o x y g e n a s e s p r o d u c i n g l l (R) - and 12(R) -hydroxy-

e i cosa - t e t r aeno ic acids ,~l and g o r g o n i a n coral has an 8-

l i p o x y g e n a s e p r o d u c i n g 8 ( R) - oxygena t ed a rach idon ic

ac id 12 (Fig. 4) . C y t o c h r o m e P-450 , a h e m o p r o t e i n mo-

n o o x y g e n a s e , oxygena t e s var ious pos i t ions o f a rach idon ic

acid. 13 The p roduc t s are a mix tu re o f R- and S -hydroxy

acids. 14

S T E R E O S P E C I F I C H Y D R O G E N A B S T R A C T I O N

L o n g - c h a i n unsa tu ra t ed fa t ty acids w i th a s t ruc ture o f

1-cis, 4-cis-pentadiene are the subs t ra tes o f l i poxygena -

ses (Fig. 3). T w o cis-double b o n d s are in t e r rup ted wi th

a m e t h y l e n e g roup . W h e n a c a r b o n a tom is oxygena t ed ,

the e n z y m e l ibera tes one o f the two h y d r o g e n a toms o f

the m e t h y l e n e g roup f l anked by the d o u b l e bonds . Ste-

reose lec t iv i ty o f the h y d r o g e n l ibe ra t ion was s h o w n

by an i so tope t racer e x p e r i m e n t wi th s o y b e a n

l i p o x y g e n a s e 2 and cyc looxygenase . ~ S te reospec i f i ca l ly

t r i t i um- labe led subs t ra te , [ 13DR-all] or [ 13Ls-aH]8 ,11 ,

14-e icosa t r ieno ic acids , was a l l owed to reac t w i th soy-

b e a n l i poxygenase 2 or the m i c r o s o m e s o f o v i n e semina l

ves ic le , 3 and the p roduc t was ana lyzed for the 3H-con-

tent . On ly the t r i t ium of L con f igu ra t i on (proS hydro-

gen) was r e m o v e d , and the o the r h y d r o g e n was re ta ined .

T h e c o n v e r s i o n o f [13Ls-aH]subs t ra te was a c c o m p a n i e d

by an i so tope effect . S ince 3H e n r i c h m e n t was found in

the p recurso r acid , the s te reospec i f ic h y d r o g e n r e m o v a l

was p r e s u m e d to occu r at the ini t ia l s tep o f the reac t ion .

As s u m m a r i z e d in T a b l e 1, such a s te reospec i f ic hydro -

gen l ibe ra t ion was also r epor ted for o the r l i poxygena -

ses; 7 -proS h y d r o g e n in 5-1 ipoxygenase reac t ion x5 and

Table 2. Substrate Specificities of Lipoxygenases. Vm, x values are compared with arachidonic acid as 100%

Substrates 5-Lipoxygenase 12-Lipoxygenase 15-Lipoxygenase

Carbon Double RBL Guinea Pig Porcine Bovine Bovine Bovine Rabbit Number Bond Cell Leukocyte Leukocyte Leukocyte Platelet Trachea Soybean Reticulocyte

18 9,12 8 13 208 69 11 5 2 51 130 310 6,9,12 -- -- 172 100 32 14 2 72 51 280 9,12,15 7 11 109 56 17 5 2 46 103 200

20 8,11 -- -- 125 -- -- 26 . . . . 11,14 -- -- 92 44 15 5 2 29 113 380 5,8,11 82 - - 1 3 0 - - - - 8 1 - - - - - - 60 7,10,13 . . . . . . . . . 590 8,11,14 II 43 144 55 78 60 7 98 -- 300 11,14,17 -- -- -- 36 28 -- 2 32 73 220 5,8,11,14 100 100 100 100 100 100 100 100 100 100 5,8,11,14,17 177 116 36 67 73 60 94 57 119 --

22 4,7,10,13,16,19 2 -- -- 56 92 14 7 91 122 80

20 5-HETE -- -- 87 50 15 -- 2 -- -- -- 5-HPETE -- -- -- 127 . . . . . . 12-HETE -- -- -- 1 . . . . . . 12-HPETE -- -- -- 1 . . . . . . 15 - HETE -- -- -- 2 . . . . . 7 15-HPETE -- -- -- 58 18 -- 6 -- -- --

Reference: 22 23 27 26 25 5 25 29 30 31

152 S. YAMAMOTO

8~ISS-dlHPETE 14R,15S-diHPETE

8S-OxygenoN ~ ~lAR-Oxygenose ~__~OOH

60H 15S-HPETE

14jlS-LTA .~1 symhose ~

p:~coo.

~.mS~ETE "'- \ HO ~ H~ bH 14S,15S-dlHETE 14R,15S-dlHETE

8S,15S~IHETE

Fig. 5. Reactions catalyzed by arachidonate 12-1ipoxygenase.

10-proS hydrogen in 12-1ipoxygenase reaction, a6,17 An antarafacial relationship was pointed out between the hydrogen abstraction and the oxygenation. ~6 A hydrogen atom leaves from one side of the substrate molecule, and an oxygen molecule enters from the other side.

Identification of the hydrogen-abstracting group of cyclooxygenase and lipoxygenases has been a subject of discussions and experimental approaches. As described below (p. 7), soybean lipoxygenase is an iron-containing enzyme, and its ferric form as an active enzyme was presumed to be reduced to the ferrous state by the substrate fatty acid with concomitant production of a fatty acid free radical, a8,19 Analysis of EPR spectra observed in the cyclooxygeenase reaction suggested the formation of a tyrosyl radical which may be involved in the hydrogen abstraction at C-13 of arachidonic acid. 2° A similar spectrum was also described by other investigators. 21

SUBSTRATE SPECIFICITY

As examined with two 5-1ipoxygenase preparations from rat basophilic leukemia cells 22 and guinea pig peritoneal leukocytes 23, 5,8,11,14,17-eicosapentaenoic acid is as active as arachidonic acid (Table 2). 5,8,11-Eico- satrienoic acid is oxygenated at C-5 almost as actively as arachidonic acid, while 8,1 l, 14-eicosatrienoic is oxygenated at C-8 at a lower rate as earlier described. 6 5- Lipoxygenase is almost inactive with Cx8 and C22 polyenoic acids.

Studies on the substrate specificity of 12-1ipoxygen- ases of bovine platelets and leukocytes brought about an interesting and important finding. 24 As listed in Table 2, 12-1ipoxygenases of platelets and leukocytes exhibit different substrate specificities. Bovine platelet 12- lipoxygenase is much more active with C2o acids than C18 and C22 acids. As tested with a purified enzyme of bovine platelets, linoleic acid and or- and v-linolenic acids are almost inactive. 25 In sharp contrast, 12-1ipoxy- genase of porcine 26'27 and bovine 25 leukocytes oxygenate C18 polyenoic acids as active substrates. C22 acid is also oxygenated at a considerable rate. Thus, there are two types of 12-1ipoxygenase in terms of substrate specificity. These two types of enzyme are also immunologically distinguishable as demonstrated by the use of monoclonal antibodies against leukocyte and platelet enzymes, z5 Leukocyte-type 12-1ipoxygenases were recently found in porcine pituitary 2a and bovine trachea. 29

Substrate specificity of soybean lipoxygenase was quantitatively described in reference 30. The enzyme is active with a variety of Cla, C2o, and C22 fatty acids with double bonds at the 6th and 9th carbon atoms counting from the methyl group. For reticulocyte 15-1i- poxygenase arachidonic acid, 8,11,14-, 11,14,17- and 5,8,11-eicosatrienoic acids and 11,14-eicosadienoic acid are active substrates. Linoleic acid and ct- and ~/-linolenic acids are more active than arachidonic acid. 31

A unique catalytic property of 15-1ipoxygenase is its

~ _ ~ H ~ cOOH

LXA4 oH +HzOf+ H LXi4 5-Lipcmyge.ale .~ - , ^~.

OOH l +H '5,.-LTA ) S y ~ e ) ~ ~ - ~ v v . H --RIO OOH +02 . / ~ ~ O O H 15.flydroperoxy-5,6 -LTA 4

,.-.,,..,,o,,,,'..,. ,,,,--...., ) (14R- -----) , , ,~,,,- ~ v v - , - o x y g m II-L~xygl~Qli 5-~b~troperoxi-11,15-LTl 4 (14,15-LTA Synthose)

Fig. 6. Lipoxin syntheses by 12-, 15- and 5-1ipoxygenases.

Mammalian lipoxygenases 153

OH" ~ C O O H 1 ooo.__ ,°, X/X/ W ' W V ' v

n ta.e 13-Oxo.iaeca-gZJIE- oo. / o

13-Hydroperoxyoctodeco- h ()o. " 9Z, I IE-dienoic ocid dienoic ocid

COOH

12-Hydroperoxyeicosa- 5Z,8Z, IOE,14Z-tetroenoic ocid

2-Octene 12-Oxododeco-SZ, 8Z,IOE- trtenoic ocid

Fig. 7. "Lipohydroperoxidase" reaction catalyzed by 15- and 12-1ipoxygcnases.

ability to oxygenate the unsaturated fatty acid moiety of phospholipid. Arachidonic acid and linoleic acid esteri- fled in phosphatidyl-choline were allowed to react with soybean lipoxygenase, and converted to 15(S)-HPETE and 13(S)-hydroperoxy-9,11-octadecadienoic acid. 32 15- Lipoxygenase preparations of human polymorphonuclear leukocyte, soybean and rabbit reticulocyte oxygenate phosphatidyl-choline at carbon-15 of the arachidonate moiety. 33 In contrast, no oxygenation of esterified arachidonic acid was observed with rat basophilic leukemia cell 5-1ipoxygenase and rabbit platelet 12- lipoxygenase. 33 A functional role of 15-1ipoxygenase was earlier proposed for the degradation of reticulocyte mitochondria in red cell maturation.34 The lipid-peroxi- dizing effect of lipoxygenase was shown by an in vivo experiment. Namely, bleeding anemia was induced in rabbit, and the red cell membranes were analyzed for the lipoxygenase products. The oxygenated products

from linoleic acid and arachidonic acid appeared in the course of reticulocytosis. 35

LIPOXYGENASES AS MULTIFUNCTIONAL ENZYMES

A multifunctional nature of lipoxygenase was first demonstrated with plant enzymes. In addition to a dioxygenation producing a monohydroperoxy product, the enzyme catalyzes a double dioxygenation producing a dihydroperoxy acid from unsaturated fatty acid. The hydroperoxy product is further metabolized anaerobically demonstrating a lipohydroperoxidase activity. Reference 36 is a review article describing catalytic properties of plant lipoxygenase.

12-Lipoxygenase of porcine leukocytes When the purified 12-1ipoxygenase from porcine 37

and bovine 25 leukocytes reacted with arachidonic acid,

E~Oxygenaeo ,..,.+..,~COOH ~,,,,m,,.,,.~OOH ~ ' | - - HO0.'-~..~ Aroohldonic AoId 12S-HPETE

4[. 15S-Oxygenou 8S-Oxygenose 5S-Oxy9 enase OOH

OOH OOH 15S'HPETE 5~ISS-dlHPL~rE 89,15S-diHPETE J4(- 14115-LTA Synmase

14tlS"LTA4 8,15-dlHETE

Fig. 8. Reactions catalyzed by arachidonate 15-1ipoxygenasc.

154 S. YAMAMOTO

ookl

~ 5S-Oxygermse

5S-I-IPETE

~COOH ~COOH I - T A 4 5S,6R'diHPETE

Fig. 9. Reactions catalyzed by arachidonate 5-1ipoxygenase.

a small amount of 15-HPETE was produced in addition to 12-HPETE as a predominant product. Thus, 12-1i- poxygenase functions as 15-1ipoxygenase with a part of substrate.

Earlier work by Brash and associates demonstrated that whole cells of porcine leukocytes produced a variety of dihydroxy acids from arachidonic acid, 17,38 and the formation of these dihydroxy acids from 15-HPETE was predicted to be "catalyzed by an enzyme having many mechanistic features in common with the 12- lipoxygenase. '']7 This prediction was shown to be true by the use of a purified preparation of 12-1ipoxygenase from porcine leukocytes. 26

The porcine leukocyte 12-1ipoxygenase reacts with 15-HPETE at a rate comparable with that of the arachidonate 12-oxygenation. The enzyme transforms 15- HPETE to a mixture of several dihydroperoxy and dihydroxy acids each with a conjugated triene. The for- marion of these products are rationalized as illustrated in Fig. 5. When the enzyme functions as 14(R)-oxygenase and 8(S)-oxygenase with 15-HPETE as substrate, the products are 14(R), 15(S)- and 8(S), 15(S)-dihydroperoxy acids, respectively. Alternatively when the enzyme cleaves the bond between the two oxygen atoms of 15-hydroperoxide, the product is a compound with a 14,15-epoxide and a conjugated triene (14,15-1eukotriene A4), which is readily hydrolyzed nonenzymatically to a dia- stereomeric mixture of 8,15- and 14,15-dihydroxy acids.

When whole ceils of porcine leukocytes were incubated with either 10-Ls-3H - or 10-DR-all-labeled 15- HPETE and the above-mentioned dihydroxy acids were analyzed for the retention and loss of 3H, about 98% of tritium was removed from 10-Ls-3H-15-HPETE, indi- cating a stereoselective abstraction of proS hydrogen from the position-10.17

Lipoxins are trihydroxy metabolites of arachidonate with a conjugated tetraene. 39 The purified 12-1ipoxyge- nase of porcine leukocytes was shown to produce lipoxins from 5(S),15(S)-diHPETE. 4° As illustrated in Fig. 6, the production of a hydroperoxy derivative

of lipoxin B 4 (5(S),14(R),15(S)-trihydroxy-6E,8Z, lOE, 12E-eicosatetraenoic acid) is attributable to the 14(R)- oxygenase activity of 12-1ipoxygenase. The hydroperoxy product is reduced to lipoxin B 4. The 14,15- leukotriene A synthase activity is responsible for the formation of 5-hydroperoxy- 14,15-1eukotriene A4, which is readily hydrolyzed to various isomers of lipoxins A 4

and B 4. In the presence of excess arachidonic acid and under

anaerobic conditions, 12-1ipoxygenase transforms 12- HPETE to 12-oxododeca-5Z,8Z, lOE-trienoic acid and presumably to 2-octene (Fig. 7). 41 This activity corresponds to the lipohydroperoxidase activity demonstrated with soybean lipoxygenase and 13-hydroperoxy-9,11- octadecadienoic acid. 42 As illustrated in Fig. 7, either an alkoxy radical (a) or a peroxy radical (b) was presumed as an intermediate. 43 13-Scission of an intermediate alkoxy radical was proposed in the reaction of 10-hydroperoxy-8-octadecenoic acid catalyzed by hematin. 44

15-Lipoxygenase of rabbit reticulocytes

A multifunctional nature was also described for 15- lipoxygenase of rabbit reticulocytes. As shown in Fig. 8, the purified enzyme also exhibits 12-1ipoxygenase activity transforming arachidonic acid partly to 12- HPETE (5-10% of 15-HPETE). 45'46 The produced 15- HPETE is subjected to 5(S)-oxygenation and 8(S)- oxygenation. 15-HPETE is also converted to 14,15- leukotriene A4, which is degraded to 8,15-diHETE with an all-trans conjugated triene. 47 An enzyme of human leukocytes was purified. 47a

Anaerobic reaction of rabbit reticulocyte 15-1ipoxy- genase with 13-hydroperoxy-9,11-octadecadienoic acid released pentane 48 (Fig. 7). As described above, this reaction corresponds to the lipohydroperoxidase reaction described for soybean lipoxygenase.

The purified 15-1ipoxygenase of rabbit reticulocyte produces lipoxin B 4 by either 14(R)-oxygenation of 5,15-diHETE or double oxygenation of 15-HETE or tri- ple oxygenation of arachidonic acid 49 (Fig. 6).

5-Lipoxygenase of human, porcine and rat leukocytes

A potato lipoxygenase with arachidonate 5-oxygenase activity was purified to apparent homogeneity. 5° The enzyme oxygenates the position-8 of 8,11,14-eicosatrienoic acid. The same purified enzyme converts 5-HPETE to 5(S), 12(S)-diHPETE and leukotriene A 4 with a 5,6-epoxide. The latter product is unstable, and its degradation products (6-trans-leukotriene B 4 and its 12-epimer) are actually detected. The leukotriene A synthase activity is about one fifth of the arachidonate 5-oxygenase activity. Experiments with inhibitors and heat inactivation suggest that the two enzyme activities are attributed to a single enzyme protein.


Mammalian 5-1ipoxygenases also show both the 5- oxygenase and leukotriene A synthase activities. 51-54 When the enzyme is allowed to react with a saturating concentration of arachidonic acid, the predominant product is 5-HPETE and only a part of the 5-HPETE is further converted to leukotriene A 4. The leukotriene A synthase activity with 5-HPETE as substrate is only about 5% of the arachidonate 5-oxygenase activity. The rate of leukotriene A 4 production from arachidonic acid is higher than that from exogenously added 5-HPETE. 52 This observation is in agreement with the result of an experiment using deuterated arachidonic acid (to endogenously generate deuterated 5-HPETE) and exogenous nonlabeled 5-HPETE. 55 As shown in Table 1, experiment with stereospecifically tritiated arachidonic acid or 5-HPETE demonstrated a stereoselective loss of proR hydrogen at the carbon 10 in the leukotriene A synthase reaction. 53,56-58

When the purified 5-1ipoxygenase reacts aerobically with 5-HPETE, 5(S),6(R)-diHPETE is produced in addition to leukotriene A4 .59 5-HETE is oxygenated to 5(S)-hydroxy-6(R)-hydroperoxy acid at 1-2% the rate of arachidonate 5-oxygenation. The activator requirement and the effect of selective inhibitors of 5-1ipoxygenase indicate the association of the 6(R)-oxygenase activity with the 5(S)-oxygenase activity.

As in the catalysis of 12-1ipoxygenase, 5,15-diHPETE is a substrate of 5-1ipoxygenase to produce various lipoxin isomers including lipoxin A46° (Fig. 6). Since the product profile is essentially the same in both aerobic and anaerobic reactions, the production of various lipoxin isomers is mostly attributable to the anaerobic 5,6-epoxide pathway. Namely, 5,15-diHPETE is trans- formed to 15-hydroperoxy-5,6-epoxide by the 5,6-1eu- kotriene A synthase activity of 5-1ipoxygenase. There is only a minor contribution of the 6(R)-oxygenase activity of 5-1ipoxygenase s9 to the aerobic multiple oxygenation pathway. The rate of the lipoxin synthesis is only 2% of the arachidonate 5-oxygenation.

COFACTORS OF MAMMALIAN LIPOXYGENASES

Iron

The content of non-heme iron as an essential component of soybean lipoxygenase was earlier reported. 61~53 The purified 12-1ipoxygenase of porcine leukocytes was reported to contain 0.45 atom of iron per mole of enzyme, 26 and about 1 atom of iron was found per mole of rabbit reticulocyte 15-1ipoxygenase. 64 The hemopro- tein nature of cyclooxygenase has been extensively investigated. 65'66 A significant quantity of nonheme iron was found in the enzyme preparation of Hemler and others 67 while only 0.1568 or 0.04--0.1269 mol of

iron per mol of enzyme was observed by Van der Oud- eraa et al.

The hydroperoxide requirement was reported much earlier for soybean lipoxygenase, 7° and its role was further studied. 71 Since the cyclooxygenase reaction was found to be triggered and accelerated by the endogenously produced prostaglandin G (a hydroperoxy prostaglandin) or by the added hydroperoxide of arachidonic acid, 72 other mammalian lipoxygenases have also been shown to require hydroperoxide to abolish the lag phase of the reaction. The reaction of 12-1ipoxygenase of porcine leukocytes with arachidonic acid starts with a lag phase of up to 30 s, and the lag is abolished by the addition of 1 ~M 12-HPETE. 26 The effect of hydroperoxy fatty acid to activate the initial reaction velocity is also observed with 5-1ipoxygenase of human leukocytes 73 and 15-1ipoxygenase of rabbit reticulocytes. 74

The requirement of hydroperoxide for the lipoxygenase reaction has been discussed in terms of the valence state of the iron contained in the enzyme. On the basis of an EPR study on soybean lipoxygenase-1, Vliegent- hart and associates proposed a mechanism that the enzyme alternatively exists in the ferric and ferrous states and the ferric enzyme is an active species which is generated by interaction of the ferrous enzyme and hydroperoxide, is Reference 71 summarizes discussions on the role of hydroperoxide activator in the lipoxygenase reaction. As for the role of the cyclooxygenase heme, Hemler and Lands proposed a hydroperoxide reduction of the native ferric enzyme to the active ferrous enzyme concomitantly generating a peroxy radical. 75 According to more recent investigations on the prostaglandin hydroperoxidase reaction, 21,76,77 the addition of hydroperoxide to the ferric enzyme did not produce the ferrous enzyme, but higher oxidation states of the enzyme were observed. Spectral changes of the enzyme-bound heme were followed by a rapid reaction technique. A spectral species similar to compound I of horseradish peroxidase appeared first, and then was converted to a species spectrally similar to compound II. Thus, the prostaglandin hydroperoxidase reaction seems to involve a cycle of a native ferric enzyme, compound I and compound II.

Calcium

The requirement of calcium ion for the 5-1ipoxyge- nase reaction was first demonstrated when a crude cell- free enzyme preparation of rat basophilic leukemia cells was incubated with arachidonic acid. 78 The conversion of arachidonic acid is markedly stimulated by the addition of mM concentrations of CaCI2. The calcium requirement was later confirmed with the purified 5- lipoxygenases from various sources .22,23,52-54,79-81 Unlike most 12-1ipoxygenases, the enzyme in the cytosolic

156 S. YAMAMOrO

fraction of rat basophilic leukemia cells was stimulated by the addition of Ca 2+, Mg 2÷, or Mn2+. 82 These divalent cations at mM concentrations were required for the 15-1ipoxygenase in the cytosol of rabbit leukocytes, but the purified enzyme did not show such a divalent cation dependence, a

ATP

ATP is another stimulator of 5-1ipoxygenase. 23 The arachidonate 5-oxygenation of a partially purified enzyme from guinea pig peritoneal polymorphonuclear leukocytes requires the presence of calcium ion. The calcium-dependent 5-1ipoxygenase activity is stimulated about 4-fold by the addition of ATP. The stimulatory effect is not specific for ATP. ADP and AMP and other nucleoside triphosphates also stimulate the calcium-dependent 5-1ipoxygenation, but ATP is the most prominent stimulator. Although the role of ATP is poorly understood, its stimulatory effect has been observed with other purified 5-1ipoxygenases. 22'52-54'81 It should be noted that the requirement of calcium and the stimulation by ATP are observed not only in the arachidonate 5-oxygenase activity but also in the leukotriene A synthase activity: 1-54

Other factors

In addition to calcium ion and ATP, maximal activity of human leukocyte 5-1ipoxygenase requires at least two other soluble high molecular weight factors derived from the high-speed supernatant of human leukocytes 81 and at least one membrane-bound component. 8t'83 The latter component is replaced by phosphatidylcholine vesicles. 84

CLONING OF COMPLEMENTARY AND GENOMIC DNAs

FOR M AM M AL IAN LIPOXYGENASES

The cDNA for cyclooxygenase was first isolated from human vascular endothelial cells by screening a kgt l l cDNA expression library with the aid of a polyclonal anticyclooxygenase antibody.S5 However, the nucleotide sequence of the cDNA remained unclarified. Later, three research groups cloned the cDNA for the enzyme of ovine seminal vesicles, and the primary structure of the enzyme was deduced from the nucleotide sequence of the cDNA, giving a sequence of 600 amino acids with a signal peptide of 24 amino acids 86'87 or that of 599 amino acids with a 23-amino acid signal sequence. 88 The site of acetylation by aspirin was located at the serine (#530) near the C-terminus. 86-88 The site-directed mu- tation from serine-530 to alanine did not affect the enzyme activity, suggesting the non-involvement of the

serine residue in the enzyme catalysis. 89 A 40-kb genomic DNA encoding the human cyclooxygenase was cloned, and its nucleotide sequence was determined. 9° The region encoding 599 amino acids is distributed into 11 exons. The human enzyme exhibits 91% amino acid identity with the sheep enzyme. According to a com- puter-assisted homology search, the N-terminal region of the ovine cyclooxygenase shares a sequence homology with epidermal growth factor. 91

A cDNA clone for 5-1ipoxygenase was isolated from human cDNA libraries by two research groups, 92'93 and the primary structure of a 673 -92 or 674 -93 amino acid protein was deduced. The 5-1ipoxygenase cDNAs were expressed in mammalian 94 and insect 95 cells. The re- combinant enzyme transforms arachidonic acid to 5- HPETE and LTA 4, and shows a dependence on calcium ion and ATP. 94'95 This finding serves as independent evidence that both 5-1ipoxygenase and LTA synthase activities are attributed to a single enzyme protein. His- tidine-362 and histidine-372 as potential iron-atom ligands when changed to serine residues, did not affect enzyme activity. 95 A genomic DNA of human 5-1ipoxygenase was isolated and found to consist of 14 exons. 96

cDNA clones for plant lipoxygenases were isolated, and their primary structures were deduced from the nucleotide sequences: soybean lipoxygenase-1, 97 soybean lipoxygenase-2, 98 soybean lipoxygenase-3, 99 and pea seed lipoxygenase, lOO-lO2 A cDNA encoding rabbit reticulocyte 15-1ipoxygenase was isolated, lO3-1o6 A genomic DNA with 14 exons for the rabbit reticulocyte 15-1i- poxygenase was isolated, lO7 A full-length cDNA for human reticulocyte 15-1ipoxygenase was also isolated, 1°8 and expressed in osteosarcoma cells and E. coli. 1°9 Comparison of these reticulocyte 15-1ipoxygenases with human 5-1ipoxygenase and soybean lipoxygenase indi- cated the presence of a cluster of 5 histidine residues which is conserved in these lipoxygenases and is predicted to chelate non-heme iron in the active site. lO6,1os

cDNA clones for porcine leukocyte 12-1ipoxygenase were recently isolated, and from their nucleotide sequences the primary structure of the enzyme protein was deduced and shown to consist of 662 amino acid residues. 11° Site-directed mutagenesis experiments with these lipoxygenase cDNA clones and analyses for the 5'-upstream regions of the genomic DNA's will lead to a better understanding of the catalytic and regulatory mechanism of lipoxygenase enzymes. References 1,36,64,65,71, and 111-120 are review articles dealing with lipoxygenases.

Acknowledgemen t s - - The author is grateful to Dr. Cecil R. Pace- Asciak, University of Toronto, for his critical reading of this manu- script, and to Dr. Alan R. Brash, Vanderbilt University, and Dr. Richard J. Kulmacz, The University of Texas, for their kind valuable advices and discussions about the reaction mechanism of lipoxygenase and cyclooxygenase.


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“enzymatic” lipid peroxidation: reactions of mammalian lipoxygenases

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