stereochemistry of the active site of &hymotrypsin

THE JOURNAL OF BIOLOGICAL C~~I~TRY Vol. 244, No. 15 Issue of August 10, BP. 4158-4167, 1969

Printed in U.S.A.

Stereochemistry of the Active Site of &hymotrypsin

THE BINDING GEOMETRY OF TRYPTOPHAN DERIVATIVES*

(Received for publication, March 13, 1969)

YUJI HAYASHI~: AND WILLIAM B. LATVSON

From the Division of Laboratories and Research, New York State Department of Health, Alban.y, New York 12201

SUMMARY

Chymotrypsin (EC 3.4.4.5) hydrolyzes methyl DL-l,Z-dihydronaphtho[Z , 1 -b]furancarboxylate (IV) very rapidly and with high D specificity. The isomeric methyl DL-dihydronaphtho[l ,2-b]- and -[2,3-b]furan-2-carboxylates are much poorer substrates than IV, and are hydrolyzed with less stereospeciticity. The binding site has been “mapped” to identify areas displaying steric hindrance to binding and areas of a polar and nonpolar nature. The results confirm Niemann’s hypothesis that the aromatic binding site in chymotrypsin is approximately planar, elongated, and curved. Methyl N-acetyl-L-3-(1-naphthyl)alaninate (VII) was also found to be a very good substrate, in contrast to its 2-naphthyl analogue, which is about 100 times less reactive than VII. A likely conformation of methyl N-acetyl-L-tryptophanate during hydrolysis at the active site of chymotrypsin is proposed.

Since the discovery (1) that the sterically restricted substrate, methyl dihydroisocarbostyril-3-carboxylate (Compound I in Fig. l), is rapidly hydrolyzed by chymotrypsin (EC 3.4.4.5) with D specificity, considerable effort has been expended to deduce the conformation of typical acyclic substrates, such as methyl N-acetyl-L-phenylalaninate (II) from results obtained with I and other substrates of restricted conformation (2-16). Much controversy has centered about the question of whether the ester group of I is “axial” (3-6, 11, 16) or “equatorial” (2, 7-10, 12-15) during hydrolysis at the active site of chymotrypsin. The arguments have proceeded from axial or equatorial hypotheses to consideration of likely conformations of the acetamido, carbomethoxy, and beneyl groups about the asym- metric carbon atom of II during its enzymatic hydrolysis. In contrast, the steric relationship of the aromatic binding site to the catalytic site in chymotrypsin has received little attention. The binding site is well known to have a broad specificity for aliphatic and especially aromatic groups. Binding affinity increases with size of the “hydrophobic” group, and molecules as large as anthracene can be accommodated (17-20). Wallace,

* This work was supported in part by Grant AM 05299 from the National Institutes of Health.

1 Present address, Department of Chemistry, Faculty of Science, Osaka City University, Sugimotocho, Sumiyoshiku, Osaka, Japan.

Kurtz, and Niemann (20) found that several benzoquinolines are efficient reversible inhibitors of the enzyme, benzo[jjquinoline being especially good, and concluded that the binding site “has greater length than breadth and with reference to its longest dimension is not straight but curved.”

I X=NH Ia x=0

III

4

V : R=CHs Va R*H

4

@&CO,. 4

VI : R=CH3 Via : R-H

VII : R’=CH,. R*=COCH, VIII R’*CH, . R*=COCH,

VIla R’= H , R2=COCH3 VIIICI : R’= R*= H

VIIb R’= R*= H

FIG. 1. Compounds I to VIII

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In phenylalanine and tyrosine derivatives, the benzene ring is symmetrical with respect to the rest of the molecule. For any given conformation, a rotation of the benzene ring through 180” results in the same conformation. The P-substituted indole nucleus of a tryptophan derivative is unsymmetrical with respect to any given conformation, and rotation of the indole ring through 180” produces a different conformation. Because of this rotational isomerism, and since N-acyl-n-tryptophan derivatives are hydrolyzed faster and more specifically by chymotrypsin than the corresponding phenylalanine and tyrosine derivatives (21, 22), we investigated the symmetry of the aromatic binding site and its relationship to the catalytic site to determine simultaneously the preferred conformation of tryptophan derivatives as they bind productively to the enzyme during hydrolysis.

The present paper reports the remarkably high reactivity and stereospecificity toward chymotrypsin of methyl 1,2-dihydronaphtho[2,1-blfuran-2carboxylatc (IV), in contrast to the other two naphthalene analogues, V and VI, of methyl hydrocoumarilate (III) (11). The results led to a correct prediction of the reactivity of methyl N-acetyl-3-(l- and 2-naphthyl) alaninates (VII) and (VIII), and to a proposal concerning the conformation of N-acyltryptophan derivatives as they interact with the active site of chymotrypsin. The implications of this model for the stereochemical relationship of the binding and catalytic sites are discussed.

EXPERIMENTAL PROCEDURE

Three isomers (Compounds IX, X, and XI in Fig. 2) of naphthofurancarboxylic acid and two isomers (VIIb and VIIIa) of 3.naphthylalanine were synthesized as described by Emmott and Livingstone (23) and Dittmer, Herz, and Cristol (24), respectively. D- and n-Hydrocoumarilic acids were prepared as described by Bonner et al. (25) and Bowen et nl. (26). Salt- free cu-chymotrypsin, recrystallized three times (Lot CDI- 7CD), was obtained from Worthington. The concentration of chymotrypsin solutions was determined spectrophotometrically at 280 nip by the method of Wu and Laskowski (27). Anhy- drous dimethgl sulfoxide was purchased from Matheson Cole- man and Bell @1X1458), and silica gel for column chromatography from Mallinckrodt (100 mesh). All other materials were high quality commercial products.

Melting points were uncorrected. Ultraviolet, infrared, and nuclear magnetic resonance spectra were determined with Cary model 14, Perkin-Elmer model 21, and Varian model A-60 spectrometers, respectively. The spectral data are given as ~$2”~ in the ultraviolet, vE:F in the infrared, and asolvent (tetramethylsilane as internal reference; coupling constants are in cycles per set) in nuclear magnetic resonance spectra. Optical rotations were measured in a Rudolph model 80 polarimeter with a model 200 photoelectric attachment. Vapor phase chromatography was carried out at 100” with SE-30 on 80 to 100 mesh Diatoport s in an F and M model 402 gas chromatograph. A Radiometer autotitrator, model TTTlc, with a GK 2021 C combination electrode was used for kinetic studies. Electrodes were standardized with Mallinckrodt standard aqueous buffer solutions of pH 7.0 and 10.0. Ozonolyses were carried out with an Orec model 03VI Ozonator.

Elementary analyses were performed by the Schwarzkopf Microanalytical Laboratory, Woodside, New York, or by Dr.

Carl Tiedcke, Laboratory of Microchemistry, Teaneck, New Jersey.

Methyl DL-i ,2-Dihydronaphtho[d, l-b]furan-2-carboxylate (IV) -To a stirred suspension of naphtho[2,1-blfuran-2-carboxylic acid (IX, 20 g, 0.095 mole) in alkali (800 ml of water and 40 ml of 6 N NaOH) were added 326 g (0.284 g atom of sodium) of 2% sodium amalgam. The mixture was stirred for 18 hours at room temperature, and the clear aqueous layer was acidified with concentrated HCl and cooled for 1 hour at 0”. Precipitated crystals were filtered, washed with water, and dried to give 16.2 g of colorless product, m.p. 160-165”. From this crude material two isomers of dihydroacid were isolated by repeated recrystallization from 70% ethanol, and finally from benzene: DL-1,2- dihydronaphtho[2,1-blfuran-2-carboxylic acid (IVa) : 5.06 g (25%); m.p. 174-176”; ,$Fol 270, 280.5, 292, 328, and 341.5 rnp (log E: 3.54, 3.64, 3.55, 3.39, and 3.44)

GJLoO~ Calculated: C 72.89, H 4.71 Found : C 72.97, H 4.94

and 4,5-dihydronaphtho[2,1-b]furancarboxylic acid (XII) : 1.11 g (5.5%) ; m.p. 224-225” (with decomposition); Xziy’ 269, 279, and 302 rnp (log E: 4.10, 4.08, and 3.97)

CdLoO~ Calculated: C 72.89, H 4.71 Found: c 73.18, II 5.03

1,2-Dihydroacid (IVa, 5.0 g) was treated with an excess of ethereal diazomethane at 0” to give the methyl ester, which crystallized from methanol: 4.05 g (76%); m.p. 103.5-104’; BCDC13 3.69 ppm (broad doublet, 2H, J = 9 cps), 3.81 ppm (singlet, 3H), and 5.37 ppm (triplet, lH, J = 9 cps)

CdL20z Calculated: C 73.67, II 5.30 Found : C 73.34, H 5.14

4,5-Dihydroacid (XII) also gave the corresponding methyl ester on treatment with diazomethane: m.p. 83-83.5”; X~~~” 268, 279, and, 303 rnp (log e: 4.10, 4.08, and 4.03); 6CDC1~ 2.97 ppm (multiplet, 4H), 3.84 ppm (singlet, 3H), 7.0 to 7.3 ppm (multiplet, 4H), and 7.37 ppm (singlet, 1H)

GJLzOz Calculated: C 73.67, H 5.30 Found : C 73.31, H 5.19

Methyl DL-2,3-Dihydronaphtho[l,d-blfuran-Z-carboxylate (V)- A suspension of naphtho[l ,2-blfuran-2-carboxylic acid (X; 34.1 g, 0.161 mole) in alkali (1500 ml of water and 68 ml of 6 N NaOH) was treated with 595 g (0.517 g atom of sodium) of 2% sodium amalgam as described above in the preparation of IVa. The crude product was recrystallized from 50y0 methanol to give 28.9 g (84%) of pure dihydroacid, m.p. 166-168”

C&H1003 Calculated: C i2.89, H 4.71 Found: C 72.57, H 4.53

This acid (11.05 g) was treated with an excess of ethereal diazomethane to give the methyl ester, which was recrystallized from methanol: 11.17 g (95%); m.p. 91”; ,g:Fol 301.5, 316, and 331 nip (log E: 3.64, 3.57, and 3.56); aCDC13 3.59 ppm (double doublet, 2H, J = 8, 9.5, and 2 cps), 3.79 ppm (singlet, 3H), and


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41G0 Stereochemistry of A ctiw Xitc of Chqmotrgpsi?~ Yol. 244, No. 15

5.38 ppm (double doublet, lH, J = 8 and 9.5 cps)

CLIH~ZOJ Calculated: C 73.67, H 5.30 Found: C 73.26, H 5.24

Methyl DZ-2,3-Dihydronaphtho[b,S-b]juran-2.carboxylate (VI) --5aphtho[2,3-blfuran-2-carboxylic acid (XI; 1.259 g, 5.94 mmoles) was reduced with 15.50 g (13.5 mg atom of sodium) of 2% sodium amalgam in alkali (60 ml of water and 4 ml of 6 N KaOH) for 7 hours at room temperature. The clear aqueous layer was acidified with concentrated HCl to precipitate the product (1.19 g, m.p. 214-220”). This was separated into two main fractions by four recrystallizations from methanol: one, which was less soluble in methanol, 588 mg (46%), m.p. 281-284”; the other, more soluble in methanol, 500 mg, m.p. 203-206”. The latter was still impure and was purified further after esterification with diaeomethane in ether. The crude methyl ester was recrystallized from methanol and then from ether-petroleum et,her to give pure VI: 205 mg (15.2%); m.p. 98-98.5”; ,~~~o1 255.5, 265, 276, 287.5, 317, and 331.5 rnp (log 6: 3.58, 3.70, 3.74, 3.60, 3.39, and 3.54); gcDc’ 3 3.54 ppm (broad doublet, 2H, .I = 8 to 9 cps), 3.78 ppm (singlet, 3H), and 5.23 ppm (double doublet, lH, J = 7.5 and 9 cps)

CdL~Oa Calculated: C 73.67, H 5.30 Found: C 73.28, H 5.53

b The less soluble component, m.p. 281-284”, was characterized as 4,9-dihydronaphtho[2,3-b]furanZcarboxylic acid (XIII) by elementary analysis and spectra of its methyl ester: m.p. 119”; X~~~nol 267 and 272.5 rnp (log E: 4.15 and 4.18); ScDc’3 3.75 to 4.11 ppm (two pairs of AU quartet, 4H, J = 6.5 and 12.5 cps, and 6 and 13.0 cps), 3.91 ppm (singlet, 3H), and 7.14 ppm (singlet, 1H)

CdL~O~ Calculated: C 73.67, H 5.30 Found: C 73.84, H 5.70

Illethyl o-llydrocoumarilate (D-III)-n-Hydrocoumarilic acid (0.93 g, 5.67 mmoles) was esterified (28) in 10 ml of anhydrous methanol with 0.5 ml (0.82 g, 6.88 mmoles) of thionyl chloride. The neutral fraction was distilled in a vacuum to give 0.83 g (8756) of colorless oil with an unpleasant odor: b.p. 99” (0.75 mm) ; [a]$ -13.3” (c, 1.47, in heptane)

CIOHIOOS Calculated: C 67.40, H 5.66 Found : C 67.21, H 5.58

Illethyl z-llydrocoumarilate (L-III)--L-Hydrocoumarilic acid (2.79 g, 17 mmoles) was esterified (28) in 30 ml of anhydrous methanol with 1.5 ml (2.46 g, 20.6 mmoles) of thionyl chloride, and the neutral fraction was distilled in a vacuum to give 2.79 g (92%) of colorless, almost odorless oil: b.p. 107” (1.5 mm) ; [a]; +13.6” (c, 2.36, in heptane)

GOHIOOJ Calculated: C 67.40, H 5.66 Found: C 66.99, H 5.66

Methyl N-Acetyl-oz-b-(I-naphthyZ)aZaninate (VII)---,4 solution of 3-(1-naphthyl)alanine hydrobromide (5.0 g, 23.2 mmoles) in 80 ml of 2 N NaOH was treated with 20 ml of acetic anhydride

for 1 hour at -5”. The crude N-acetylamino acid, 1n.p. 174- 178“, was dissolved in 50 ml of anhydrous methanol and treated with 2.5 ml (4.1 g, 37.6 mmoles) of thionyl chloride at -10” (28). The mixture was kept at room temperature for 1 hour and then treated as usual to give 3.62 g of ester (83.45;). Recrystalliza- tion from ether-petroleum ether gave 2.94 g of pure crystals; m.p. 98”; v”m;; 3256, 1745, 1638, and 1547 cm-’

CdL~NO~ Calculated: C 70.83, H 6.32, N 5.16 Found : C 70.48, II 6.14, N 5.16

Methyl N-:lcetyZ-oz-S-(Z-naphthyZ)aZaninate (VIII)-DL-3-(2- Saphthyl)alanine was acetylated and then esterified as described for t,he l-naphthyl derivative, VII. The crude product was recrystallized from ether-petroleum ether: m.p. 81-82”; VEX; 3334, 1724, 1643, and 1527 cm-l

Calculated: C 70.83, H 6.32, ?j 5.16 Found: C 70.88, H 5.94, N 5.19

Resolution of Methyl DL-1 ,2-Dihydronaphtho[Z,i-b]furan-2- carboxylate (IV) by Chymotrypsin (Fig. S)-To a stirred solution of DL-IV (3000 g, 13.2 mmoles), in a mixture of 2400 ml of 50% aqueous dimethyl sulfoxide and 60 ml of 1.2 N NaCl, was added a solution of 180 mg of ac-chymotrypsin in 60 ml of distilled water at room temperature (substrate concentration, 5.24 mM; enzyme concentration, 2.86 PM). Hydrolysis was followed by titration with 0.1 N NaOH at pH 7.0 apl)nrent. .ipproximatjcly 75 m1 (5.5 mmoles of NaOH, 56.87; for DL) of alkali were consumed in 20 min, a11d the reaction practically stopped. The mixture was diluted with 1200 ml of water and extracted four times with ether. The combined ether layer was dried (XIgSOh) and evaporated to give 1.238 g (41.3%) of I,-IV, m.p. 82%83”, which was recrystallized from methanol: m.p. 82-83”; [a];* +116.2” (c, 1.07, in ethanol)

C1*1~1203 Calculated: C 73.67, H 5.30 Found: C 73.25, H 5.09

The aqueous solution was acidified to pII2 with concentrated HCl and extracted four times with ether. The ether solution was dried and evaporated to give 1.441 g (51.27,) of crystals: m.p. 1555162”; [&* -59.9” (c, 0.98, in ethanol). A purer sample of IVa was obtained by recrystallization from 707; ethanol: m.p. 165-168”; [a Ii* -75.5” (c, 1.00, in et,hanol). The crude acid was treated with etheral diazomethane at room temperature to give D-IV, which was recrystallized from methanol: m.p. 83”, [(Y]? -110.0” (c, 1.01, in ethanol)

C1aIL203 Calculated: C 73.67, H 5.30 Found: C 73.31, H 4.92

Resolution of Methyl oz.2,3-Dihydronaphtho[l ,2-b] juran-d- carboxylate (V) by Chymotrypsin (Fig. 4)--9 stirred solution of DL-V (2.5 g, 10.95 mmoles) in 1500 ml of 507, aqueous dimethyl sulfoxide and 150 ml of 1.2 N NaCl was treated at room temperature with a solution of 225 mg of Lu-chymotrypsin in 150 m1 of water at 1~11 7.0 apparent (substrate concentration, 6.1 rnM; enzyme concentration, 5.0 PM). Sfter addition of the enzyme, 2.0 ml of 2 N K:\OH (4.0 mmoles of NaOH, 36.574 hydrolysis) were


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consmrrc~l ill 15 min. The mixture was diluted with 1500 ml of ice water; the neutral com1)onent was cxtractcd with ether as quickly as possible a~rd thcJr worked ulJ as usual. The crude JlCUtJXl l,OrtiOl~, 1.46 a (58.4oi), 1U.p. 7590”, [Cy];’ -39.4” (C,

1.07, in ethanol), was recrystallized repeatedly fro111 nlethanol to give L-V: 111.1). ii”, [(Y]:* - 143.0” (c, 0.99, in ethanol)

CdLzOa Calculated: C 73.67, H 5.30 Found: C 73.28, H 5.68

The crude acidic portion (0.87 g (37.1%), Jn.p. 150-162”, [a]:’

65.1” (c, 0.98, in ethanol)) was purified in the same may, after esterification u-ith diazomethane, to give D-V: m.p. $7”; [a]:* +145.5” (c, 0.94, in ethanol)

Calculated: C 73.67, 11 5.30 Found : C 73.06, H 5.10

Resolution 0s Xethyl N-Acetyl-o&-(1 -naphtl~yl)alaninafe (VII) by Ch2/motrypsin-un-VII (6.37 g, 23.5 mmolcs) was hydrolyzed in a solution containing 1650 ml of 505;) aqueous dimethyl sulfoside and 150 ml of 1.2 K N&l with 100 rng of LY- chymotrypsin at 1)H 7.0 apparent (substrate concentration, 1.3 x 1OP M; enzyme concentration, 2.2 PM). After 5.6 ml of 2 N ~&J-I (11.2 JJlJJJOlW of NaOH, 47.7’;; for DL) had bee11 con- sun~d in 60 min, the reaction stopped. The mixture was di- lntcd lvith 1500 ml of water and extracted with ethyl acetate as usual. The neutral part, 2.85 g (44.77{), m.1’. 9X-100”, was rrcrystallized from ethyl acetate-petroleulJ1 ether to give D-VII: m.p. 9X-98.5”, [oI]; $17.4” (c, 1.28, ill ethanol)

Calculated: C 70.83, H 6.32, N 5.16 Found : C 70.89, II 6.14, N 5.16

The acidic part, 2.51 g (41.5%), m.p. li9”, [c$’ -30.3” (c, 0.90, in ethanol), was esterified with 35 ml of methanol and 1.5 ml (2.1 eq) of thionyl chloride (28) to give 2.41 g of L-VI, m.p. IOl-102”, which was recrystallized from ether: m.p. 102-103”, [cu]:’ -18.1” (c, 1.59, in ethanol)

C~~HUNOZ Calculated: C 70.83, H 6.32, N 5.16 Found : C 70.88, H 5.89, N 5.12

Another san~ple (600 mg) of the acidic part described above was hydrolyzed by refluxing with 15 ml of 6 N IICl for 4 hours. After standing overnight at room temperature, crystals of t,he amino acid hydrochloride were filtered, dissolved in 10 ml of hot water, and neutralized with pyridine to give the free L-amino acid, VIIb, as a crystalline precipitate, 205 mg, m.p. 226-227” (with decoml”)sit,ioli). The amino acid was purified by dissolving in hot 2 N IICl and subsequent neutralization with pyridine, m.p. 229” with decomposition, [cy]:’ -13.1 (c, 0.154, in 90y0 aqueous dimethyl sulfoside), [M]n -28.2”; [a$!,’ +72.5” (c, 0.196, in 1.2 x lI(‘l ill 907; aqueous dimethyl sulfoside), [X], +156.0”; [X], in HCl - [Xl, in II20 = +184.2”.’

13-(1.Naphthyl)alanine (VIIb) is too insoluble in water to measure rotations accurately. We assume that the use of 90yG nqueo~~ dimethyl sulfoxide does not affect the optical correlations (29). In any event the difference in [Ml, values is similar for L-phenylalanine, L-tryptophan, and the amino acid considered to be L-VIIb.

GJLaNOx

Calculated: C 72.54, H 6.09, N 6.51 Found : C 72.72, H 6.14, N 6.48

[IX]~ values for L-tryptophan and L-phenylalanine were determined in the same solvents. n-Tryptophan: [LY]~* -27.0” (c, 1.06, in 90% aqueous dimethyl sulfoxide), [Ml,, -55.1”; [LY]~* +61.9” (c, 1.00, in 1.2 N HCl in 90% aqueous dimethyl sulfoxide), [A& = +126.3”; [MID in HCl - [Ml, in Hz0 = +181.4”. L- I’henylalanine: [cu]$’ -26.6” (c, 0.265, in 90(% aqueous dimethyl sulfoxide), [M]n -43.8”; [LX]~* +50.7” (c, 0.99, in 1.2 1\~ HCl in 50% aqueous dimethyl sulfoxide), [M], +83.6”; [Ml, in HCl - [Ml, in Hz0 = +127.4”.’

Ozonolysis of Methyl L-I ,2-Dihydronaphtho[Z, 1 -b] juran-Z-car-

boxylate (z-IV)-A solution of L-IV (1.100 g, [ali f111.5’) in 22 ml of glacial acetic acid and 22 ml of ethyl acetate was ozonized (25) at 0” for 12 hours. The reaction mixture was eva1)orated under vacuum to approximately 10 ml, and then diluted to 25 ml with glacial acetic acid. After the addition of 7 ml of 30~~ HzOs, the mixture was allowed to st’and at room temperature for 2 days. Excess peroxide was decomposed by stirring with 200 mg of 10 c/o palladium on charcoal for 30 min and then adding 1.2 g of NaHSO3 (until the KI-starch reaction was negative). The mixture was evaporated to dryness and the residue was extracted with methanol. The methanol solution was concentrated to approximately 10 ml and treated with an excess of diazomethane solution at room temperature for 1 hour. The yellow oil obtained by evaporation of solvent was distilled in vacuum (12 mm Hg) at 120-160” (bath temperature) to give 538 mg of liquid. This was placed on a 25-g column of silica gel and the coIumn was developed with ether-petroleum ether (1:2). After elution of most of the byproducts with 300 ml of ether-petroleum ether (1:2) and 200 ml of ether-petroleum ether (1.5:2), dimethyl malate was cluted with ether-petroleum ether (1: 1). The malate-containing fractions were dried by evaporation, and the remaining oil was distilled under vacuum to give 40 mg of dimethyl I,-malate as a colorless liquid, [a]: - 10.2” (c, 1.12, in acetone) (lit,erature (30), [o(]t’ -11.58” (c, 4.23, in acetone)). This product showed a single peak on vapor phase chromatography and gave an infrared spectrum identical with that of authentic dimethyl nn-malate.

Ozonolysis of Methyl D-I ,2-Dihydronaphtho[l ,Z-bljuran-Z-carboxylate (D-V)-A solution of D-V (1.500 g, [(Y]!’ +lOS”) in 30 ml of glacial acetic acid and 30 ml of ethyl acetate was ozonized at 0” for 18 hours. The mixture was evaporated under vacmm~ and the residue was heated with 60 ml of 0.5 N HCl at 100” for 30 min. NaHS03 was added until the KI-starch reaction was negative, and the mixture was treated as described for the deg- radation of L-IV. The colorless liquid (303 mg) obtained by distillation of the crude product was chromatographed on 15 g of silica gel. Evaporation of the ether-petroleum ether (1: 1) fraction and subsequent distillation gave 90 mg of dimethyl n-malate, [o(]$ +8.35” (c, 1.72, in acetone), which had an infrared spectrum identical with that of dimethyl nn-malate.

ZCinetic Procedure-Kinetic studies were carried out at 25”, $1 8.0 nl)parent, in aqueous dimethyl sulfoxide under nitrogen. The reaction mixture, consisting of 10 ml of substrate solution, 1 ml of I .2 N NaCl, and 1 ml of enzyme solution, was titrated with 0.1 N NaOH (for reactive substrat’es) or 0.05 pi NaOH (for less reactive substrates) delivered from the automatic burette (0.5.n,l volume) of the Radiometer titrator. Each substrate


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4162 Stereochemistry of Active Site of Chymotrypsin Vol. 244, No. 15

was dissolved in 507; dimethyl sulfoxide (final concentration of dimethyl sulfoxide in the reaction mixture of the total volume 12 ml was 41.7%) at a concentration of 3 to 10 mM (2.6 to 5.5 mM for II, III, IV, and V, and 4.7 to 10.0 m&f for the amino acid derivatives). The initial hydrolysis rates were determined at 1, 0.5, 0.25, 0.15, and 0.10~ concentrations of the original substrate solution. The enzyme solution was made by dissolving cr-chymotrypsin in distilled water, and the enzyme concentration in the reaction mixture was 3 to 4 X 1OP M for react,ivesubstrates and 3 to 9 x 1O-5 M for less reactive substrates. The kinetic parameters obtained are summarized in Table I.

The rates of hydroxide ion-catalyzed hydrolysis of all substrates were determined by the same titration method (with 0.200 N NaOH) with 10 ml of substrate solution at pH 10.0 apparent for methyl hydrocoumarilate and methyl dihydronaphthofurancarboxylates and at pH 11.0 apparent for methyl N- acetylnaphthyalaninates and methyl A-acet,ylphenylalaninate. The substrate concentration was 4 to 10 rnnf in 50y0 aqueous dimethyl sulfoxide containing 0.1 M NaCl. Half-times (t+) of the hydrolysis reactions obtained from the tit,ration curves led to the first order kinetic con&ants ken--, which were compared at pH 10.0 for all compounds, assuming that JZon- (pH 11.0) = 10koH- (pH 10.0). This assumption is reasonable since t’he pH dependence of the chymotryptic hydrolysis of methyl N-acetyl- L-phenylalaninate in this solvent bet)ween pH 6 apparent and pH 10 apparent is completely normal. Relative ratios (intrinsic reactivities) of hydroxide hydrolysis rates are lisbed in Table II (Column 3).

RESULTS

Preparation of Substrates-Reduction of naphthofurancarboxylic acids (23), IX, X, and XI, with sodium amalgam in aqueous alkali gave the corresponding dihydroacids, IVa, Va, and Via, respectively. While X gave exclusively the desired dihydroacid Va, reduction of IX and XI led to the formation of isomeric dihydroacids, XII and XIII, in addition to IVa and Via (Fig. 2).

The structures of the dihydro compounds were established by spectroscopy of the acids or their methyl esters as follows. (a) Ultraviolet spectra of IV and VI showed typical fi-naphthyl ether type absorption (32, 33) at 260 t,o 290 rnp and 315 to 340 rnp, while t,he spectrum of V displayed cu-naphthyl ether type absorption (33) at 300 to 330 mp. (b) Very similar ABX type resonance signals (3.4 to 3.8 ppm (HA and HB), and 5.2 to 5.4

IX X XI

KY XII XIII

FIG. 2. Compounds IX to XIII

ppm @Ix)) appeared in the nuclear magnetic resonance spectra of IV, V, and VI. (c) The methyl esters of XII and XIII showed furancnrboxylic ester type absorption (34) at approximately 260 to 280 mp in the ultraviolet, and a group of signals at 2.8 to 3.2 ppm (413) for XII (as ester) and at 3.7 t,o 4.1 ppm (4H) for XIII (as ester) in nuclear magnetic resonance spectra, which were not inconsistent with these structures.

The methyl esters of t’he above compounds, as well as the other substrates, were prepared by conventional methods as described under “Experimental Procedure.”

Enzymatic Experiments--Aqueous dimethyl sulfoxide (40 to 50%) was the medium for the enzymatic reactions (done in a pH- stat) because this solvent is excellent for many substrates having low water solubility and does not appear to inactivate chymotrypsin, at least during the course of the enzymatic hydrolyses. The apparent pK, for the hydrolysis of methyl N-acei&,- phenylalaninate (II) by chymotrypsin in 50% aqueous dimethyl sulfoxide is 6.72, which is quite similar to a pK, of 6.74 determined (35) for the hydrolysis of ethyl N-acetyl-L-tyrosinate in water. The rate of hydrolysis of II by chymotrypsin is about 16 times slower in aqueous dimethyl sulfoxide than in water (see References 11 and 13, and Table II). The slower rate in the former is caused primarily by an increase in K,, which is to be expected in mixtures of organic solvents and water (36).

In exploratory experiments at pH 8.0 apparent, hydrolysis of one enantiomer of DL-IV (3.75 mM) was practically finished in 30 min (t+ &Z 7 min) at an enzyme concentration of 2.4 X 10’ M,

when alkali consumption practically stopped. This result shows the extremely high reactivity and stereospecificity of the hydrolysis of IV. Controls with methyl N-acetyl-L-phenylalaninate (II; 2.62 mM) and methyl N-acetyl-L-tryptophanate (XIV; 2.51 m&f) gave half-times (t;) of 14 and 11 min, respectively, at the same enzyme concentration.

DL-V is hydrolyzed at a slower rate and with much less stereospecificity than IV. The initial hydrolysis rate was estimated to be approximately one-fifteenth that of DL-IV, and the titration curve indicated little stereospecificity. DL-VI is even less reactive than V, and t,he initial hydrolysis rate was approximately 150 times slower than that of IV.

Methyl A-ace@-uL-3-(l-naphthyl)alaninate (VII) is only 3 to 4 times less reactive than methyl N-acetyl-L-tryptophanate (XIV) f and its hydrolysis is highly stereospecific (DL substrate, 4.92 mnl; enzyme, 2.4 X 10e7 M; t+ E 40 min), while methyl N- acetyl-m-3-(2.naphthyl)alaninate (VIII) is about 100 times poorer as a substrate than VII.

Compounds IV, V, and VII were resolved with chymotrypsin, and t,he absolute configurations of the enantiomers were determined as follows.

When UL-IV was hydrolyzed at pH 7.0 apparent, the reaction became very slow after an uptake of 0.5 eq of sodium hydroxide. Workup of t,he reaction mixture gave unhydrolyzed (+)-ester (4lcj, yield), [o(JD +116.2”, and (-)-acid, [a& -75.5”, which afforded a levorotatory methyl ester, [LY]D -110.0”. This enzymatic hydrolysis proceeds with D specificity, as shown by ozonolysis (25) of t,he unhydrolyzed (+)-ester and conversion of the product to dimethyl L-malate (L-XV), [aID -10.2” (reported [& -11.58” (30)) (Fig. 3).

DL-V was hydrolyzed by the enzyme to the extent of 35 to 40%, and the crude neutral fraction was purified by repeated recrystallization from methanol to give a sample of (-)-V, [LY]D -143”, which was approximately 98% optically pure from the results of


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D-mnlate (D-XV), [& +8.35”. The optical purity of this D-

CH2N2 3. D-IV malnte, and accordingly that of (+)-V, [LY],, +106O, was cal-

culated to be S6yc,, based on the reported rotation of dimethyl L-

malate (30). The chymotryptic hydrolysis of DL-V thus pro-

? DL-IV cT

pH7.0

D-IVa

ceeds wkh modest D specificity (Fig. 4). Methyl N-acety1-or,-3-(l-naphthyl)alaninate (VII) was resolved

TABLE II

CO,CH, CO,CH, Reactivity (k,,,/li,) and stereospecificity in chymotryptic hydrolysis Reactivity (k,,,/li,) and stereospecificity in chymotryptic hydrolysis

HO-G-H HO-G-H of substrates of substrates

It is not intended that these numbers represent accuracy be- It is not intended that these numbers represent accuracy be-

C"'2 C"'2 pond two significant figures. pond two significant figures.

CO,CH, CO,CH, I I

L-IV L-XV

FIG. 3. Resolution of Compound DL-IV by chymotrypsin (CT)

1. Substrate

2 &at/K

41.7% dimethyl sulfoxide-water: 5. Water:

--I- b/Km

‘m 3. Intrinsic 1. kc,tIKm correctedC reactivity” corrected*

6. Specificity

cH2Nz, D-7

1 I) 0, 2) CH2N, D-Va I

2°2cH3 =

H-C-OH :

iH2 I CO2CH3

L-V iJ-xv

FIG. 4. Resolution of Compound DL-V by chymotrypsin (CT)

D-I L-I D-Ia L-Ia L-II D-II D-III L-III D-IV L-IV D-V L-V DL-VI D-VI L-VI L-VII D-VII DL-VIII L-XIV D-XIV

3,800 -0 416

5 6,900

0. 306

73 8

1,960 <o.

1G 5,200

-0

8.46 (DL) 8.46 (DL)

27.2 (DL) 27.2 (DL)

1.0 (L) 1.0 (L)

34 CDL) 34 CDL) 50 CDL) 50 CDL) 35 CDL) 35 b-1 42 42 CDL) 42 (=‘L)

1.0 (DL) 1.0 (DL) 1.0 1.0 (L)

5,070d 1.25d

2,210d 23.5d

,800 62,000” -0 -0e

12.2 650f 0.15 8.0f

338 17 .OOOQ 0.01 0.5s 8.7 4350 2.1 1059 0.2 100

-100 .h

-0.2Qe’ ,960

(0.1 31yoooi <li 10 250i

1,200 425,OOOi 1.0 (L) -0

I 1-O

4,050

940

>62,000

82

34,000

4

1 -50

1 >31,000

>425,000

TABLE I a Intrinsic reactivity is the rate of the first order hydrolysis of

Kinetic parameters for hydrolysis of substrates by chymotrypsin at an ester by hydroxide ions relative to the rate of hydrolysis of

pH 8.0 apparent and 26” in 41.7% dimethyl sulfoxide-water methyl N-acetyl-L-phenylalaninate (II), which is assigned an intrinsic reactivity of 1.0. These values were obtained in 50%

It is not intended that these numbers represent accuracy be- dimethvl sulfoxide-water and 0.1 M NaCl. yond two significant figures. -

--

-

- * Corrected for intrinsic reactivity (Column 3). c Obtained or calculated as indicated. These values are pre-

Substrate

L-II ........... D-III .......... L-III .......... D-IV. ......... L-IV .......... D-V. .......... L-V ........... DL-VI ......... L-VII. ........ D-VII. ........ DL-VIII ....... L-XIV. ........

101 [El koat .-

db se0 ntM

3.27 40.8 10.8 20.2 5.1 12.3

517 0.069 12.7 2.91 21.8 1.29

700 0.001 2

34 24.5 80.2 102 1.31 17.8 299 0.38 47.0

3.79 49.2 25.1 907 <O.OOl <lO 307 0.0013 0.83

3.24 28.3 5.5 -

3,800 416

5 lG,900

0.5 306

73 8

1,960 <O.l 16

5,200

sented for comparison of some literature values, obtained in water, with those of the present work, obtained in dimethyl sulfoxide- water.

d Estimated from the literature values for I (5) and Ia (13), with the use of the intrinsic reactivities in Column 3.

c Cohen and Schultz (13), at 25”, pH 7.8, in 0.1 M NaCl. f Calculated from the value for D-III (ll), an intrinsic reactiv-

ity of 21.5 in water, and the specificity D-III/L-III obtained in 41.7yo dimethyl sulfoxide-water.

0 Obtained by multiplying k&K,,, (app) (corrected) (Column 4) by the difference in k.,t/K,,, (app) for methyl o-hydrocoumarilate in going from dimethyl sulfoxide-water to water. The factor is about 50.

h Estimate based on the specificity in the chymotryptic hydrolysis of III, and the symmetry of VI.

i Obtained as in Footnote g, with L-II as the standard; the factor is about 16.

the ozonolysis described below. Esterification of the crude acid fraction and Purification gave (t-)-V, [QIIIJ 4-145.5” (optical

i Peterson (22), at 25”, pH 7.90, in 0.1 M NaCl. A value of 83,000 is obtained by the method described in Footnote i. An-

purity, 99”/Q. A less highly purified sample of (+)-V, [& other value of 703,000, at 25”, pH 8.25, in 0.05 M CaCl,, may be

+106”, was ozonized and the product was converted to dimethyl calculated from the data of Bender and Hamilton (31).


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4164 Stereochemistry of A ctive Site of Chymotrypsin Vol. 244, No. 15

similarly to the (+)-ester ((+)-VII), [aID +17.4” and (-)-acid (( -)-VIIa), [& -30.3”, which gave the enantiomeric (-)-ester (( -)VII) of [& -18.1”. Acid hydrolysis of the (-)-acid led to an enantiomer of 3-(l-naphthyl)alanine (VIIb), m.p. 229” (with decomposition), the absolute configuration of which was assigned to the L series by comparison of its molecular rotation value (29) (see “Experimental Procedure”) in acidic and neutral 9O70 aqueous dimethyl sulfoxide.

Kinetic studies of the chymotryptic hydrolysis of all substrates were carried out at 25“ and pH 8.0 apparent in 41.7”/” aqueous dimcthyl sulfoxide under a nitrogen atmosphere. Linewcavcr- Burk plots of the initial hydrolysis rates at various substrate concentrations gave the kinetic parameters, kcat and K, (app). The reactivities of all substrates were compared by means of kcat I&, (app) ratios (37) (Table I).2 Kinetic data for the methyl hydrocoumarilates (III), methyl N-acetyl-L-phenylalaninatc (II), and methyl N-acetyl-L-tryptophanate (XIV) were also obtained for reference under the same conditions. The intrinsic reactivities (Table II) for hydroxide-catalyzed hydrolysis of the substrates were determined in the same solvent system by comparison of the first order rate constants at pH 10.0 apparent, and normalized with methyl N-acetyl+phenylalaninnte (II) as a standard, to which a reactivity of 1.0 was assigned (Table II, Column 3). Reactivity (kc&L (app)) values for the enzymatic hydrolysis were corrected for the intrinsic reactivitics (Table II, Column 4). For comparison with enzymatic reac- tirities (k,,JK, (app)) in water reported in the literature fol other substrates, the former were corrected by factors obtained from the measured differences in enzymatic reactivity in the two solvent systems of methyl N-acetyl-L-phenylalaninate (II) and methyl DL-hydrocoumarilate (D-III) to give calculated enzymatic reactivities in water. Column 5 of Table II contains a collec- tion of measured chymotryptic reactivities in water and values from Column 4 corrected by these factors. Because the differences in enzymatic reactivity in the two solvent systems were not studied extensively, values in Column 4 are probably more reliable than the corrected values in Column 5, which arc given only for purposes of rough comparison.

DISCUSSION

It is necessary to examine the validity of the dihydronaptho- furancarboxylic ester systems as models for N-acyl-L-tryptophnn esters. Naphthalene is isosterica with indole, and some work on naphthalene derivatives as tryptophan antagonists has been

2 Reactivities of the two rather unreactive substrates VI and VIII were obtained with DL compounds. In each case the less reactive enantiomer is expected to inhibit competitively the hydrolysis of the more reactive enantiomer. The binding constants (K, and R;) for many enantiomeric substrate-inhibitor pairs are quite similar in magnitude (cf. the discussion by Zerner and Bender (38)). I f this is true also for the DL compounds studied here, the chymotryptic reactivities of the more reactive enantiomers of our DL pairs should be about twice as large as the values in Table I. Such differences are not expected to change the order of reactivities in Table III or the conclusions of this paper.

3 Friedman (39) has described the evolution of the concept of isosterism sinre its introduction by Langmuir (40) in 1919. A definition by Erlenmeyer and Leo (41) w&s translated (39) as follows. “Atoms, ions, or molecules in which the peripheral layers of electrons can be considered to be identical are termed isosteres.”

reported. Not enough has been done, however, to give a clear picture of the biological effects of interchanging the two ring systems. Obviously, a naphthalene ring might substitute for an indole ring in some situations but not in others, because of its inability to undergo the metabolic changes typical of indole rings. Bloch and Erlenmeyer (42) found that 3-(1-naphthyl) acrylic acid inhibits the growth of Escherichia coli, and that the inhibitory effect is reversed by tryptophan. On the other hand, Dittmer (43) was not able to inhibit the growth of several micro- organisms with either of the two isomeric 3-naphthylalanines, or to substitute them for t)ryptophan with organisms that require the latter amino acid. In the case of chymotrypsin, the high degree of reactivity (with L-specificity) in the hydrolysis of mebhyl N-acetyl-3-(1-naphthyl)alaninate (VII; Tables I and II) allows us to assume with some confidence that, for this enzyme, indolc and nsphthalene rings similarly situated in substrates are roughly equivalent.

A second problem concerns the operational equivalence of the heterocyclic part of naphthofurancarboxylic esters with the substituents about the a carbon atoms of tryptophan derivatives, such as methyl N-acetyl-L-tryptophanate (XIV). It is clear from work with bicyclic analogues of methyl N-acetyl-L-phenylalaninate (II) that, whatever the stereochemical significance, the analogues arc hydrolyzed with n-specificity in all cases in which specificity has been determined: methyl dihydroisocarbostyril- 3.carboxylate (I (5, 6))) methyl 3,4-dihydroisocoumarin-3-carboxylate (Ia (10, 13)), p-nitrophenyl 1,2-dihydro-2-naphthoate (9), and methyl hydrocoumarilate (III (11)). The novel 2,2’- bridged biphenyl analogue of benzoyl phenylalanine methyl ester described by Belleau and Chevalier (16) is hydrolyzed with L specificity, but the stereochemistry of this compound is quite different from that of those analogues just mentioned. We think it reasonable to assume that, just’ as methyl n-hydrocoumarilate (III) is an analogue of methyl N-acetyl-L-phenylalaninate (II), the three isomeric methyl dihydronaphthofurancarboxylates described here are analogues of methyl N-acetyl-L- tryptophanate, in which the naphthalene rings are rigidly fixed in various positions relative to the ester group and the ether oxygen atom of the dihydrofuran moiety. They thus correspond to conformat,ions of methyl N-acetyl-L-tryptophanate (XIV) in which the posit,ion of the indole ring is fixed relative to the other groups about the o( carbon atom. We anticipated that one of the isomeric DL pairs, IV or V, would be hydrolyzed rapidly and with good D specificity, and that the stereochemistry of the most reactive D isomer would correspond approximately to that of methyl N-ncetyl-L-t,ryptophannte (XIV) as it binds productively to the active site of chymotrypsin. It appeared likely that the less reactive isomer, V or IV, might show some inversion of specificity in the direction 11 + L. Isomer VI was expected to be fairly unreactive, since Peterson (22) has shown that bulk in the para position of methyl N-ncetyl-I,-phenylalalinates is not well tolerated by the enzyme. These predictions were substantiated by the results.

Of all of the isomeric methyl dihydronaphthofurancarboxylates, D-IV exhibited the highest reactivity (k,,JK, (app) = 17,000) and stereospecificity (34,000) in its hydrolysis by chymotrypsin. These values are several times better than those for methyl dihydroisoca,rbostyril-3-carboxylate (I; Table II), which is the best substrate previously described with the exception of natural amino acid derivatives. It appears that the stereo-


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J D-IV 0

FIG. 5. Left, methyl D-1,2-dihydronaphtho(2,1-b)furan-a-carboxylate (D-IV) at the active site of chymotrypsin. The areas enclosed by dotted lines indicate moderate steric hindrance in Direction @ and considerable steric hindrance in Direction 0. Right, benzolflquinoline (XVI).

chemistry of D-IV is especially satisfactory for productive binding at the active site of the enzyme.4 From the k,,JK,,, (app) values

in Table III, a reactivity sequence D-IV > D-III > D-V > L-V > D-VI(?) > L-III > L-IV > L-VI(?) was obtained for the enantiomeric methyl dihydronaphthofurancarboxylates and hydrocoumarilates. Although the enantiomers of VI were neither resolved nor tested, because of the comparative inaccessibility of the DL compound, the reactivities of D- and L-VI are expected to be as indicated, provided that the stereospecificity of the hydrolysis of VI is about 50, which would be comparable to that of III (the symmetry of both compounds is the same). The largest difference between successive members in the sequence was observed between D-IV and D-111 (approximately 30 times in reactivity and 420 times in stereospecificity). Therefore, the addition of a benzene ring to position 4,5 of D-III produced the greatest effect toward increasing the reactivity and specificity of the basic structure, III. Thus, the dihydrofuran moiety and the l-substituted /3-naphthol moiety must operate in D-IV to bring

the susceptible ester group into correct juxtaposition with the catalytic site. From this point of view, let us consider four structural elements, @ to 0, as shown in Fig. 5 and Table III. In Table III, the formulas of the substrates are drawn so that the ester groups are located above the plane defined by the ring system, and the structural elements are weighted as follows: 0, f2; 0, +I; 0, -I-; and 0, -2. I f Element 0 is unfavorable, it is assigned a weight of -1. These values allow correlation of the reactivities from $3 (highest) to -3 (lowest). When the

difference in the reactivities of enantiomers is calculated, the re- sulting number is a measure of specificity, ranging from 5 (IV, highest specificity) to 1 (V, lowest specificity).

This analysis permits the conclusion that the reactivity of any enantiomer in the series arises from a combination of these structural elements. The primary positive factor is a D configuration of the ester group in the dihydrobenzofuran portion of the substrates, as seen from the specificity of hydrolysis of IV, III, and V (and probably VI). The secondary positive factor is the addition of a benzene ring at position 4,5 of D-III, and shows its effect in the extreme reactivity of D-IV, as well as in the partial inversion of specificity observed with V. In spite of the unfavor-

4 For a discussion of the relevance of kinetic constants, particularly the k&K,,, ratio, to correlations between structure and snecificitv. see Bender and KBzdv (371. I . r

able configuration of the dihydrobenzofuran part of L-V, the positive influence of the extra benzene ring makes it a fairly good substrate, while the hydrolysis of its n-enantiomer is retarded by the unfavorable position of its extra benzene ring.

The prediction by Wallace, Kurtz, and Niemann (20) of the shape of the aromatic binding site is substantiated by the data in Table III, and other features of the site can be described. Let us refer again to Fig. 5 and consider the site to mirror the positive features in the structure of D-IV, as well as negative features in the other substrates. It is clear that the best inhibitor described by Wallace, Kurtz and Niemann (20), benzo[f]quinoline (XVI), should easily fit a binding site that accommodates D-IV, with respect to over-all shape, planarity of the ring systems, and the presence of a unique heteroatom (oxygen or nitrogen). The site must be roughly planar, elongated, and curved (L shaped). In terms of Fig. 5, there is no indication as yet how far the planar

TABLE III

CORRELATION OF REACTIVITY AND SPEClFfClTY WITH THE STRUCTURAL ELEMENTS DEPICTED IN FIGURE 5

i- ,TRUCTURAL ELEMENT+

3

+2

+2

+2

-I

+2

-I

-I

-I

0 From Table II, Column 5. The relative reactivities are about the same if values from Table II, Column 2 or 4, are used instead of these values.

Copy 17000

D-III 650.

D-VI

0

+I

+I

0 -

-I

-I

ZA~n;lVITY

PEClFIClTYC)

+3(5)

+2(3)

+I(11

0

0 (3)

-I

-2

-3

tive one of each pair.

b Element @ is assigned a value of +2 when present and -1 when absent. The remaining elements are given positive or negative values only when present, as shown.

c Reactivity is represented by the sum of values for a given compound. Specificity is calculated by subtracting the react.ivity values of two enantiomers, and is given only after the more reac-


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4166 Stereochemistry of Act&e Site of Chymoirypsin Vol. 244, So. 15

Possible site of a groupwhich could form a hydrogen bond with the OH group of tyrosine derivatives or the NH group of tryptophan derivatives. d, moderate steric hindrance. e, polar side of the short section. f, nonpolar side of the short section.

hydrophobic region extends in Direction 2, but steric hindrance is moderate in Direction 3, and considerable in Direction 4. A hydrogen bond acceptor is possibly located in Direction 4, where it could interact with the para-OH group of tyrosine derivatives and the ring NH group of tryptophan derivatives. Deductions about the location at which the heterocyclic ring of D-IV binds to the enzyme cannot be made from the data in this paper alone. In bicyclic substrates like I and III, high reactivity with good specificity has been found when an oxygen atom (III (ll)), an NH group (methyl indoline-2-carboxylate),5 an amide group (I (5,6)), a lactone group (Ia (10, 13)), or a carbon- carbon double bond (p-nitrophenyl and methyl 1,2-dihydro- naphthoates (9, 13))5 occupies the position analogous to that of the oxygen atom in D-IV. In this region, the binding site appears to prefer a polar, planar grouping on one side, and a nonpolar grouping on the other side (compare the reactivities of D-IV and L-V in Table III). The polar side of the site is expected to bind the ar-acylamido substituents of normal substrates.

A further question is the axial or equatorial position of the ester group in substrates of restricted conformation (2-16). Cohen and Schultz (13) have correctly pointed out that the heterocyclic ring of methyl hydrocoumarilate (III) is likely to be puckered (on the basis of cyclopentene as a model), contrary to the assertion of Lawson (11). Recent work on the conformation of cyclopentene (44, 45) confirms Cohen’s thesis, but indi- cates that the energy of the barrier to ring inversion is sufficiently small6 so that the angle between the ester group and the ring system of III could conform to any reasonable requirement of the enzyme. This makes the actual conformation in solution

5 Y. Hayashi and W. B. Lawson, unpublished observations. 6 The barrier to ring inversion in cyclopentene (44,45) is 232 f

5 cm-i, or 0.664 kcal. In III, in which the number of interactions is fewer than in cyclopentene, the barrier is very likely of the order of 0.3 kcal. The barrier to olanaritv in the closelv related 2,3-dihydrofuran has recently been found-to be 83 cm-< or 0.238 kcal (46).

FIG. 7. Comparison of methyl S-acet,yl-n-3(1- and 2-naphthyl)- alaninates (L-VII and L-VIII) with methyl n-1,2-dihydronapht,ho- (2,1-b, 1,2-b, and 2,3-b)furan-2-carboxylates (n-IV, D-V, and D-VI). In each pair, attention is drawn to the similar positioning of the naphthalene rings and the oxygen atoms indicated by the arrotus. a, L-VII and D-IV. b, I,-VIII and D-V. c, L-VIII and D-VI.

of the ester group of III or IV irrelevant, and seems to re. duce the conformational question again to an indeterminate state. However, other arguments (11) in favor of the axial hypothesis are still valid, and the high specificity (D/L, 34,000) in the chymotryptic hydrolysis of IV supports the hypothesis that its ester group is quasiaxial’ or axial7 during hydrolysis by the enzyme. If the ester group were equatorial: the shape of D-IV would become rather similar to that of L-IV.8 However, as

7 “Quasiaxial” refers to the conformation of the ester group when the dihydrofuran ring is planar, while “axial” and “equatorial” correspond to forms obtained by ring puckering, which are similar in geometry to axial and equatorial forms in cyclo- hexane systems.

* Additional support for the axial hypothesis comes from the chymotryptic hydrolysis of the methyl esters of 1,2,3,4-tetrahydro-4-keto-2-naphthoic acid (A) and 1,2,3,4-tetrahydro-2- naphthoic acid (B). B is hydrolyzed 3.5 times more slowly than A, but the specificity (~10) is about the same as with B. (Rates were corrected for the intrinsic reactivities of the ester groups.) It seems reasonable to explain the difference in rate between A and B on the basis of steric hindrance by an axial hydrogen atom in position 4 to an axial ester group in B (W. B. Lawson, unpublished observations). However, we believe that the axial and


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previously proposed (II), we do not favor a too literal carryover of the axial hypothesis to acyclic systems. d conformation of methyl car-acctyl-L-phen~lalaninatc (II) similar to that of Hein and Nicmann (4), in which the ester group is not strictly like an asial or an equatorial one but intermediate between these es- tremes, seems most probable8 A model of methyl N-acetyl-n- tryptophanatc (XIV) in such a conformation, depicting also some of the previous conclusions about the binding site, is shown in Fig. 6.

The results with methyl dihydronaphthofurancarbosylates tempted us to predict the reactivity of the isomeric methyl N- ncet3-l-3-(naphthyI)alaninates VII and VIII toward chymotrypsin. Further, the reactivity of these esters in comparison with methyl N-acetyl-L-trS~pto1)h:lnate (XIV) was important from the standpoint of whether indole and naphthalene rings are similarly bound to the active site (see above). Finally, dhnond, i\jIan- ning, and Niemann (47) had predicted that methyl N-acetyl-L- 3-(2.naphthyl)alaninatc (L-VIII) “would be a particularly effec- tive substrate of a-chymotrypsin and would bc hydrolyzed at a rate faster than any hitherto evaluated oc-N-ilcet~l-L-oc-alnino acid methyl ester.”

Careful consideration of molecular models of both compounds indicated that L-VII can assume a conformation similar to that of D-IV with respect to the naphthalene ring, the ester group, and the oxygen atom of the cu-acetylamido group, as shown in Fig. 7a, while L-VIII is similar to D-V or D-VI, as shown in Fig. 7, b and c. It appeared that L-VII should be a better substrate than L-VIII, and the prediction was borne out perfectly by the results. In fact, L-VII exhibited very good reactivity with the enzyme (k&R, (api)), -31,000; stereospecificity, >30,000) which is comparable to the reactivity of methyl iV-acetyl-t- phenylalaninate (II), while the reactivity of or,-VIII is comparable to that of D- or L-V and about 100 times less than that of L-VII. The prediction of Almond, i\Ianning, and Niemann (47) was not far off, but they had selected the wrong isomer.

REFERENCES

1. HEIN, G. li:., MCGRIFF, It. B., AND NIEUNX, C., J. Amer. Chem. Sot., 82, 1830 (19GO).

2. WILSOS, I. B., XYD EHLII\‘GER, B. F., J. Amer. Chem. Sot., 82, 6422 (19GO).

3, Awm, E. S., NEUUTH, II., .\ND H.%RTLEY, B. S., J. ni0!. Chem., 235, PC35 (1960).

4. HEIS, G. E., AND NIEBL~NN, C., Proc. I\;&. flcad. Sci. U. S. il., 47, 1341 (1961).

5. HEIS. G. E., AND KIEM~XN, C., J. Amer. Chem. Sot., 84, 4487 (19G2).

6. HEIS, G. E., AND NIEK\NN, C., J. Amer. Chem. Sot., 84, 4495 (1962).

7. COIIEX, 8. G., KLEE, I,. II., BND WEINSTEIN, S. Y., J. Amer. Chem. Sot., 88, 5302 (19GG).

S. SILVER, M. S., J. Amer. Chem. Sot., 88, 4247 (196(i). 9. SILVER, 31. S., AND SONE, ‘I’., J. Amer. Chem. Sot., 89, 457

(1967).

:quatorial hypotheses are both rather artificial with respect to the conformations accessible to typical acyclic substrates. In spite of its comparative steric homogeneity, the corrected reactivity of I is much lower than that of II (Table II) toward chymotrypsin. Further work on this aspect of the subject is in progress.

10.

11. 12. 13.

14.

15.

1G.

17.

18.

19. 20.

21.

22.

23. 24.

25.

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Yuji Hayashi and William B. LawsonGEOMETRY OF TRYPTOPHAN DERIVATIVES

-Chymotrypsin: THE BINDINGαStereochemistry of the Active Site of

1969, 244:4158-4167.J. Biol. Chem.

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stereochemistry of the active site of &hymotrypsin

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