conformational analysis of the enzyme-substrate complex in the

THBI JOURNAL OP BIOLOGICAL CHEMISTRY Vol. 246, No. 3, Issue of February 10, pp. 561368, 1971

Printed in U.S.A.

Conformational Analysis of the Enzyme-Substrate Complex in the Dehydrogenation of Sterols by Tetrahymena pyriformis*

(Received for publication, July 27, 1970)

WILLIAM R. NES AND P. A. GOVINDA MALYA

From the Department of Biological Sciences, Drexeb University, Philadelphia, Pennsylvania 19104

FRANK B. MALLORY, KAREN A. FERGUSON, JOSEPHINE R. LANDREY, AND ROBERT L. CONNER

From the Departments of Chemistry and Biology, Bryn Mawr College, Bryn Mawr, Pennsylvania 19OiO

SUMMARY

Both ergostaJ,24(28)-dien-d/?-o1 and stigmastad, frans- 24(28)-dien-3/3-ol were metabolized in Tefrahymena pyriformis to give the respective A5.1,22.24(28)-tetTaenes, vis. ergostad,7,frans-22,24(28)-tetraen-30-01 and stigmastad,‘7,- trans-22, frans-24(28)-tetraen-3P-ol. While stigmasta-5, frans-24(28)-dien-dfl-ol yielded some triene (stigmasta3,7, trans-24(28)-trien3@-ol), stigmasta-5, c&24(28)-dien- 3p-01 was converted ahnost entirely to the triene (stigmasta- 5,7, -cis-24(28)-trien-3P-01). No dealkylation at C-24 occurred. The results are interpreted to mean that, in the enzyme-substrate complex for the introduction of a AZ2 bond, the side chain assumes a conformation in which C-20, C-22, C-23, C-24, C-25, C-28, and (if present) C-29 lie in a plane, and one side of the plane is exposed to the enzyme with cis- elimination of 2 hydrogen atoms. Such a conformation is thermodynamically favorable with the 24-methylene and frans-24-ethylidene sterols, allowing 22,23-dehydrogenation to occur, but unfavorable with the cis-24-ethylidene sterol, thereby restricting 22,23-dehydrogenation. Since in all three cases the A’ bond was introduced, the capacity of the side chain to assume one or the other of these conformations

appears to be irrelevant in the formation of a complex with the AT-oxidoreductase.

The discovery (1) that the ciliated protozoon, Tetrahymena pyriformis, metabolizes cholesterol to cholestad ,7, truns-22- trien+-ol has made it possible to investigate the stereochemis- try of dehydrogenation at C-7, C-S (2, 3) and at C-22, C-23 (3). Among the recent findings is that the 22-pro-R- and 23-pro-S- hydrogen atoms are removed when the AZ2 bond is introduced into

* This investigation was supported by Grants AM-12172 and HE-12372 from the National Institutes of Health and GB-12133 from the National Science Foundation and comprised part of the requirements for the degree of Doctor of Philosophy of P. A. G. M. One of us (K. A. F.) thanks the United States Public Health Service for Predoctora.1 Fellowship lFOl-GM43,591-01.

cholesterol. Since the A22 bond is also known (1) to possess the bans configuration, we came to the conclusion that an analogous planar conformation in the side chain should exist in the enzyme- substrate complex at the moment of dehydrogenation at C-22, C-23. This would require that only substrates capable of such a conformation could undergo the reaction readily.

In order to test this hypothesis, we chose three sterols as substrates, only two of which could readily assume the correct conformation. These two were ostreasterol,’ which bears a 24-methylene group, and isofucosterol, which bears a trans-24-ethylidene group. Both sterols should be capable of metabolism to their respective AS*7*n*24(28)-tetraenes. The third substrate, fucosterol, bearing a cis-24-ethylidene group, cannot readily assume the correct conformation and should proceed instead to the As*r*24(*)- triene (on the assumption that the character of the side chain does not materially interfere with dehydrogenation in Ring B). The results of incubations of these three sterols with T. pyrijormis were in essential agreement with the predictions.

EXPERIMENTAL PROCEDURE

Materials and Methods-Fucosterol (m.p. 123-124”) was isolated from Fucus vesiculosus which was collected on the beach at Atlantic City, New Jersey. It was converted to ostreasterol (m.p. 145-146”) via ozonolysis to 24-ketocholesterol and introduction of the methylene group by the Wittig reaction as previously described (4,5). Isofucosterol (m.p. 134-136”) was isolated from Ulva, Zuctuco (6) which was collected by Dr. G. F. Gibbons near

r The nomenclature used is: ostreasterol, ergostad,24(28)dien- 30-01; isofucosterol, sti.gmastad,trans-24(28)-dien-3&ol: fucnn- terol, stigmastaS,&s-24(28)dieu-30-01; desmosterol, cholesta- 5,24(25)-dien-3p-01; 24-ketocholesterol, 24-oxocholestb-en-3p-01. In keeping with the trivial name (7,22-bisdehydrocholesterol) recently used (1) for the metabolite (cholesta-5,7, trans-22-trien- 3@-01) of cholesterol in T. pyriformis, we assign t&ial names, e.g. 7,22-bisdehydroostreasterol, to the nolvenes derived from the ~-- --- metabolites used in the present study. - The terms cis or tram indicate the configuration about acyclic double bonds which, in the present case, are located in the side chain. The largest substituents on the 2 unsaturated carbon atoms are denoted by those prefixes. The term methylene refers to the =CHs group, and ethylidene refers to the =CH-CR8 group. A further discussion of nomenclature of Azr(zs)- and related sterols is found elsewhere (4). The nomenclature is illustrated in Scheme 1.

561

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562 Dehydrogenation of Xterols Vol. 246, No. 3

Ostreasterol (ergosta-5,24(28)-dien-3p-ol)

I

7,22-Bisdehydroostreasterol (ergo&a-5,7-trans-22,24(28)-

tetraen-30-01)

Isofucosterol (stigmasta-5, trans.24(28)-dien-3p-ol)

1

7,22-Bisdehydroisofucosterol (stigmasta-5,7,trans-22,

trans-24(28)-tetraen-3p-ol)

Ftxosterol (stigmasta-5,cis-24(28)-die~~-3P-o1)

7-1)ehydrofncosterol (stigmasta-5,7,&s-24

(28) -t rien-3p-01)

SCHEME 1. Major metabolites of Aa4@)-steroIs in Tetrahymena pyriformis.

Rockport, Massachusetts. Sitosterol was a mixture of 24- methyl- and 24-ethylcholesterol purchased from Calbiochem. 7-Dehydrocholesterol, ergosterol, and stigmasterol were obtained from Nutritional Biochemicals.

Argentation thin layer chromatography was carried out by im- pregnation of Silica Gel G with 5 % silver nitrate (w/w) . The developing solvent was composed of hexane, chloroform, acetic acid in a ratio of 75:25: 0.6. The chromatoplates were dried at room temperature for 15 to 30 min and redeveloped a second time. The spots were visualized by a spray of rhodamine 6G dye (0.1% in acetone). Sterols were eluted with ether. The Rs values for the acetates of several authentic sterols were as follows (where Rs = distance moved after two developments/distance moved by cholesteryl acetate) : Sitosterol, 1.00; 7-dehydrocholesterol, 0.39; ostreasterol, 0.39; fucosterol, 0.78; isofucosterol, 0.70; stigmasterol, 0.98; and ergosterol, 0.35. RF values are reported for nonargentation thin layer chromatography on Silica Gel G developed with methylene chloride. Gas chromatography was performed at 235” with 0.94% of silanized XE-60 (Analabs, Inc.) on Chromosorb W. Helium was used as carrier gas. The retention times are given relative to cholesterol which was removed from the column in 8 to 10 min. Representative relative retention time values are: Sitosterol, 1.27 and 1.54 representing 24- methyl- and 2Cethylcholesterol; 7-dehydrocholesterol, 1.20; stigmasterol, 1.30; fucosterol, 1.58; isofucosterol, 1.61; ostreasterol, 1.34; ergosterol, 1.34; and tetrahymanol, 2.74. The relative retention times of the acetates were 50/, larger than those of the corresponding alcohols. Other chromatographic details are given in earlier publications from these laboratories (1,4). Mass spectroscopy was performed by Morgan-Schaffer Corporation, Montreal, Canada, with a direct inlet at 90”. Ultraviolet spectra were obtained in an ethanolic solution and infrared spectra in KBr pellets with Perkin-Elmer instruments, models 202 and 137, respectively. Nuclear magnetic resonance spectra were obtained with a Varian A-60A instrument in deuterated chloroform

by Mrs. M. Lalevic. A Kofler block was used for melting point determinations.

Incubations-T. pyriformis, type W, was cultured in a medium of distilled water containing 2% (w/v) of proteose peptone (Difco) and 0.1% (w/v) of yeast extract (Difco), and it was 9 X l,O+ M in iron-EDTA complex (7). The substrates were added to the culture medium in a solution of ethanol as previously described (1). Approximately 3.0 mg of sterol per 500 ml of medium were incubated at, 28” for 40 hours. The cells were separated from the medium by centrifugation in a manner reported earlier (8). The cells and medium were separately lyophilized prior to extraction.

The sterols were isolated from the lyophilized culture medium by direct saponification (250 ml of 10% ethanolic KOH per orig- inal 500 ml of culture medium; reflux 1 hour), dilution with water, and extraction with ether. They were separated from tetrahymanol and other materials by preparative thin layer chromatography (0.5-mm thick plates) on Silica Gel G with methylene chloride as the developing solvent. The eluted sterols were used for analysis by gas-liquid chromatography.

The sterols and their esters were isolated from the lyophilized cells by continuous extraction with acetone in a Soxhlet appara- tus. The residue from the acetone extract was chromatographed on a column of alumina. Material eluted by hexane was dis- carded, and the fractions from elution with hexane-benzene (1: 1, w/v), benzene and benzene-ether (1: 1, w/v), and ether were examined. Two peaks of substance were observed, probably corresponding to esters and free alcohols. Fractions were combined from the beginning of the least polar to the end of the most polar peak, the latter being crystalline. The purpose of this chromatogram was to separate sterols and their esters from phospholipids. The sterols were then separated from tetrahymanol and similar materials by a second chromatography on alumina after saponification (5 y0 ethanolic KOH) of the combined fractions corresponding to the two peaks in the first chromatogram. The sterols and


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tetrahymanol formed crystalline residues in two peaks, the latter being eluted first. Tetrahymanol (1.3 i 0.1 mg/500 ml of incu- bate corresponding to 52% inhibition2 of biosynthesis) was identified in the cellular material by its lesser polarity on the alumina column, an Rp of 0.67 on thin layer chromatography as expected for a triterpenoid, and its relative retention time of 2.74 in gas- liquid chromatography. An authentic sample (1) possessed the same properties. No tetrahymanol was found in the aqueous media. The sterols were examined analytically by gas-liquid chromatography and, except for the mixture obtained from isofucosterol, were crystallized from methanol and methanol-water. A portion was acetylated (acetic anhydride-pyridine, room temperature, 15 hours) and chromatographed preparatively on a 0.5-mm thin layer of Silica Gel G impregnated with silver nitrate. The remaining substrates (as acetates) from the ostreasterol and fucosterol incubations were reisolated in this way and identified by gas liquid chromatography (relative retention time, 1.43 and 1.70, respectively). Since the amount of substrate in the isofucosterol incubation was only a few per cent (judged by gas- liquid chromatography) and since the two metabolites did not separate sufficiently when the mixture was submitted to argentation chromatography under analytical (small amounts) conditions, the sterol mixture from isofucosterol was not submitted to preparative argentation chromatography. The amounts (from measurements of weights or peak areas (or both) in gas-liquid chromatography) of tetrahymanol, substrates, and metabolites from the various incubations are summarized in Table I. The major physical constants of the metabolites and their acetates are summarized in Tables II, III, and IV. Additional data are given under “Results.”

RESULTS

Quantitative Aspects-Most of the sterols found in the media of the various incubations were unchanged substrate. The other sterols possessed the same retention times in gas liquid chromatography as did the respective metabolites which were isolated by further procedures from the cells. The average substrate to metabolite ratio in the media was 2.2: 1.0. The sterols isolated from the cells were composed principally of metabolites, the average substrate to metabolite ratio being 0.1O:l.O. In no case did a ratio differ by more than 10% from the average value. This leads us to believe that the sterols that we isolated are actually representative of the metabolic capabilities of the organism and that differing absolute yields have their origin in unaccounta- ble losses during isolation. The total yields of recovered sterol usually were about 55%, but in some cases they were much less. There was no obvious correlation between recovery and the substrate used. The quantitative data are summarized in Table I. The values recorded were taken from a set of experiments in which a special attempt was made to maintain all of the conditions uni- form. However, while in this series the total recovery of sterol from the fucosterol incubation was 61%, in another closely similar experiment it was only 24%. The latter value is similar to the yield (21%) obtained from isofucosterol in the incubation reported in Table I. Since variations in total recovery appeared to be capricious and unrelated to the substrate used, we doubt that they are attributable to catabolic processes selectively re-

2 The term “52% inhibition” means that the amount of tetrahymanol isolated was 48% of the amount obtained after a similar incubation in which no sterol was added.

TABLE I

Sterols isolated from Tetrahymena pyriformis after incubation with A24@s)-sterob

Substrate

Amount incubated. Substrate recovered. 7,22-Bisdehydro me-

tabolite . 7-Dehydro metabo-

lite . .

From aqueous medium

m&T

35.0 40.0 20.0 0.7 0.6 0.9

0.3 0.2 None

None 0.1 0.4

From cells

17.8

None

moving the metabolites of one rather than another substrate. Consequently, it is reasonable to assess the nature of the enzyme- substrate complex by an analysis of the products obtained, but there must be some slight reservation in view of the lack of 100% accountability for the mass of sterol incubated.

Ostreasterol and fucosterol led essentially to single metabolites, while isofucosterol led to a pair of metabolites. All of the metabolites retained the R group which was originally present at C-24 in the substrate. Had desmosterol or cholesterol been formed, they should have been converted to their respective 7,22-bisdehydro metabolites. None of these four compounds was evident in the gas-liquid chromatograms of the cellular materials at levels (1 to 2%) which we could have readily detected. However, the water medium from ostreasterol did yield a peak (lOyo of the total aqueous sterol or 1% of the substrate incubated) corresponding to cholesterol. Since cholesterol was absent from the substrate at a concentration as high as l%, the nature and origin of the aberrant peak are unclear. The problem was not further in- vestigated.

IdentiJication of ?‘,Z%Bisdehydroostreasterol-7,22-Bisdehy- droostreasterol has previously been isolated from yeast (9, 10). Definitive structural information was obtained by Petzoldt et al. (10) through the ultraviolet and nuclear magnetic resonance spectra of the alcohol and its derivatives. The metabolite of ostreasterol which we obtained in T. pymformis had a melting point identical with that obtained by the latter authors (10) and nearly the same as that of Breivik, Owades, and Light (9). In addition, the ultraviolet and infrared spectra of the metabolite or its acetate were in agreement with the literature (9, 10) as well as with expectation (11). The A5s7 system was evident in a band at 282 nm with appropriate fine structure and with the expected extinc- tion coefficient. Similarly, a strong band at 232 nm identified the presence of the A22*24(28) system. Bands at 967 and 888 cm-1 confirmed the presence of trans-AZ2 and A%@) bonds, respectively. Bands at 805 and 838 cm+ confirmed the Ass7 system. That it was a polyene was confirmed by a slow rate of movement in argentation and gas-liquid chromatography. The molecular weight and empirical formula (394, and CZ8He0 for the alcohol) were determined from the strong M+ peak in the mass spectrum of the alcohol and from the peak for M+ less CH&OOH in the spectrum of the acetate. The capacity to form an acetate and for the latter to lose CH&OOH confirmed the presence of an alcoholic function. The peaks corresponding to M+ less CHa- COOH, to side chain (C9Hlo+), and to M+ less CH&OOH less side chain confirmed the presence of two double bonds in the ring


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564 Dehydrogenation of Sterols

TABLE II

Vol. 246, No. 3

Physical constants of 7$X2-bisdehydroostreasterol derived from incubation of ostreasterol Unless otherwise noted, data given are for the acetate. For experimental conditions, see “Experimental Procedure.”

Property

Melting point Melting point (free alcohol)

Infrared bands (cm-l) Ultraviolet bands (nm, C)

Ultraviolet bands (free alcohol) (nm, E)

Mass spectrum peaks (m/e)

MWS spectrum peaks of free alcohol (m/e)

Rs in AgNOa thin layer chromatography (cholesteryl acetate = standard)

Relative retention time in gas-liquid chromatography (cholesterol = standard)

Relative retention time in gas-liquid chromatography of free alcohol (cholesterol = standard)

Observed

118-119” 1X-116’

805, 838, 888, 967 232 (19,000), 272 (ll,OOO), 283 (12,660),

294 (7,000) 232 (lS,SOO), 272 (11,500), 283 (11,800),

294 (7,100) 376 @I+ less CHsCOOH) 253 (M+ less CHICOOH less CoHo) 158, 157, and 143 (for As*‘) lo? (CQHIS+)

394 (M+> 253 (M+ less Ha0 less C8H16) 251 (M+ less IIt0 less CQHXS less 2) 158, 157, and 143 (for As*‘) 123 (CQHU+)

0.22

1.63

1.51

Literature (reference)

141-144O (9) 116-117O (10) 118-120” (9) 800,835,885,967 (9)

system and two in the side chain. Exact values for the pertinent physical constants are summarized in Table II.

Since the substrate possessed the alcoholic function in the 3fi position, such a group is implied for the metabolite. The data then require that the metabolite be 7,22-bisdehydroostreasterol (ergosta-5,7, trans-22,24(28)-tetraen-3/l-01), if we also assume that the methylene group is C-28 as in the substrate. Direct evidence for this was available. Since an infrared band at 887 cm+ for a methylene group was actually observed, the side chain could possess the Aa’JJz, AmJJ(B), AH@)@, or the A23,2s structures. The latter two structures are eliminated by the absorption in the infrared spectra for a trans-ethylenic double bond. In addition, only one of the four structures should undergo facile allylic cleavage at C-17, C-20, yielding a peak for M+ less side chain. This is the A22*24(28) structure. The other three by allylic cleavage should not lose the entire side chain but instead should cleave at C-24, C-25 (for the A20*% case), C-22, C-23 (for the A”lcra) case), or C-20, C-22 (for the AzrJ6 case). Actually both the metabolite and its acetate underwent very marked allylic cleavage at C-17, C-20. This was evident in the mass spectrum of the alcohol from peaks for the side chain and for M+ less the side chain at m/e 123 and 271 and an analogous peak at m/e 253 corresponding to M+ less side chain less HzO. The three peaks each varied from half as strong to as strong as the M+ peak.3 The spectrum of the acetate also showed the peaks at m/e 123 and 253 corresponding to side chain and to M+ less CH&OOH less side chain, and they were stronger than the peak for M+ less CHcCOOH. All of the properties therefore agree with the assigned structure as well as

3 Since the most intense peaks in the mass spectrum of AK,‘- sterols are associated with fragments corresponding to Rings A and B, the relative amounts of cleavages of the side chain are best assessed by comparison with the molecular ion.

with expectations derived from the structure of the substrate and the known (1) metabolic processes of T. pyriformis.

There is, nevertheless, one discrepancy. The melting point of our acetate (118-119”) was lower than that (141-144’) obtained by Breivik et at. (9) for the yeast product. Whether this is due to polymorphism, impurities, or an alternative structure for the sterol obtained by Breivik et al. (9), etc., has not been further examined. Petzoldt et al. (10) unfortunately did not report the acetate. It is also possible that the acetate obtained by Breivik et al. (9) had undergone some oxidation or rearrangement (or both), since their acetylation method involved heating to 199” for 1 hour and their carbon analysis was 0.86% lower than the calculated value. Petzoldt et al. (10) actually found that the A22,24(28)- diene system underwent facile (acid-catalyzed) rearrangement. Finally, Breivik et al. (9), unlike Petzoldt et al. (10) and ourselves, obtained no information to discriminate between alternative structures.

Icknti,ficattin of 7-Dehydroisofucosterol and 7,2.%Bisdehyclroiso- fucosterol-The pair of metabolites of isofucosterol possessed molecular weights of 410 and 408, indicating a mixture of a triene and a tetraene with empirical formulas of C&I~O and C~QHUO,

respectively. This was arrived at from the peaks corresponding to M+ less CH&OOH in the mass spectrum of the mixed acetates. The intensities of the two metabolite peaks in gas-liquid chromatography and of the two major M+ less CH&OOH peaks in mass spectroscopy yielded values corresponding, respectively, to 30 and 45 y. of triene and 70 and 55 To of tetraene. There was also a small peak at m/e 394 for M+ less CH&OOH for the substrate which was also observed in gas-liquid chromatography. A peak at m/e 137 corresponding to a diunsaturated side chain (CloH&+ appeared in the mass spectrum and was half as strong as the peak for M+ less CHGOOH. The fragment for M+ less


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TABLE III

565

Physical constants of mixture (7-dehydroisofucosterol and ?‘,.%?-bisdehydroisofucosterol) derived from incubation of isofucosterol Unless otherwise noted, data are for the acetate. The mixture contained 5 to 10% of isofucosteryl acetate. For experimental con-

ditions, see “Experimental Procedure.”

Observed Expected by analogy (a) or calculation (c)

Melting point Ultraviolet bands (nm, E)

Ultraviolet bands of free alcohol (nm, B)

Infrared bands (cm-*) Mass spectrum peaks (m/e)

Rs, argentation thin layer chromatography (cholesteryl acetate = standard)

Relative retention time, gas-liquid chromatography (cholesterol = standard)

120-124' 237 (8300), 271 (7800), 282 (9500), 293

(6400) 237 (8600), 271 (7500), 282 (9200), 293

(6400) 802, 834, 967 390 and 392 (I@+ less CHrCOOH) 253 @I+ less CH&OOH less C~OHI,) 158, 157, and 143 (for As*') 137 KkdG++) 0.20

1 1.91 and 2.64

237 (c) 271, 282, 293 (a) 237 (c) 271, 282, 293 (a) 800, 835, 967 (a) 390 and 392 (a, c) 251 and 253 (a, c) 158, 157, and 143 (a, c) 137 (a, c) 0.22 and 0.27 (c)

1.96 and 2.03 (c)

CHrCOOH less CrJ& appeared at m/e 253, and it was nearly

dienic substrate, 38% trienic metabolite, and 52yo tetraemc

as strong as the peak for M+ less CHICOOH. This is consistent with the presence of a A~*~@@-dienic side chain which underwent allylic cleavage at C-17, C-20. In the fragmentation of isofucosteryl acetate, by contrast, very strong allylic cleavage has been observed in this and other laboratories (6, 12-15) at C-22; C-23 through the appearance of a dominant peak at m/e 296 for M+ less CH&OOH less C,H14. Thus, the ratios of the intensities of the peaks at m/e 296 to m/e 253 were 0.21 in the mixed acetates of the metabolites and 7.7 in isofucosteryl acetate, showing clearly that C-17, C-20 cleavage was dominant in the former and C-22, C-23 cleavage was dominant in the latter. A As,‘-dienic structure for the ring system was evident from strong peaks at m/e 143, 157, and 158 as expected (16). Corroboration of this was available from the ultraviolet spectrum which showed the typical (11) band for the AS*’ system at 282 nm with appropriate fine structure. The apparent intensity (E 9,500) was on the low side of the acceptable range of 10,000 to 15,900 (ll), but, when the gas-liquid chromatographic data indicating the presence of 5 to 10% of substrate are taken into consideration, the e value for the two metabolites is brought above 10,000. This indicates that both metabolites possessed the ASJ system. The ultraviolet spectrum also showed a band at 237 nm. By the use of Wood- ward’s rules (17,lS) the addition of 5 nm for C-29 to the observed value (232 nm) for the A~.~@) system in 7,22-bisdehydroostreasterol yields a calculated value of A,,,, 237 nm for the A22**4@) system in a sterol bearing a Cc group at C-24. Consequently, the ultraviolet data also corroborate the mass spectral data for the presence of the A22*24(2s) system. Similarly a band at 967 cm+ in the infrared spectrum was consistent with this assignment and, further, proved that the A** bond was trans-oriented. The intensity (t 8300) of the band at 237 nm indicated that the sterol with the Az*r@) diene system in the side chain was present as a 44% component, and by difference the one with only one double bond (A*J@@) in the side chain must be a 46% component, if the substrate is taken to be a 10% component. If we average the quantitative figures from gas-liquid chromatography and mass and ultraviolet spectroscopy we obtain a composition of 10%

I _ _ .

Identification of 7-Dehydrojucosterolter incubation of fucosterol, the sterol fraction was isolated by chromatography on alumina. The ultraviolet spectrum of the sterol was immediately determined at this stage to avoid any loss of metabolites. The spectrum showed only the absorption maximum at 282 nm with annronriate fine structure exnected (11) for a As*7 svstem. A

metabolite. The structural data (summarized in Table III) from spectroscopy, together with the assumption that the alcoholic group is 3&oriented and that the configuration about the A24(*) bond is the same as in the substrate, prove that the metabolites are 7-dehydroisofucosterol (stigmastad, 7, trans-24(28)-trien-3& 01) and 7,22-bisdehydroisofucosterol (stigmasta-5,7, truns-22, truns-24(28)-tetraen-3p-01). These assignments were further cor- roborated by the rates of movement in chromatography.

The relative retention time of the triene should be 1.69 (relative retention time for isofucosteryl acetate) times the increment (1.20) for the Ass7 grouping (relative retention time of 7-dehydrocholesterol) or 2.03. The observed value was 2.04. The relative retention time of the tetraene should be 1.63 (relative retention time of 7,22-bisdehydroostreasteryl acetate) times the increment for C-29 (1.20 = relative retention time of isofucosteryl acetate/relative retention time of ostreasteryl acetate) or 1.96. The observed value was 1.91. The calculated value for Rs for the triene in argentation thin layer chromatography is 1.00 (& of 24-ethylcholesteryl acetate) times the increment for the A24(2s) bond (0.70 = Rs of isofucosteryl acetate) times the increment for the As.7 group (0.39 = Rs of 7-dehydrocholesterol) or 0.27. Similarly, the tetraene should have an Rs of 1.00 (Re of 2Cethylcholesteryl acetate) times the increment for the Aa*r@) group (0.56 = Rs of 7,22-bisdehydroostreasterol/Rg of 7-dehydrocholesterol) times the increment for the AS.7 group (0.39) or 0.22. The metabolites gave a single spot with an Rs of 0.20. Presumably with Re values as small and as close together as these no significant separation occurred. It was, incidentally, for this reason that isolation of each individual component of the mixture was not further pursued. Preparative gas-liquid chromatography also showed no promise. Although two peaks for the metabolites were clearly observed, the two bands overlapped extensively.


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566 Dehydrogenation of Xterols

TABLE IV

Vol. 246, No. 3

Physical constants of 7-dehydrofucosterol deGved from incubation of fucosterol

Unless otherwise noted data given are for the acetate. For experimental conditions, see “Experimental Procedure.”

Property Observed Expected by analogy (a) or calculation (c)

Melting point Melting point of free alcohol Ultraviolet bands (nm, c) Ultraviolet bands of free alcohol (nm) Infrared bands (cm-l) Mass spectrum peaks (m/e)

Rs in AgNOa, thin layer chromatography (cholesteryl acetate = standard)

129-131° 119-121” 271 (lO,SOO), 282 (11,200), 294 (7,560) 271, 283, 294 (E not measured exactly) 805, 822, 836 392 (M+ less CH&OOH) 158, 157, 143 (for As*‘) 0.25

271, 282, 293 (a) 271, 282, 293 (a) 800, 817, 835 (a) 392, 158, 157, 143 (a, c)

0.30 (c)

Relative retention time in gas-liquid chromatography (cholesterol = standard)

Relative retention time in gas-liquid chromatography of free alcohol (cholesterol = standard)

Nuclear magnetic resonance peaks (ppm)

1.95

1.87

1.55, 1.61 -

2.00 (c)

1.90 (c)

1.54 and 1.60 (a)

minimum of absorption occurred at 220 nm. In gas-liquid chromatography, except for a few per cent of a peak (relative retention time 1.54) corresponding to fucosterol, a single maximum (relative retention time 1.87) was observed. A small (approximately 5%) shoulder which has not been identified also appeared at relative retention time 2.19. 7-Dehydrofucosterol would have been predicted to have a relative retention time of 1.58 (relative retention time of fucosterol) times the increment for the As,7 grouping (relative retention time of 7-dehydrocholesterol 1.20) or 1.90. The agreement of the latter value with that observed (1.87), the fact that only a single major peak appeared in gas-liquid chromatography, and the presence of a single ultraviolet absorption band proved that the Ar bond had been introduced with little or no introduction of a An group. However, a small amount of the 7,22-bisdehydro product might have been formed, since a small shoulder in the ultraviolet spectrum appeared at 237 nm, and the minimum of absorption in authentic As*‘-sterols (e.g. ergosterol) appeared at 235 nm. The difference in intensity (e 2400) at 237 nm between the E values for Ass’- sterols and the metabolite indicated the possible presence of about 11 y. of a sterol bearing a AzzJ4@) system, i.e. 7,22-bisdehydro- fucosterol, but the major metabolite was clearly only the triene, 7-dehydrofucosterol.

Acetylation and purification of the sterols (from another incubation) on a column of silica gel impregnated with silver nitrate freed the metabolite from unchanged substrate which was recovered in appropriate fractions. The ultraviolet spectrum of the purified acetate exhibited less absorption at 237 nm and the minimum of absorption was at 225 nm. There still, however, may have been small amounts of tetraene present.

The mass spectrum of the purified acetate showed a peak for M+ less CH&OOH at m/e 392 corresponding to a molecular weight of 452 (or 410, C29H460, for the alcohol) in agreement with the structure of 7-dehydrofucosteryl acetate. The Aas7 system was evident in peaks at m/e 143, 157, 158, and only a very small peak (6% of the peak for M+ less CH&OOH) appeared at m/e 253 for M+ less side chain which would have been expected for the tetraene. The absence of a band in the infrared spectrum at 967 cm+ also showed that the major component lacked the A22 bond required by the tetraenic structure. There was, however,

in agreement with the ultraviolet data, a small peak in the mass spectrum for M+ less CHICOOH at m/e 390. The intensity of this peak was higher than would be expected for isotopic distribu- tion. We estimate that it corresponded to 8% of the tetraene.

The presence of the 24-ethylidene group in the metabolite was shown in the nuclear magnetic resonance spectrum by a doublet at 1.58 ppm displacement from the peak for tetramethyl silane with a coupling constant of 6.5 Hz, corresponding to the absorption of the 3 hydrogen atoms on C-29 which are split into a doublet by the single hydrogen atom on C-28 (6, 19, 20). The Az40s) bond was also evident in a band at 822 cm+, corresponding to the vibration of the hydrogen atom on C-28 in the 24-ethylidene group which is observed in fucosteryl acetate at 817 cm+. Bands at 805 and 836 cm-1 confirmed the presence of the A5+7- diene system. Analytical argentation chromatography of the metabolite acetate yielded an Rs value of 0.25. The calculated value for the A5*7J4(s)-triene is 0.78 (Rs of fucosteryl acetate) times the contribution for the AK,’ group (0.39 = Rs of 7-dehy- drocholesteryl acetate) or 0.30. All of the data are, therefore, consistent with the major (approximately 90%) metabolite’s being stigmasta-5,7, c&24(28)-trien-3/3-ol (7-dehydrofucosterol), if we assume only that the hydroxyl group is at C-3 and that the configurations at C-3 and C-28 are the same as in the substrate. The important physical constants are summarized in Table IV.

DISCUSSION

The introduction of a A7 bond into all of the observable metabolites of the three substrates indicates that steric and n-electronic phenomena in the vicinity of C-24 do not materially influence the A’-oxidoreductase. While this is not surprising in view of the large distance between C-24 and Ring B, it could not have been predicted with any degree of certainty. On the other hand, a priori considerations do lead one to a semiquantitative prediction of the steric influence at C-24 on the rate of dehydrogenation at C-22, C-23.

It is known from the present and previous (1) investigations that the A22 bond introduced by T. pyriformis has the trans-configuration, i.e. C-20 and C-24 lie on opposite sides of the inter- nuclear line between C-22 and C-23, and these 4 carbon atoms all lie in a plane. It is also known (3) that the 22-pro-R- and


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Issue of February 10, 1971 Nes, Malya, Mallory, Ferguson, Landrey, and Conner

23-pro-S-hydrogen atoms are removed from cholesterol4 On the assumption (a) that the enzyme and its associated cofactors do not constitute an energy source to counteract conformational preferences in the substrate, (b) that consequently the enzyme can adsorb only a conformation which is already feasible in the un- perturbed substrate, and (c) that no movement of carbon atoms occurs in the substrate during dehydrogenation, we arrive at a conformation (Conformer 1, Scheme 2) for adsorption and reaction in which, from the absolute configuration of the hydrogen atoms removed, a &s-elimination must occur from the backside (viewed as shown in Scheme 2) of the plane containing C-20, C-22, C-23, and C-24. This being so, to allow close approach of the side chain to the dehydrogenating groups of the enzyme (or enzyme-cofactor system), C-28 and C-25 must lie, as implied in Conformer 1, in the plane of C-20, C-22, C-23, and C-24. This is readily possible for ostreasterol, since the left-hand hydrogen atom (R = H, Conformer 1, Scheme 2) on C-28 would lie between the hydrogen-atoms of C-22 and the right-hand hydrogen atom (R’ = H) would bifurcate the angle between one of the CHz groups and the hydrogen atom on C-25. Ostreasterol should, therefore, readily undergo dehydrogenation at C-22, C-23. In fact, it proceeded to a single metabolite, 7,22-bisdehydroostreasterol, in which the At2 bond had been introduced.

If R in Conformer 1 were changed from H, as in ostreasterol, to CHI, as in fucosterol, a very large change in the stability of the conformer should occur. A strong nonbonded interaction should ensue between this CH, group and the hydrogen atoms on C-22. The alternative structure (Conformer 2) derived primarily by rotation of 180” about the C-23, C-24 bond is also unfavorable, since C-26 and C-27 protrude to the sides of the central plane, and this should destabilize the enzyme-substrate complex. Any other conformation, which might be preferred by the energetics of the substrate, would be still worse in terms of effective binding to the enzyme, since in all of the other cases C-28 and C-25 would protrude, along with their associated substituents, on both sides of the central plane inhibiting formation of the substrate.enzyme complex. Consequently, fucosterol should undergo dehydrogenation at C-22, C-23 much less readily than ostreasterol. In fact, at least 90% of the metabolic mixture from incubation of fucosterol was the triene, 7-dehydrofucosterol, lacking the AZ2 bond.

In between the extremes of ostreasterol and fucosterol is iso-

4 It should be noted that the Cahn-Ingold-Prelog sequence rule (24, 25) requires that C-22 and its system of substituents take priority over C-24 and its system of substituents in cholesterol, but that unfortunately the reverse is true in Az4@*)-sterols. Con- sequently, the nomenclature for the prochirality of C-23 (but not of C-22) is reversed in the two cases, the 23-pro-S hydrogen of cholesterol being equivalent to the 23-pro-R hydrogen of a A24@8)- sterol. In the latter case, it would be the 22-pro-R and 23.pro-R hydrogen atoms which are eliminated to form the AZ* bond. The designations of the hydrogen atoms are shown below.

Cholesterol side chain A24(2*)-Sterol side chain

Conformer 1 Conformer 2

SCHEME 2. Conformations considered for formation of enzvme- substrate complex during dehydrogenation at C-22, C-23: In A6 series: R = R’ = H, ostreasterol; R = CHI, R’ = H, fucosterol; R = H, R’ = CH,, isofucosterol.

fucosterol in which R’ in Conformer 1 is CH,. While the problem of the opposition between the R group and the hydrogen atoms on C-22 is the same as in ostreasterol, it is difficult to make a quantitative assessment of the remaining effects. One would not expect them to be large, since 1 of the hydrogen atoms in the CHa group (R’) could bifurcate the CH,-C-H angle of the isopropyl group without much change in the latter. Such a conformation would place the other 2 hydrogen atoms of C-29 on opposite sides of the central plane, a steric increase over ostreasterol but still not a large increase. Consequently, one would predict that Conformer 1 is feasible for isofucosterol and that dehydrogenation would occur to a greater or lesser extent, probably as the major process. In fact, the principal metabolite was 7,22-bisdehydroisofucosterol, although nearly half of the metabolic mixture was the triene, 7-dehydroisofucosterol. Appa- rently, the effect of the CH, group is quite significant. Never- theless, the results are qualitatively within expectation. Since the extreme examples, the metabolism of ostreasterol and fucosterol, were well within the limits of qualitative prediction, we believe on balance that the a priori view of the nature of the substrate-enzyme complex is validated.6

It is of interest to draw attention to the fact that ostreasterol, fucosterol, and isofucosterol display other properties in which steric differences are manifest. In particular, they are distin- guishable through their rates of movement in gas-liquid and argentation chromatography as described in the present work and elsewhere (4, 12), and they each display characteristic nuclear magnetic resonance spectra (6, 19, 20) and mass spectra (6, 12). Moreover, the closer proximity of C-29 to C-22, C-23 in fucosterol as opposed to isofucosterol is manifest in a smaller binding constant (faster rate of movement in argentation chromatography) with silver ion.

The dehydrogenation of cholesterol itself is also amenable to analysis through Conformer 1 (CRR’ replaced by Hz). In this case the hydrogen atoms on C-24 should lie on either side of the central plane and offer no more steric hindrance than the hydro-

6 The model consisting of a fully planar receptor site on the enzyme “surface” is the simplest of several alternatives. A more complicated one would be the existence of a definite “hole” which would accept, for example, the isopropyl terminal group of a nonplanar conformation, but the present work indicates that, at least as a first approximation, such a hole does not contribute to the situation. While it is conceivable that some degree of folding could account for the detailed ratio of triene to tetraene, it is impossible at this time to discriminate at such a precise quantitative level between the conformational requirements of the steroid and those of the enzyme.


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568 Dehyclrogenation of Sterols Vol. 246, No. 3

gen atom on C-25 in ostreasterol. In fact, cholesterol readily undergoes dehydrogenation at C-22, C-23 (1).

The failure to observe dealkylation of any of the substrates is somewhat surprising. The Aza(@ bond is formed during alkylation (21, 22), and reversal of the process might have been expected, since 24ethylcholesterol actually undergoes dealkylation in T. p~rifomis (23). A possible explanation is that, during dealkylation, the A24(28) intermediates are ensyme or particle bound and not in significant equilibrium with the unbound compounds. Perhaps a more sophisticated explanation is that true Au@)- sterols are not actually involved in dealkylation in T. pyrifomnis and that instead an entirely different process, e.g. hydroxylation and reduction or recycling of electrons, occurs. An example would be the formation of a 29-hydroxylated compound which, after phosphorylation of the hydroxyl group and attack at C-24 by hydride ion (from reduced pyridine nucleotide), would elimi- nate ethylene and yield cholesterol.

REFERENCES

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2. CASPI, E., AND ZANDER, J. M., Chem. Commun., 1141 (1969). 3. BIMPSON. T.. GOAD. L. J.. AND GOODWIN. T. W.. Biochem. J.,

116,85? (1969). ’ . 4. VAN ALLER, R. T., CHI~MATSU, H., DE SOUZA, N. J., JOHN,

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Corporation, New York, 1959, pp. 17-18. 19. NES, W. R., CASTLE, M., MCCLANAHAN, J. L., AND SETTINE,

J. M., Steroids, 8,655 (1966). 20. FROST, D. J., AND WARD, J. P., Tetrahedron Lett., 3779 (1968). 21. RUSSELL, P. T., VAN ALLER, R. T., AND NES, W. R., J. Biol.

Chem., 242, 5802 (1967). 22. LEDERER, E., Quart. Rev., 23, 453 (1969). 23. MALLORY, F. B., AND CONNER, R. L., Lipids, in press. 24. CAHN, R. S., J. Chem. E&c., 41, 116 (1964). 25. CAHN, R. S., INQOLD, C. II., AND PRELOO, V., Anger. Chem.

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R. Landrey and Robert L. ConnerWilliam R. Nes, P. A. Govinda Malya, Frank B. Mallory, Karen A. Ferguson, Josephine

Tetrahymena pyriformisDehydrogenation of Sterols by Conformational Analysis of the Enzyme-Substrate Complex in the

1971, 246:561-568.J. Biol. Chem.

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