“Enzymatic” lipid peroxidation: Reactions of mammalian lipoxygenases

Free Radical Biology & Medicine, Vol. 10, pp. 149-159, 1991 Printed in the USA. All rights reserved.

0891-5849/91 $3.00 + .00 Copyright © 1991 PergamonPress plc

- ~ ReviewArticle "ENZYMATIC"

LIPID PEROXIDATION: REACTIONS OF MAMMALIAN LIPOXYGENASES

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 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 human4 and bovine5 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, 11Z, 14Z-eicosatetraenoic acid (5-HETE). 6 This finding implicated the occurrence of a 5-1ipoxygenase 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)-hydroperoxy5Z,8Z, 11Z, 13E-eicosatetraenoic acid.S

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 Faculty 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 University, School of Medicine, since 1979. His current research interests are enzymology and molecular biology of lipoxygenases, lipoxygenase inhibitors, and immunoassays of oxyeicosanoids. 149

150

S. YAMAMOTO

~ v' ~'x~COOH

OH

'~ /~

HO0" 12 12-HPETE

i ~

,I

~__~__%/%/00H

~

PGG2

15-HPETE

" ~ ~ x j ~ j GOOH 5-HPETE

4

1,4-pentodlene

•H ,

.H HO-O" ~H Fig. 3. Oxygenasereactioncatalyzedby lipoxygenases.

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 15-1ipoxygenase 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-

'0"0 ~ O O H ArochldonlcAcid Forty Acid Cyclooxygenose

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 illustrates 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 lperoxyarachidonic acid is presumed as an enzymebound 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 5-1ipoxygenase, 12(S)-HPETE by 12-1ipoxygenase, and 15(5")HPETE by 15-1ipoxygenase. In the cyclooxygenase reaction, the presumed 11-peroxy intermediate is of Rconfiguration, and the final product (prostaglandin G2) has a 15(S)-hydroperoxy group. Sea urchin eggs contain

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

~ Proetoglandln Hyclroperoxldase

OOH 5S-HPETE

PGH2

andprostaglandinhy-

Fig. 2. Reactionsof fatty acid cyclooxygenase droperoxidase.

OOH

OOH 15S-HPETE

12S-I'~ETEcooH j

bOH PGG2

8R-HPETE

OOH 12R'HPETE

OOH I IR-HPETE

Fig. 4. Stereospecificlipoxygenasereactions.

151

Mammalian lipoxygenases Table 1. Stereoselective Hydrogen Liberation in Lipoxygenase-Catalyzed Reactions

Enzyme

Source

Substrate

Product

Eliminated hydrogen

Cyclooxygenase

Seminal vesicle

Bishomo-?-linolenic acid

PC_K31

13-proS(L)

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

Leukocyte Arachidonic acid 5-HPETE

5-HPETE 5,6-LTAa

7-proS(D) 10-proR(D)

15 53,56--58

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

Platelet Arachidonic acid 15-HPETE

12-HPETE 14,15-LTA 4

10-proS(L) 10-proS(L)

16,17 17

15-Lipoxygenase

Soybean

Bishomo-'y-linolenic acid

15-Hydroperoxyeicosatrienoic acid

13-proS(L)

acids. 14

H Y D R O G E N

3

2

r e o s e l e c t i v i t y o f the h y d r o g e n l i b e r a t i o n w a s s h o w n by an isotope tracer experiment with soybean lipoxygenase 2 and cyclooxygenase. ~ Stereospecifically t r i t i u m - l a b e l e d substrate, [ 13DR-all] or [ 1 3 L s - a H ] 8 , 1 1 , 1 4 - e i c o s a t r i e n o i c acids, w a s a l l o w e d to r e a c t w i t h soyb e a n l i p o x y g e n a s e 2 or the m i c r o s o m e s o f o v i n e s e m i n a l v e s i c l e , 3 a n d t h e p r o d u c t w a s a n a l y z e d for the 3H-content. O n l y t h e t r i t i u m o f L c o n f i g u r a t i o n ( p r o S h y d r o g e n ) w a s r e m o v e d , a n d the o t h e r h y d r o g e n w a s r e t a i n e d . The conversion of [13Ls-aH]substrate was accompanied b y a n i s o t o p e effect. S i n c e 3H e n r i c h m e n t w a s f o u n d in the p r e c u r s o r acid, t h e s t e r e o s p e c i f i c h y d r o g e n r e m o v a l w a s p r e s u m e d to o c c u r at t h e initial step o f the r e a c t i o n . A s s u m m a r i z e d in T a b l e 1, s u c h a s t e r e o s p e c i f i c h y d r o g e n l i b e r a t i o n w a s also r e p o r t e d for o t h e r l i p o x y g e n a ses; 7 - p r o S h y d r o g e n in 5 - 1 i p o x y g e n a s e r e a c t i o n x5 a n d

lipoxygenases producing l l(R)- and 12(R)-hydroxye i c o s a - t e t r a e n o i c acids,~l a n d g o r g o n i a n coral h a s a n 8lipoxygenase producing 8(R)-oxygenated arachidonic acid 12 (Fig. 4). C y t o c h r o m e P - 4 5 0 , a h e m o p r o t e i n m o nooxygenase, oxygenates various positions of arachidonic acid. 13 T h e p r o d u c t s are a m i x t u r e o f R- a n d S - h y d r o x y

STEREOSPECIFIC

References

ABSTRACTION

L o n g - c h a i n u n s a t u r a t e d fatty acids w i t h a s t r u c t u r e o f

1-cis, 4-cis-pentadiene are the s u b s t r a t e s o f l i p o x y g e n a ses (Fig. 3). T w o cis-double b o n d s are i n t e r r u p t e d w i t h a m e t h y l e n e g r o u p . W h e n a c a r b o n a t o m is o x y g e n a t e d , the e n z y m e l i b e r a t e s o n e o f t h e t w o h y d r o g e n a t o m s o f the m e t h y l e n e g r o u p f l a n k e d b y the d o u b l e b o n d s . Ste-

Table 2. Substrate Specificities of Lipoxygenases. Vm,x values are compared with arachidonic acid as 100% Substrates Carbon Number 18 20

22 20

Reference:

5-Lipoxygenase

12-Lipoxygenase

Double Bond

RBL Cell

Guinea Pig Leukocyte

9,12 6,9,12 9,12,15 8,11 11,14 5,8,11 7,10,13 8,11,14 11,14,17 5,8,11,14 5,8,11,14,17 4,7,10,13,16,19

8 -7 --82 . II -100 177 2

13 -11 ---

208 172 109 125 92

--

130

5-HETE 5-HPETE 12-HETE 12-HPETE 15-HETE 15-HPETE

.

.

Porcine Leukocyte

Bovine Leukocyte

69 100 56 -44 --

15-Lipoxygenase

Bovine Platelet

11 32 17 -15

5 14 5 26 5

--

81

43 -100 116 --

. 144 -100 36 --

. 55 36 100 67 56

.

.

-------

-------

87 ------

50 127 1 1 2 58

15 . . . . 18

22

23

27

26

25

2 2 2 .

. -5

--

--

--

--

7 --

29

30

31

-. . .

. 6 25

310 280 200

-73 100 119 122

--

98 32 100 57 91

2

Rabbit Reticulocyte

380 60 590 300 220 100 -80

--

. . .

.

29

. 7 2 100 94 7

--

130 51 103 . 113

.

2 --

. . .

Soybean

51 72 46

.

. 60 -100 60 14

78 28 100 73 92

Bovine Trachea

. . .

. . .

.

152

S. YAMAMOTO SUBSTRATE SPECIFICITY

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-dlHETE14R,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

As examined with two 5-1ipoxygenase preparations from rat basophilic leukemia cells 22 and guinea pig peritoneal leukocytes23, 5,8,11,14,17-eicosapentaenoic acid is as active as arachidonic acid (Table 2). 5,8,11-Eicosatrienoic 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 5Lipoxygenase is almost inactive with Cx8 and C22 polyenoic acids. Studies on the substrate specificity of 12-1ipoxygenases 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 12lipoxygenase 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, 12-1ipoxygenase of porcine26'27 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 15-1ipoxygenase 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

oH

LXi4 OOHl +H

5-Lipcmyge.ale .~ '5,.-LTA ) S y ~ e ) ~ H

+02 . / ~ ~ O O H

~

cOOH

LXA4 - , ~-~vv.

^~.

+HzOf+ H

--RIO

OOH

15.flydroperoxy-5,6 -LTA4

,.-.,,..,,o,,,,'..,. ,,,,--...., ), , (14R~vv-, - o x y -----) gm II-L~xygl~Qli ,~,,,5-~b~troperoxi-11,15-LTl 4 (14,15-LTASynthose)

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

Mammalianlipoxygenases

OH" ~

ooo.__ oo. 13-Hydroperoxyoctodeco9Z, I IE-dienoic ocid

C

O

O

153

H

1

,°, / h

X/X/ W ' W V ' v o n"

()o.

ta.e 13-Oxo.iaeca-gZJIE-

dienoic ocid

COOH

2-Octene 12-Hydroperoxyeicosa5Z,8Z, IOE,14Z-tetroenoic ocid

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

Fig. 7. "Lipohydroperoxidase"reactioncatalyzedby 15- and 12-1ipoxygcnases.

ability to oxygenate the unsaturated fatty acid moiety of phospholipid. Arachidonic acid and linoleic acid esterifled 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 15Lipoxygenase 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 12lipoxygenase. 33 A functional role of 15-1ipoxygenase was earlier proposed for the degradation of reticulocyte mitochondria in red cell maturation.34 The lipid-peroxidizing 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 LIPOXYGENASESAS MULTIFUNCTIONALENZYMES 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.'-~..~ | - AroohldonicAoId 12S-HPETE 4[. 15S-Oxygenou 8S-Oxygenose 5S-Oxy9 enase

89,15S-diHPETE

OOH

OOH OOH 15S'HPETE 5~ISS-dlHPL~rE J4(-14115-LTA Synmase 14tlS"LTA4 8,15-dlHETE

Fig. 8. Reactions catalyzedby arachidonate 15-1ipoxygenasc.

154

S. YAMAMOTO

ookl

~ 5S-Oxygermse 5S-I-IPETE

~COOH ~COOH I-TA4

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, 12-1ipoxygenase 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 12lipoxygenase. '']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 15HPETE to a mixture of several dihydroperoxy and dihydroxy acids each with a conjugated triene. The formarion 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 diastereomeric 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 15HPETE 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, indicating a stereoselective abstraction of proS hydrogen from the position-10.17 Lipoxins are trihydroxy metabolites of arachidonate with a conjugated tetraene. 39 The purified 12-1ipoxygenase 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 14,15leukotriene 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 12HPETE 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-9,11octadecadienoic 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 15lipoxygenase of rabbit reticulocytes. As shown in Fig. 8, the purified enzyme also exhibits 12-1ipoxygenase activity transforming arachidonic acid partly to 12HPETE (5-10% of 15-HPETE). 45'46 The produced 15HPETE is subjected to 5(S)-oxygenation and 8(S)oxygenation. 15-HPETE is also converted to 14,15leukotriene 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 15-1ipoxygenase with 13-hydroperoxy-9,11-octadecadienoic acid released pentane48 (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 triple 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 lipoxygenases Mammalian 5-1ipoxygenases also show both the 5oxygenase 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 transformed to 15-hydroperoxy-5,6-epoxide by the 5,6-1eukotriene A synthase activity of 5-1ipoxygenase. There is only a minor contribution of the 6(R)-oxygenase activity of 5-1ipoxygenases9 to the aerobic multiple oxygenation pathway. The rate of the lipoxin synthesis is only 2% of the arachidonate 5-oxygenation.

COFACTORS OF MAMMALIANLIPOXYGENASES

155

iron per mol of enzyme was observed by Van der Ouderaa 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 leukocytes73 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, Vliegenthart 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.

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 hemoprotein 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

Calcium

The requirement of calcium ion for the 5-1ipoxygenase reaction was first demonstrated when a crude cellfree 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 5lipoxygenases from various sources .22,23,52-54,79-81 Unlike most 12-1ipoxygenases, the enzyme in the cytosolic

156

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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 leukocytes81 and at least one membrane-bound component. 8t'83 The latter component is replaced by phosphatidylcholine vesicles. 84

C L O N I N G O F C O M P L E M E N T A R Y AND G E N O M I C DNAs F O R M A M M A L I A N LIPOXYGENASES

The cDNA for cyclooxygenase was first isolated from human vascular endothelial cells by screening a kgtll 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 mutation 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 computer-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 insect95 cells. The recombinant enzyme transforms arachidonic acid to 5HPETE 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. Histidine-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, lO3-1o6A genomic DNA with 14 exons for the rabbit reticulocyte 15-1ipoxygenase 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 indicated 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. author is grateful to Dr. Cecil R. PaceAsciak, University of Toronto, for his critical reading of this manuscript, 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.

Acknowledgements -- The

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