Eur. J. Biochem. 84, 293-299 (1978)

Conversion of the Active-Site Cysteine Residue of Papain into a Dehydro-serine, a Serine and a Glycine Residue Peter I. CLARK and Gordon LOWE The Dyson Perrins Laboratory, Oxford University (Received September 28, 1977)

Photolysis of papain which had been inhibited with 2-bromo-2’,4‘-dimethoxyacetophenone regenerated papain, but also formed [ A Ser2j]papain (i.e. papain in which the active-site cysteine residue 25 was replaced by dehydroserine) via the intermediate dehydrocysteine analogue, [ACys2’3papain. Reduction with sodium borohydride gave [Ser2’]papain. Both [Ser2’]papain and [ A Ser2j]papain had binding properties similar to those of papain, but were devoid of enzymic activity. Their fluorescence properties were also investigated. Incubation of [ A Ser2j]papain at p1-I 9.0 gave [Gly2 ]papain.

Kinetic specificity or the manifestation of enzyme specificity in the maximum velocity is a frequently encountered phenomenon in enzymology [I]. It is not explicable in terms of a simple structural complementarity between enzyme and substrate [2] and several alternative concepts have been suggested [ 3 - 61. In order to gain a deeper understanding of the causes of kinetic specificity it is necessary to have detailed structural information of the enzyme-substrate complex. X-ray crystallography is at the present time the only method capable of providing sufficiently detailed structural information to determine the cause of kinetic specificity. Low-temperature X-ray crystallography is a promising approach for the study of enzyme-substrate complexes [7,8], but for the proteolytic enzyme, papain, where suitable crystals for highresolution X-ray crystallography were obtained only from methanol/water mixtures [9], a different approach seemed necessary. It was considered that if the active-site thiol group of Cys-25 in papain could be converted to a hydroxyl group, the modified enzyme (henceforward referred to as [ S e ~ ~ ~ I p a p awould i n ) retain its binding properties for peptides, but would be incapable of hydrolysing ___.

~

Abbreviations. [SerZs]papain,[ ASerzS]papainand [Glyzs]papain; papain in which the active-site cysteine residue 25 has been replaced by serine, dehydroserine and glycine, respectively. Enzyme. Papain (EC 3.4.22.2).

them. The chemical modification envisaged had the desirable feature of slightly reducing the steric requirement of the essential thiol group and therefore would not lead to steric exclusion of the substrate. Moreover the replacement of a thiol group by a hydroxyl is electronically the closest that could be envisaged and had the further desirable feature of leading to a natural amino acid, a process which has been referred to as ‘chemical mutation’ [lo]. It was expected that such a modification would not significantly perturb the tertiary structure of the enzyme. The conversion of the active-site serine residue of subtilisin into a cysteine residue was achieved [lo- 121 without perturbation of the tertiary structure of the enzyme (J. Kraut, private communication). The conversion of a cysteine residue to a serine residue in a protein molecule has not hitherto been reported. The chemical strategy envisaged for this transformation is outlined in Scheme 1. a-Bromoketones selectively alkylate the active-site cysteine residue of papain. By using a phenacyl bromide with an absorption band at longer wavelength than that of the enzyme, photolysis should lead to Norrish type II cleavage 1131, provided that the protein would allow the required conformation to be adopted by the alkylated cysteine residue. Thiols have been converted into thioaldehydes by this method in non-hydroxylic solvents [13- 151, but it was anticipated that steric restriction arising from the protein structure [9] would

294

Chemical Mutations of Papain

-E

i E-CH~.SH

Reagents.

i. B r C H COAr; 2

-c.

\'.H ' s - b r H

ii

-

\>

E-c

Ar

H'

HO

-

CH3COAr

ii, hy; iii. H20; i v . NaBH 4

Scheme 1

prevent oligomerisation of the thioaldehyde and favour addition of water to give a hemithioacetal. Spontaneous loss of hydrogen sulphide would then lead to the corresponding aldehyde. Reduction of the aldehyde to a hydroxyl group should be possible with sodium borohydride. [SerZ5]Papain has been obtained in this way and its properties investigated. MATERIALS AND METHODS Papain

Papain was prepared from dried papaya latex [16] (we gratefully acknowledge a generous gift of this material from Travenol Laboratories, Inc., Cleveland, Mississippi 38732, U.S.A.) and purified by affinity chromatography [17]. The enzyme was assayed spectrophotometrically with N-benzyloxycarbonyl-glycine y-nitrophenyl ester as substrate [18].

water (4/1, v/v) to give colourless crystals of 2-bromo2',4'-dimethoxyacetophenone (1.62 g) m.p. 100101 "C, A,,, (EtOH) 230 nm ( E = 14600 M-' cm-', 274 nm (I: = 14500 M-' cm-I), 307 nm ( E = 12550 M-' cm-'); z (C2HC13) 2.13 (lH,d,J = 9 Hz, 6'ArH), 3.35-3.60 (2H,m,3' and 5'-ArH), 5.49 (2H,s, CHZ),6.11 and 6.17 ppm (2 x 3H, 2s, 2' and 4'-OMe). Found: C, 46.3; H, 4.3; Br, 30.6; CIOHl1BrO3 requires C, 46.3; H, 4.3; Br, 30.8 A stock solution of the bromoketone in dry acetonitrile (7.32 mM) was stored at -20 "C in a sealed flask.

x.

S- (2,4Dimethoxyphenacyl)-papain

A solution of 100% active papain (166.4 mg) in water (325 ml) was adjusted to pH 7.5 by dropwise addition of 1 M hydrochloric acid to the stirred solution. Addition of an aliquot of the stock solution of 2-bromo-2',4'-dimethoxyacetophenone in acetoAffinity Chromatography nitrile (1.05 ml, 1.1 mol/mol of enzyme) completely Sepharose-aminohexanoyl-glycyl-L-phenylalanyl- inhibited the enzymic activity. The solution was dialysed and concentrated under nitrogen (to 110 ml) L-arginine previously prepared in this laboratory gave by ultrafiltration. less tailing and hence a sharper peak in the affinity chromatography of papain (Bendall, Hawcroft and Lowe, unpublished results) than Sepharose-glycylPhotolysis of S- (2,4-Dimethoxyphenacyl) -papain glycyl-L-(O-benzy1)-tyrosine-L-arginine[17]. It was Photolysis was performed in a cylindrical quartz used therefore for comparing the binding properties apparatus with a medium-pressure mercury lamp of chemically modified papain with the native enzyme. (Hanovia 450 W, type 2521) along the axis with the inner compartment containing circulating tap water 2-Bromo-2',4'-dimethoxyacetophenone as coolant, the middle compartment containing 0.1 M 2',4'-Dimethoxyacetophenone (Aldrich Chemical naphthalene in iso-octane to cut out irradiation below 320 nm and the outer compartment containing the SCo., Ltd) was brominated by the method of King (2,4-dimethoxyphenacyl)papain solution with nitrogen and Ostrum [20]. A hot solution of 2',4'-dimethoxyacetophenone (3.6 g) in chloroform (25 ml) was added bubbling through to agitate and de-oxygenate it. to a stirred suspension of cupric bromide (8.93 g) in Samples were withdrawn at intervals and assayed for refluxing ethyl acetate (50 ml). The reaction was comenzymic activity. Activity was progressively regeneratplete in 1 h. The reaction mixture was filtered through ed up to 76% of the equivalent concentration of the celite, the solvent removed under reduced pressure native enzyme. An aliquot of the 2-bromo-2',4'and the residual solid recrystallized from ethanol/ dimethoxyacetophenone solution (0.8 ml, 1.1 mol/mol

P. 1. Clark and G. Lowe 10 201

295

n

c

60r

Fraction number

Fig. I. The chromaio,orumof phoiolysetl S-(2,4-dimetho.u~phenucyl)papain on a column of Sepharose-aminohexanoyl-glyc~l-r.-phenylalanyl-r.-arginine (24 x I . 7 cm). Initial fractions were eluted with 25 mM EDTA (pH 4.3); after fraction 45, an exponential gradient of 25 mM EDTA (54 ml, pH 4.3) and water was used; 12.5 rnl fractions were collected

\

1000

2

4

6

8

10 12 14 16 18 20 22 24 26 2 8 30 32 Fraction number

Fig. 2. The chromatogram of ( A ) [Ser25Jpupain,( B ) [ASer2’ Jpapain and ( C ) native papain om a column of Sepharose-aminohexanoylglycyl-L-phenylalanyl-L-arginine (27 x 0.9 cm). The column was preequilibrated with 20 mM EDTA (pH 4.3) and eluted with an exponential gradient generated from 20 mM EDTA (34 ml, pH 4.3) and water: 10-ml fractions were collected

of enzyme) was added to the photolysate. The photolysis-inhibition cycle was repeated three times, assays at the end of each photolysis (about 1 h) showing 57 %, 38 o/, and 26 % respectively of the equivalent concentration of the original native enzyme. The photolysate The solution was applied to a column of Sephadex was inhibited again with bromoketone and dialysed G-25 and eluted with water. The fractions containing against 25 mM EDTA, pH 4.3, for 24 h, concentrated protein were lyophilised, hydrolysed and submitted to 15 ml by ultrafiltration, filtered (by Millipore) and to amino acid analysis. The radiochromatogram is applied to a column of Sepharose-aminohexanoshown in Fig. 3A. yl-glycine-L-phenylalanyl-L-argininepre-equilibrated The amount of protein used, assuming an absorpwith 25 mM EDTA, pH 4.3. The chromatogram is tion coefficient of 58500M-’ cm-’, i.e. the same shown in Fig. 1. Fractions 59 - 84 contained [ ~ l S e r ~ ~ ] as that of papain [21,22], was 153.8 nmol. The papain. amount of protein collected from the Sephadex column, based on the valine analysis and a papain (Ser25]papain content of 18 valine residues, was 41.64 nmol. The specific activity of the sodium b~ro[~H]hydride deter[dSerzS]Papain (11.2 mg in 3 ml) was dialysed mined experimentally was 1170 counts min-’ nmol-’. against 10 mM sodium phosphate buffer (pH 7.5) for If there is no kinetic isotope effect for the reduction 24 h. Aliquots of sodium borohydride in methanol of [dSer2’]papain, 47 500 counts/min should be present (4 x 5 yl; 26.3 mM) were added at 10-min intervals. in the serine fraction. The total activity in the serine After 1 h, the pH was adjusted to 5.0 with 0.1 M fraction after subtraction of background was 9712.5 hydrochloric acid and the solution dialysed against counts/min. This corresponds to 9712.5/47 500 or 20 mM EDTA (pH 4.3) for 24 h. The solution was 0.204 residues of serine/mol of protein generated on filtered (by Millipore) and applied to a column of reduction with sodium b~ro[~H]hydride, assuming Sepharose - aminohexanoyl- glycyl- L - phenylalanyl- L no kinetic isotope effect. arginine pre-equilibrated with 20 mM EDTA (pH 4.3). The chromatogram is shown in Fig.2A. Fractions 23 - 29 contained the [Serz5]papain. For comparison Immediate Reduction of Photolysate [dSerZs]papain (Fig. 2B) and native papain (Fig. 2C) wiith Sodium Bovohydride were run on the same column with the same elution A solution of S-(2,4-dimethoxyphenacyl)-papain conditions. was photolysed as described above, but instead of the photolysate being chromatographed, a portion (2 ml, [3H]Serine25-[Ser2s JPapain 7.2mg) was treated with aliquots of sodium boro[ ~ l S e r ~ ~ ] P a p(1 a iml, n A:;&,,, = 9.0) was added [3H]hydridein methanol (2 x 20 pl, 13.1 mM, 2.878 Ci/ to 10 mM sodium phosphate buffer (1 ml, pH 7.5). mol) with a 10-min interval between additions and Aliquots of sodium b~ro[~H]hydride in methanol the solution stirred for 30 min. The protein was (2x 10 yl, 13.1 mM, 1.90 Ci/mol) were added with desalted by passing through Sephadex G-25 with a 10-min interval and the solution stirred for 30 min. water and the fractions containing protein were lyo-

Chemical Mutations of Papain

296 500

-

450

-

400

-

350

-

.

-;300 v)

c

5 250

-8

-

100

r

B

-

Fraction number

200

-

-0

4

8 12 16 Fraction number

20

24

0

4

8

12

16

20 2 4 2 8 32 Fraction number

36 40

44 48

52

F i g . 4. The rudiochromutogram .from the amino acid analysrr of the hydrolj.sute of [ ~ I S e r ~ ~ ] p a p a( iAn) that had been incubated with [ 3 H ] ~ r a t eat r p H 9.0 for three bveeks and ( B ) that had been incubated with [3H]wutrr at p H 9.0 ,for three weeks and then reduced with sodium horohydride

Photolysis of S- (2,4-Dimethoxyphenucyl)-pupain in I3H ] Water

A solution of S-(2,4-dimethoxyphenacyl)-papain (2 ml, 6.4 mg) containing r3H]water (100 pl, 1.55 Ci/ Fraction number ml) was photolysed for 3 h as described above. The Fig. 3 . The radiochromatogrum from the amino acid analyser of' the photolysate was lyophilised and taken up in water hydrolysates from the photolysis product of S-i2,4-dimethoxyphenseveral times to remove exchangeable tritium, hydroacyl)-papain ( A ) reducedwith sodium horo[3H]hydride after chromalysed and submitted to amino acid analysis. The radiotography on Sepharose-aminohexanoyl-~lycyI-L-~~henylalanyl-r~-ur~ichromatogram showed no ['Hlalanine. nine und ( B ) reduwd by sodium horo[3H]h.ydride irnnirdialely after pho to1,vsis

Fluorescence Spectra

philised, hydrolysed and submitted to amino acid analysis. The radiochromatogram is shown in Fig. 3B. [3H]Glycine25-[Gly25/Papain

A solution of [ASer25]papain (2 ml, 7.2 mg) was adjusted to pH 9.0 with 0.1 M sodium hydroxide solution. [3H]Water(50 pl) was added giving a solution with specific activity of 14.94 Ci/mol. The solution was kept at 20 "C for 3 weeks and then divided into two equal parts. To one portion was added sodium borohydride in methanol (20 pl, 13.1 mM). After 30 min both samples were adjusted to pH 5.0, lyophilised, hydrolysed and submitted to amino acid analysis. The radiochromatograms are shown in Fig. 4.

Fluorescence emission spectra were recorded on a Perkin-Elmer-Hitachi spectrofluorimeter (HPF2A) in the ratio mode with excitation at 286nm. Buffer solutions in the range pH 3 - 8 were prepared from 20 mM sodium citrate and 20 mM sodium phosphate both containing 1 mM EDTA and 0.3 M NaCI. Buffer solutions (2.7 ml) were equilibrated thermally at 25 "C in a cuvette and papain solution (100 pl, approx. 70 pM) was added and mixed. The pH of each solution was measured within 10 min of the fluorescence emission spectrum being recorded. Assay ~ f [ A S e r ~ ~ ] P a pand a i n[Ser25]Papuin for Enzymic Activity

The protein solution (10 pl, A & & , = 4.0) and N-benzyloxycarbonyl-glycine p-nitrophenyl ester in

P. I. Clark and G. Lowe

acetonitrile (50 yl, 5 mM) were added with rapid mixing to a thermally equilibrated buffer solution (3 ml, 0.1 M NaH2P04, 1 mM EDTA, pH 6.0) in a cuvette in a Unicam SP 1800 spectrophotometer. The release of p-nitrophenol was followed by the change in absorbance at 340 nm. A similar assay using N-benzyloxycarbony1-Llysine p-nitrophenyl ester in acetonitrile (50 yl, 5 mM) at pH 6.0 and pH 7.0 was also performed. An assay of the two protein solutions under the above three sets of conditions was also performed after incubation of the protein solutions for 0.5 h with 100-fold molar excess of 2-mercaptoethanol, to check if any papain-sulphenic acid was formed. N o enzymic activity was observed.

Amino Acid Ana1ysi.r and Radioactivity Measurements Protein solutions to be analysed for amino acids and radioactivity were lyophilised and redissolved in water several times in order to remove exchangeable tritium. The final lyophilisation was performed in a hydrolysis tube and the residue dissolved in 6 M hydrochloric acid (2 ml) containing phenol (2 mg). The solution was degassed and the tube sealed under reduced pressure (0.1-mm Hg, 13.3 Pa>.After hydrolysis for 20 h at 110 "C, the hydrochloric acid was removed by rotary evaporation and the residue dissolved in 0.01 M hydrochloric acid (2.5 ml). A part of this solution (1.1 ml) was injected into the long column of the Jeol JLC-5AH amino acid analyser. The eluant from the column was split into two streams. One stream (0.41 ml/rnin) was used for colorimetric analysis, the other (0.42 ml/min) was collected in fractions each of 5-min duration. Aliquots (0.1 ml) from the fractions were added to scintillation solution (6 ml) and counted in a Beckman DPM-100 liquid scintillation system. RESULTS AND DISCUSSION

2-Bromo-2',4'-dimethoxyacetophenone inhibits papain stoichiometrically and irreversibly. Photolysis of a solution of S-(2,4-dimethoxyphenacyl)-papain with a medium-pressure mercury lamp and a filter solution cutting out light below 320 nm, regenerated 76% of the original enzymic activity. Several cycles of inhibition and photolysis were necessary therefore to achieve a high degree of conversion. After three successive cycles followed by inhibition with the bromoketone, the photolysate was chromatographed on an affinity column of Sepharose-aminohexanoylglycyl-L-phenylalanyl-L-arginine [ 191. The inhibited enzyme and denatured protein were rapidly eluted and an exponential gradient of 2 5 m M EDTA with water was used to elute the bound protein (Fig. 1). Reduction of this protein with sodium borohydride

297

gave a product with a similar elution profile (Fig. 2A) to that of the unreduced protein (Fig. 2B) and native papain (Fig. 2C) on the same affinity chromatography column. It is clear that the modified papain and its reduction product have similar but not identical binding properties to those of the native enzyme for the affinity chromatography column, indicating, that the tertiary structure of the native enzyme has been preserved in the modified proteins. When the reduction of the photolysate after affinity chromatography was performed with sodium b ~ r o [ ~ H ] h y d r i dand e the "-labelled protein hydrolysed and submitted to amino acid analysis, radioactivity appeared only in those fractions containing serine (Fig. 3 A). From the radioactivity in these fractions, it was calculated that 0.204 mol of serine/mol of protein had been produced on reduction, assuming there to be no kinetic isotope effect. Although kinetic isotope effects for hydride transfer reactions are known to be low [23,24], a significant kinetic isotope effect is nevertheless expected. The primary kinetic isotope effect for the reduction of carbonyl groups by sodium b ~ r o [ ~ H ] hydride is, however, also known to be insensitive to the reactivity of the carbonyl group, a range of k,/k3, of 3.23 - 5.03 being observed for a range of reactivity of 1 :45 000, the highest value being for the very severely hindered tetramethylpropan-2-one [25]. Since the approach to the carbonyl group in [ASerZ5]papain will be severely hindered by the protein, a primary kinetic isotope effect close to 5 can be expected. This estimate for the primary kinetic isotope effect indicates that one mole of serine/mole of protein was formed on reduction with sodium borohydride. If reduction with sodium b ~ r o [ ~ H ] h y d r i dis e performed immediately after photolysis, the hydrolysed protein contained [3H]cysteic acid (derived from r3H]cysteine) and [3H]serine (Fig. 3 B). This observation confirms that the initial photolysis product is the thioaldehyde which is only slowly hydrolysed to the aldehyde. The considerable amount of enzymic activity which is regenerated on photolysis of S-(2,4-dimethoxyphenacy1)-papain suggests that the conformation required for the Norrish type I1 cleavage is not easily attained and that the more direct cleavage of the C-S bond between the phenacyl group and the sulphur atom is preferred. The possibility that cleavage of the C - S bond of Cys-25 itself had occurred was excluded by performing the photolysis of S-(2,4dimethoxyphenacy1)-papain in [ 3 Hlwater, no [ 3 H I alanine being detected in the hydrolysate of the protein. It seems probable therefore that in the photolysis of S-(phenacy1)-proteins where the thiol group is less severely hindered, the Norrish type 11 cleavage will predominate. It seemed possible that [ASer25]papain could be converted to [ G l ~ ~ ~ I p a p aunder in mildly alkaline

Chemical Mutations of Papain

298

-Q

CHO

-

+

HC02H

/OH

Ctr

OH

-CONH

-CONH

II

2 +

NH-

7 OH

0

Scheme 2 65 r conditions as outlined in Scheme 2. When [ ~ I S e r ~ ~ I p a 60 pain was incubated in [3H]water at pH 9.0 for 3 weeks and the protein hydrolysed, radioactivity was found 5 55 c only in the fractions containing glycine (Fig. 4A). 50 When an aliquot of the solution which had been incubated for 3 weeks was reduced with sodium borogP 45 hydride prior to hydrolysis, radioactivity was again 2 40 found only in the fractions containing glycine from the 35amino acid analyser (Fig. 4B). This control experi5 30 ment excluded the possibility that r3H]glycine was - n formed during hydrolysis of the protein rather than 25 during the incubation period. 20 ( 1 1 1 1 1 1 1 1 1 1 1 The fluorescence emission spectrum of a papain 2 3 4 5 6 7 PH solution at neutral pH is dominated by the contribution of Trp-177 [18]. Moreover the contribution of Fig. 5. The pH-dependence of’ the relative fluorescence intensity at the rmi.vsion maximum (excitation at 286 nm) f o r (ASer25]papain this residue is pH-dependent with a pK, about 8.6 (A) und ( S ~ . s ~ ~ ] p a i (e). ~ u i nThe theoretical curves are for p K , [26]. However papain derivatives in which the thiol values of 4.60 and 5.05 respectively group of Cys-25 is modified, for example by forming a disulphide bond with ethanethiol, show pH-dependent fluorescence emission on a group with pK, about 4.0 [27]. This pK, value has been assigned to the imidazole group of His-159. The pK, value of 8.6 344 for the active enzyme is also considered to be due to 342 the imidazole group of His-159, the change in pK, being due to the interaction between Cys-25 and His340 159 [28]. The pH-dependence of the fluorescence intensity 338 of [Ser25]papain and [ ~ I S e r ~ ~ I p a p aare i n shown in E 336 Fig. 5. The quenching of the fluorescence intensity now E depends on a group with pK, 5.05 in [Ser25]papain A 334 and pK, 4.60 in [ASer25]papain. The fluorescence 332 emission maximum of the two modified forms of papain show similar pH-dependence (Fig. 6) but the 330 accuracy of the data does not justify determining pK, 328 values from them. The interaction which occurs 2 3 4 5 6 7 8 between Cys-25 and His-159 is clearly not occurring PH between Ser-25 and His-1 59. Fig. 6 . The pH-dependence of the tvavelength of the fluorescence The distance between the S atom of Cys-25 in rmiksion mauimum (e-xcitation at 286 nm) .for [ASer”5]papain (A) rind jScr25]papain(e) papain and the nearest N atom of His-259 is 0.34 nm [29]. In [Serz5]papain the 0 atom of Ser-25 would be at a greater distance from the nearest N atom of His-159, assuming no significant change in the protein obtained from the pH-dependent quenching of the tertiary structure, because of the shorter C- 0 bond fluorescence emission can therefore be assigned to the length and slightly different bond angles. The hydroxyl ionisation of His-159. group (which also has a shorter bond length than the [Ser25]Papain and [dSerZ5]papain show no enthiol group) would not therefore be able to interact zymic activity towa.rds N-benzyloxycarbonyl-glycine with the imidazole group of His-159 by hydrogen p-nitrophenol ester or N-benzyloxycarbonyl-L-lysine bonding, the maximum distance for hydrogen bonding p-nitrophenyl ester which are good substrates for between 0 and N atoms being 0.30 nm [30]. The pK, papain [18,31]. This is not surprising since the cata%

I

0)

0)

3

0)

-

P. I. Clark and G. Lowe

lytic activity of the native enzyme is attributed to the critical alignment and interaction of the functional groups in the side chains of Cys-2.5, His-1 59 and Asn175. The lack of interaction between Ser-2.5 and His159 in [Ser25]papain as well as the probable necessity of a charge relay system to form a serine protease [32] are sufficient to cause the complete loss of catalytic ability. It is clear therefore how a single point mutation could lead to complete loss of enzymic activity. [Ser25]Papain has similar binding properties to those of native papain (vide supra) and provides the opportunity therefore of determining by X-ray crystallography the structure of a stable modified enzymesubstrate complex from which the structure of the true papain-substrate complex could be deduced. There are therefore now available stable analogues of the papain-substrate complex, the tetrahedral intermediate (papain with an N-acyl-aminoacetaldehyde) [33] and the acyl-enzyme (papain with N-acyl-aminoacetonitrile) [34- 361. A detailed knowledge of the structure of these three complexes together with that of the free enzyme should provide valuable insight into the structural changes that occur in both enzyme and substrate along the reaction pathway.

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299 11. Polgar, L. B Bender, M . L. (1966) J. Am. Ckem. Soc. 88, 31 53- 3154. 12. Polgar, L. & Bender, M. L. (1967) Biochemistry, 6,610-620. 13. Woodward, R. B. (1970) Hunbury Memorial Lecture of Roy. Pharm. Soc. quoted by Heusler, K. (1972) in Cephalosporins and Penicillins (Flynn, E. H., ed.) p. 274, Academic Press, New York. 14. Cheney, J., Moores, C. J., Raleigh, J. A., Scott, A. I . & Young, D. W. (1974) J . C. S. Chem. Commun. 47-48. I S . Brandt, A., Bassignani, L. & Re, L. (1976) Tetrahedron Lett., 3975-3978. 16. Kimmel, J. R. & Smith, E. L. (1954) J. B i d . Chem. 207, 515531. 17. Blumberg, S., Schechter, 1. & Berger, A. (1970) Eur. J. Biochem. 15,97-102. 18. Lowe, G. & Whitworth, A. S. (1974) Biochem. J . 141,503-515. 19. Reference deleted. 20. King, L. C. & Ostrum, G. K. (1964) J. Org, Chem. 29, 34593461. 21. Glazer, A. N. & Smith, E. L. (1961) J . Biol. Chenz. 236, 29482951. 22. Mitchel, R. E. I., Chaiken, I. M. & Smith, E. L. (1970) J . Biol. Cliem. 245, 3485- 3492. 23. Swain, C . G., Wiles, R. A. & Bader, R . F. W. (1961) J . A m . Cheni. Soc. 83, 1945 - 1950. 24. Wigfield, D . C. & Phelps, D. J. (1972) Cun. J . Chem. SO, 388- 394. 25. Pasto, D. S. & Lepeska, B. (1976) 1.Am. Chem. Sue. 98, 1091 1095. 26. Shyterman, L. A. E , & d e Graaf, M. J. M. (1970) Biochem. Biophys. Acta, 200, 595 - 597. 27. Bendall, M. R. & Lowe, G. (1976) Eur. 1. Biochem. 65, 481 491. 28. Drenth, J., Swen, 13. M., Hoogenstraaten, W. B Sluyterman, L. A . E . (1975) Proc. Kon. Ned. Akad. Wet. Ser. C, B i d . Med. Sci. 78, 104- 110. 29. Drenth, J., Jansonius, J. N.. Koekoek, R., Sluyterman, L. A. E . & Wolthers, B. G. (1970) Phil. Trans. Roy. Soc. Lond. Ser. B, Bid. Sci. 257, 231 -236. 30. Speakman, J. C. (1975) The Hydrogen Bond, The Chemical Society Monograph for Teachers No. 27, p. 12. (1973) Eur. J . Biochcm. 31. I3ollaway, M. R. & Hardman, M . .I. 32,537 - 546. 32. Blow, D. M., Birktoft, J. J. & Hartley, B. S. (1969) Nature (Lond.) 221, 337 - 340. 33a.Clark, P. I . , Lowe, G. & Nurse, D. (1977) J . C. S . Chcm. Cummun. 451 -453. 33 b.Bendal1, M. R., Cartwright, 1. L., Clark, P. I., Lowe, G. & Nurse, D. (1977) Eur. J . Biochem. 79, 201 -209. 34. Lucas, E. C. & Williams, A. (1969) Biochemlrtry, 8, 51255135. 35. Lowe, G . & Yuthavong, Y. (1971) Biochem. J . 124, 307- 115. 36. Sluyterman, L. A . K . & Wijdenes, J. (1973) Biochim. Biophj,.s. Acta, 302, 95 - 101 and 321, 697 - 699.

P. I. Clark. Department of Chemistry, University College of Swansea, University o f Wales, Singleton Park, Swansea, Great Britain, SA2 8PP G . Lowe*, The Dyson Perrins Laboratory, University of Oxford, South Parks Road, Oxford, Great Britain, OX1 3QY

*

T o whom correspondence should be addressed.

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Conversion of the active-site cysteine residue of papain into a dehydro-serine, a serine and a glycine residue.

Eur. J. Biochem. 84, 293-299 (1978) Conversion of the Active-Site Cysteine Residue of Papain into a Dehydro-serine, a Serine and a Glycine Residue Pe...
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