Conformation of allantoicase in aqueous solution

Conformation of allantoicase in aqueous solution

BIOCHIMICA ET BIOPHYSICA ACTA 393 BBA 35997 CONFORMATION OF ALLANTOICASE IN AQUEOUS SOLUTION E. J. 'S-GRAVENMADE*, C. VAN D E R D R I F T AND G. I)...

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BIOCHIMICA ET BIOPHYSICA ACTA

393

BBA 35997 CONFORMATION OF ALLANTOICASE IN AQUEOUS SOLUTION

E. J. 'S-GRAVENMADE*, C. VAN D E R D R I F T AND G. I). VOGELS

Department of Biophysical Chemistry and Department of Biochemistry, University o.f Nijmegen, Nijmegen (The Netherlands) (Received J u n e 25th, 1971)

SUMMARY

I. The separation of o.9-S and IO.8-S allantoicase with the aid of a 2H~O-H20 gradient was described. The resulting preparations were subjected to sedimentation equilibrium, optical rotatory dispersion (ORD), circular dichroism (CD) and infrared studies. 2. The molecular weight of o.9-S allantoicase was determined to be about I . I . I O a g/mole in studies on the sedimentation behavior, the metal content and amino acid composition. The molecular weight of IO.8-S allantoicase was about 15. 4 • lO 4 g/mole. 3. Optical rotatory dispersion, circular dichroism and infrared studies indicated that both molecules contain a-helix, fl conformation and random coil. A Cotton effect at 418 nm was ascribed to the asymmetric binding of Mn 2÷ to the enzyme. Competitive inhibitors decreased the absorption and circular dichroism bands at about 280 nm and 418 nm. These phenomena suggested that the aromatic groups m a y play an essential role in the binding of substrates and inhibitors by the Mn~+-enzyme complex. 4. Comparison of a-helical contents of metalloallantoicases showed t h a t the enzymes with low helical contents exhibited high enzymic activities. 5. The nearly identical physicochemical behavior and specific enzymic activity of o.9-S and IO.8-S allantoicase indicated that they are very similar in structure and conformation.

INTRODUCTION

Allantoicase (allantoate amidinohydrolase, EC 3.5.3.4) plays an essential role in the metabolism of purines in animals 1 and microorganisms 2. The enzyme catalyzes the reversible conversion of allantoate (diureidoacetate) into (--)-ureidoglycolate and urea, and of (+)-ureidoglycolate into glyoxylate and urea. Previous papers dealt with the non-enzymic hydrolysis of allantoate 3 and ureidoglycolate 4, and the purification 5, " Present address: L a b o r a t o r y for Materia Technica, State University of Groningen, Groningen (The Netherlands).

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E.J. 'S-GRAVENMADEet al.

the specificity of binding subsites ~ and the role of metal ions in the catalytic action and stability 7 of the enzyme from Pseudornonas aeruginosa. This paper deals with sedimentation, optical rotatory dispersion (ORD), circular dichroism (CD) and infrared studies on two enzymically active components of allantoicase. Special attention will be given to the spectropolarimetric data obtained by studies on various metalloallantoicases and on the effect of pH and inhibitors on the aromatic and intrinsic Cotton effects.

EXPERIMENTAL

Materials Allantoicase was isolated from Pseudomonas aeruginosa and purified as given previously 5. The IO.8-S and o.9-S components of allantoicase were separated by centrifugation at 200 ooo × g for 7 h at o ° to sediment the io.8-S component. The supernatant contained chiefly ( > 9 5 % ) o.9-S allantoicase and the upper two-third part was used in the study on this component. The sedimented material was resuspended in a small amount of water and separated from the larger aggregates by preparative ultracentrifugation using a linear 2H20-H20 gradient with concentration limits from 99.8% 2H20 (d = 1.1o5) to 5% 2H~O (d = 1.oo5). The separated IO.8-S material was dialyzed against 5 mM triethanolamine-HC1 buffer (pH 7.6), containing lO-4 M MnC12, to remove ~H20. Metal-free enzyme was prepared according to VAN DER DRIFT AND VOGELS7. Mn 2+-, Cd 2÷-, Cos+- and Cu~+-allantoicases were prepared by incubation of metalfree enzyme (about 50/~g) and the appropriate metal ion in a final concentration of 4.5" lO-3 M at pH 5.85 for IO rain at 3 o°. This time was enough to saturate the enzyme. Excess of metal ions was removed by exhaustive dialysis against o.oi M Tris-HC1 buffer (pH 7.5)- 54Mn~÷-allantoicase was prepared in the same way by the addition of 3.8 #C 54Mn2÷ per ml. 54Mn2÷was obtained as 54MnCl~ (O.lO3 mC/ml) from the Radiochemical Centre, Amersham, England. N-Carbamoyl-(R)-asparagine was synthesized from the corresponding amino acid and potassium cyanate according to the method of STARKAND SMYTH8. Throughout this paper the symbols S and R are used according to the nomenclature of CAHN et al. 9. (+)- and (--)-ureidoglycolate are (R)- and (S)-ureidoglycolate, respectively. Methods The optical rotatory dispersion and circular dichroism spectra were measured as given previously 1°. The circular dichroism and rotatory dispersion data of the multiple Cotton effect in the aromatic absorption region and the extrinsic Cotton effect at 416 nm were interconverted with the aid of the simplified Kronig-Kramers' transformation n. Protein concentrations were determined according to LowRY et al. 12 using bovine serum albumin as a standard and from the absorbance at 280 nm (A~s~mnm= I is equivalent to 315 /~g allant0icase per ml measured according to the method of LOWRY et al.12). Ultraviolet absorption spectra were measured with a Beckman DK-2A, ratio recording spectrophotometer. Atomic absorption spectroscopy was performed with a Biochim. Biophys. Acta, 251 (1971) 393-4o6

CONFORMATION OF ALLANTOICASE

395

Techtron Atomic Absorption Spectrophotometer Model AA-loo. 54Mnz+ was measured with a Philips scintillation detector P W 4119. Infrared spectra were recorded on the EPI-G2 Hitachi infrared spectrophotometer. Solutions in ~H~O were prepared with a lyophilized enzyme preparation, dried overnight in vacuo over P2Oa at room temperature. The solutions were measured in cells with AgC1 windows. Infrared spectra of the solid enzyme were recorded using a thin film cast on an AgC1 plate. The solution was deposited on a crystal plate of AgC1 and evaporated to dryness at room temperature in a desiccator over P2Q, first under atmospheric pressure and thereafter in vacuo overnight. Molecular weights of allantoicase were estimated according to the method of Archibald adapted to absorption techniques 13 and from studies of the sedimentationdiffusion equilibrium. The Beckman Model E analytical ultracentrifuge was used in combination with the electronic scanning system described by VAN Es AND BONT14. The sedimentation runs were performed at 20 ° in 5 mM triethanolamine-HC1 buffer (pH 7.6), containing lO -4 M MnC12, with rotor speeds of 25 980 and I I 272 rev./min for o.9-S and IO.8-S allantoicase, respectively. The partial specific volume was assumed to be 0.74 cm~/g. Amino acid analysis was performed according to the principles of SPACKMAN et al. 15 with an amino acid analyzer, extended with the automatic apparature as described by GERDING AND PETERS16. Hydrolysis was performed in 6 M HC1 (Merck suprapur, diluted with water) during 22 h. Corrections for loss in hydrolysis of threonine and serine were made according to DowNs AND PIGMAN17. Cysteic acid was determined separately according to the method of MOORETM. The tyrosine/tryptophan ratio was estimated according to the method of BENCZE AND SCHMIDTM. RESULTS

o . 9 - S and x o . 8 - S allantoicase

From sedimentation velocity studies it appeared that the purified enzyme preparation 5 contained about 75% IO.8-S, 15% o.9-S and i o % small particles and larger aggregates. The IO.8-S and o.9-S types of allantoicase were separated by centrifugation as given in EXPERIMENTAL. Sedimentation studies showed that the purified IO.8-S allantoicase was a single component and that o.9-S allantoicase did not contain IO.8-S allantoicase or larger aggregates, but still contained a small amount of another component with a low molecular weight, which was not further investigated. The molecular weights of o.9-S and IO.8-S allantoicase were estimated according to the Archibald method 18 and from studies of the sedimentation-diffusion equilibrium, and amounted to I I ooo + 300 and 154000-1-8000 g/mole, respectively. This suggests that one io.8-S particle is composed of about 14 o.9-S components. Metalloallantoicase preparations were submitted to atomic absorption spectroscopy. From the amounts of metal found and the amount of enzyme calculated on dry weight basis it followed that I / , m o l e Mn 2+ was present in I I 400 ± 6o0/*g protein. For Cu 2+-, Zn 2+- and Co~+-allantoicases this value amounted to I / , m o l e metal ion in I i IOO -4- 600, I I 40o -1- 600 and I I 500 4- 600 #g protein, respectively. I f the metal content was determined by measuring ~4Mn~+ in 54Mn2+-allantoicase a value of I/*mole Mn 2+ in IO 800 #g protein was found. Biochim. Biophys. Acta, 251 (1971) 393-4o6

396

E . j . 'S-GRAVENMADEet al.

Both IO.8-S and o.9-S allantoicases exposed an enzymic activity towards allantoate (specific activity 59 ° and 69o units per mg protein, respectively). However, we cannot exclude the possibility, that interconversion of the two components occurred during the enzymic test. Purified o.9-S allantoicase (about 3oo #g/ml) dissolved in 5 mM triethanolamine HC1 buffer (pH 7.6), containing IO-~ M MnC12, aggregated to IO.8-S and larger aggregates on standing for 3 weeks at 4 °. Sedimentation studies of the resulting solution showed the presence of about 50°/0 o.9-S allantoicase, 45% io.8-S allantoiease and 5% larger aggregates. On further incubation of this solution for 3 days at 4 ° in the presence of 2% sodium dodecyl sulfate, the IO.8-S allantoicase and the larger aggregates dissociated fully into o.9-S allantoicase. Small particles were also formed on incubation of a solution, containing about 300 #g IO.8-S allantoicase per ml in the presence of 8 M urea or 5 M guanidine. HC1 for 24 h at room temperature.

Amino acid analysis The complete amino acid analysis of the enzyme was performed with a material which contained about 3O~o o.9-S and 70% IO.8-S allantoicase. Since both components are about equally active in enzymic tests and can be interconverted it was assumed that the amino acid composition (Table I) was the same. Assuming the presence of one residue of cysteine per molecule of enzyme and normalizing the resulting values to the nearest integer, we found the following amino acid ratio : ASpl0 Thr~ Ser 5 GlUlo Pr% Glyl0 Alan(12 ) Val 8 Mete(3) I1% Leu s Tyr 2 Trp3 Phe a Lysa His s Arga Cys 1. Glucosamine (1.6 mole °/o) was found as a peak emerging between Tyr/Phe and Lys on the short column. The identity and quantitative amount of this component was determined by analysis of a calibration mixture, to which a known quantity of glucosamine was added. The amount of ammonia found (15 mole %) may be ascribed to the presence of Asp(NH2) and Glu(NH2). On adding the weight of the amino acid residues and the weights of one manganese atom and one (two) glucosamine molecule(s), the molecular weight was calculated to be between io 9oo and I I 2oo, which is in good accordance with the molecular weights obtained from sedimentation equilibrium or sedimentation studies (II ooo ± 300).

Optical rotatory dispersion and circular dichroism of o.9-S allantoicase The spectra (around 200-600 nm) of the o.9-S component of allantoicase were measured in aqueous media buffered at slightly alkaline pH. The results are shown in Fig. I. From the shallow trough at 232 nm, the pronounced peak at 206 nm, and the cross-over point at about 220 nm it was concluded that the major conformation of the o.9-S component is a/~ structure 2°. The CD spectrum shows a major band with a minimum at about 218 nm and a cross-over point at 204 nm (Fig. 2). These characteristic wavelengths correspond with those observed in various proteins and synthetic polypeptides, for which the presence of the/~-type conformation had been established by a correlation of optical rotatory 21, infrared spectroscopic 22, and X-ray diffraction data 23. The aromatic Cotton effect at 280 nm (Fig. i) is a multiple one (Fig. 3, Curve I) and was absent in solutions containing about 300/~g o.9-S allantoicase per ml 8 M urea. Biochim. I3iophys. Acta, 25I (I97 I) 393-406

397

CONFORMATION OF ALLANTOICASE [ m ' ] ( d e g . c m % declmole-l.10 "~) 0

250 t I

t

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-SO i -601 [m'

(deg.emZ.aecimole

-1)

Fig. I. Optical r o t a t o r y dispersion of o.9-S allantoicase in 5 m M t r i e t h a n o l a m i n e - H C 1 buffer (pH 7.7), c o n t a i n i n g lO -4 M MnCI~. T h e c o n c e n t r a t i o n s of allantoicase were o.21, 0.35 a n d 1.2 m g / m l in t h e t h r e e s u b s e q u e n t w a v e l e n g t h regions. T h e optical p a t h w a y w a s io.o m m in t h e m e a s u r e m e n t p e r f o r m e d in t h e u l t r a v i o l e t region a n d 50.0 m m in t h e visible region. C u r v e s i a n d 2 in t h e n e a r u l t r a v i o l e t region refer to allantoicase in t h e a b s e n c e a n d presence of 2 m M h y d a n toate. C u r v e s I a n d 2 in t h e visible region were m e a s u r e d a t p H 7.7 a n d 4.6 (o.i M acetic a c i d a c e t a t e buffer, w i t h o u t Mn~+), respectively. C u r v e 3 w a s c a l c u l a t e d f r o m C u r v e i, e l i m i n a t i n g t h e 416 - a n d 2 8 o - n m C o t t o n effects.

The Cotton effect at 416 nm (Fig. i) is a positive one as became apparent from CD measurements. It may be caused by the Mn2+-enzyme complex present in o.9-S allantoicase. The Cotton effect decreased about lO% on incubation for 5 h at 4 ° in the presence of 0.02 M EDTA, while the pH was maintained at 7.6 by addition of o.I or o.oi M NaOH. This decrease is time-dependent. Under the same conditions the absorption band at 416 nm (Fig. 4, Curve I) diminished 3, 6 and 18% after an incubation period of 20, 60 and 27 ° min at 4 °, respectively. These phenomena are consistent with the time-dependent removal of Mn 2+ and the decrease of enzymic activity in the Biochim. Biophys. Acta, 251 (1971) 3 9 3 - 4 o 6

398

E. J. 'S-GRAVENMADE el al.

[ O'] (deg, cm 2. decirnole-l-10 -3 )

[ e ' ] (deg • c r n 2 . d e c i m o l e - i )

+4-

+100

+2-

-

+80

1

+60

0

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+ 40

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A(nm)

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, 250

260

270

- - 1280 --

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310

X(nm)

Fig. 2. Circu lar d i c h r o i s m of o.9-S a l l a n t o i c a s e a t p H 7.7. The c o n c e n t r a t i o n of a l l a n t o i c a s e w a s o . i i m g / m l in 5 mM t r i e t h a n o l a m i n e - H C 1 buffer, c o n t a i n i n g i o 4 M MnCI~. The o p t i c a l p a t h w a y s were io .o a n d I.O m m . Fig. 3. Circu lar d i c h r o i s m s p e c t r u m of side c h a i n c h r o m o p h o r e s of o.9-S a l l a n t o i c a s e a t p H 7-7 (5 mM t r i e t h a n o l a m i n e - H C 1 buffer, c o n t a i n i n g lO -4 M MnCI~). C u r v e I a n d 2 refer t o o.9-S all a n t o i c a s e in t h e a b s e n c e or p r e s e n c e of i o 4 M N - c a r b a m o y l - ( R ) - a s p a r a g i n e , r e s p e c t i v e l y . Conc e n t r a t i o n of t h e e n z y m e w a s o.61 m g / m l . The o p t i c a l p a t h w a y s w e re 2o.o a n d i o . o ram.

presence of 1.25 mM E D T A observed b y VAN DER DRIFT AND VOGELS?. Half of the original activity was lost in IO h on incubation at p H 7.8 and 3 o°. No detectable optical activity accompanied the small absorption bands between 500 and 600 nm (Fig. 4, Curve I). These bands were not observed when the solution was incubated in 0.02 M E D T A for 5 h, which indicates t h a t they too m a y be ascribed to Mn2+-eomplex bands.

absorbance 1"01

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Fig. 4. A b s o r p t i o n s p e c t r a of o.9-S a l l a n t o i c a s e a t p H 7.7 (Curve I) a n d 4.6 (Curve 2). C urve 3 refers to o.9-S a l l a n t o i c a s e in t h e p r e s e n c e of o.i M glycol a t e . The s a m e buffers were used as g i v e n in Fig. I. o.3o m g / m l a l l a n t o i c a s e was present. The o p t i c a l p a t h w a y w a s i o.o ram.

Biochim. Biophys. Acta, 251 (1971) 393-406

CONFORMATION OF ALLANTOICASE

399

[ m'] (deft. cm2.deeimole-1.10 "~ ) +8

*6

+4

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250

300

350 X(nm)

Fig. 5. Optical r o t a t o r y dispersion of io.8-S allantoicase in 5 mM triethanolamine-HC1 buffer (pH 7.7), containing lO -4 M MnC12. The c o n c e n t r a t i o n of IO.8-S allantoicase was 2.8 (Curve I) or 4.7 (Curve 2) mg/ml.

Optical rotatory dispersion and circular dichroism of xo.8-S allantoicase The ORD curve of IO.8-S allantoicase is shown in Fig. 5. No absorption band and Cotton effect were observed at 416 nm for this component. Above 260 nm this compound exposed a positive residual specific rotation, which became less positive on a 2-fold dilution. The reduced mean residue rotation at 320 nm of solutions containing 4.7, 2.8 or 0.3 mg IO.8-S allantoicase per ml in 5 mM triethanolamine-HC1 buffer (pH 7.6), containing lO -4 M MnC12, amounted to + 4 0 0 °, + 1 5 °0 or - - 6 o o °, respectively. Comparison of the ORD spectra of o.9-S and IO.8-S allantoicase (Figs. I and 5), and of the CD curves (Figs. 2 and 6) revealed only minor differences. The location and depth of the minimum at 222-223 nm (compare Fig. 6) suggested that IO.8-S allantoicase contains a somewhat higher amount of a-helical structure than o.9-S allantoicase. [ o' ] (d,g.~m2.d~imol~-1.I0-~)_ _ +4*20 -2-4-6-S

x(nm) Fig. 6. Circular dichroism s p e c t r u m of io.8-S allantoicase at p H 7.7. The concentration of Io.8-S allantoicase w a s 82/~g/ml in 5 mM t r i e t h a n o l a m i n e - H C 1 buffer, containing io-* M MnCI~. The optical p a t h w a y s were io.o a n d i.o m m .

Biochim. Biophys. Acta, 25I (I97 I) 393-406

400

E.J.

'S-GRAVENMADE

et al.

Influence of preincubation at pH 4.6 on the ORD and CD of o.9-S allantoicase In a study on the enzymic activity of allantoicase VAN 9ER DRIFT AND VOGELS7 found that preincubation of the enzyme at pH 4.6-4.7 reduced the activity measured at pH 7.8 to about 5O~o of the original value. On repeating this experiment the activity was reduced to about 25~o activity. This might be accompanied by irreversible changes in the conformation or by a dissociation into subunits. Comparison of the ORD and CD data shows that preincubation of o.9-S allantoicase at pH 4.6 results in a change of conformation. The maximum of the ORD curve shifted from 206 to 200 nm and a shoulder appeared at 21o-215 nm which indicates that the amount of a-helical structure had increased during preincubation. The same conclusion can be drawn from the shift of the 218 219-nm CD band (ascribed to a fl conformation) with a minimum o f - - 5 8 0 0 degrees, cm 2. dmole -1 to 22o-221 nm specific for a-helical conformation with a minimum o f - - 7 8 0 0 degrees.cm2.dmole -1. The calculated amounts of a-helix present under these circumstances are summarized in Table I. Preincubation at pH 4.6 for more than 15 rain resulted in the formation of a precipitate which could not be dissolved by adjusting the pH to 7.6. No dissociation into smaller particles was observed after preincubation of o.9-S allantoicase at pH 4.6 for 15 rain. Measurements of the absorption spectra of o.9-S allantoicase in 5 mM triethanolamine HC1 buffer (pH 7.6), containing io 4 M MnC12, and in o.I M acetate buffer (pH 4.6) indicated a shift of the absorption band at 416 to 393 nm (Fig. 4)The absorption bands between 500 and 600 nm present at pH 7.6 were not observed in the solution at pH 4.6. These shifts in the spectra were instantaneous and reversible. The absorption band at 393 nm was not accompanied by a detectable CD band as opposed to the Cotton effect observed for the absorption band at 416 nm (Fig. I). TABLE I AMINO

ACID

ANALYSIS

OF ALLANTOICASE

Amino acid

izmoles/sample

Moles/ioo moles

Asp Thr Ser Glu Pro Gly Ala Val Met Ile Leu Tyr Phe Cys* Trp** Lys His Arg Tyr Phe

2.1o 1.o9 o.97 2.o 7 0.99 2.00 2.37 1.61 o. 51 0.80 1.59 0.62 0.78 0.20 o.61 0.98 0.48 0.84 o.65 0.80

lO.17 5.28 4.73 lO.O4 4.78 9.69 11.5o 7.81 2.49 3.86 7.73 3.Ol 3.79 0.99 2.95 4.78 2.34 4.07 3.16 3.87

" Cys determined as cysteic acid. *" Trp determined b y the m e t h o d of BENCZE AND SCHMIDTM.

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CONFORMATION OF ALLANTOICASE

401

The a b o v e results i n d i c a t e t h a t the h a l v i n g of the e n z y m i c a c t i v i t y d u r i n g p r e i n c u b a t i o n at p H 4.6 was not a c c o m p a n i e d b y a r e m o v a l of Mn 2+ from the e n z y m e or b y a dissociation of e n z y m e molecules. However, we o b s e r v e d an irreversible increase in t h e a m o u n t of a-helical s t r u c t u r e a n d a reversible a l t e r n a t i o n in the m o d e of Mn ~+ b i n d i n g a t p H 4.7-

Inhibitor-induced changes in the ORD of o.9-S allantoicase T h e i n t e r a c t i o n of N - c a r b a m o y l - ( R ) - a s p a r a g i n e (pK, = 4.o3), glycolic acid (pKt = 2.59) a n d h y d a n t o i c acid (pKi = 2.51) w i t h the e n z y m e was s t u d i e d b y m e a n s of s p e c t r o p h o t o m e t r y a n d s p e c t r o p o l a r i m e t r y . The positive Cotton effect at 418 n m d i m i n i s h e d a b o u t IO% on a d d i t i o n of the optical inactive substance h y d a n t o a t e (Fig. 7) or on a d d i t i o n of N - c a r b a m o y l - ( R ) - a s p a r a g i n e , one of t h e most p o t e n t inhibitors found s. U p o n a d d i t i o n of an excess (o.I M) g l y c o l a t e to o.9-S allantoicase t h e i n t e n s i t y of the 416 - a n d 278-nm a b s o r p t i o n b a n d s decreased b y 25~o a n d IO%, r e s p e c t i v e l y (compare Fig. 4). [0'] ( d e g . c m g ' d e c l m o l e +150 -

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*100

+ 50

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-50

360

54o

400

420

440

460

X(nm) Fig. 7- Circular dichroism spectra of o.9-S allantoicase in the absence (Curve i) and presence of 2 mM tlydantoate (Curve 2) and 2 mM N-carbamoyl-(R)-asparagine (Curve 3) at pH 7.6. Tile concentration of o.9-S allantoicase was o.35 mg/ml in 5 mM triethanolamine-HC1 buffer. The optical pathway was IO.O mm.

Since t h e CD s p e c t r a of allantoicase, m e a s u r e d in the presence a n d absence of h y d a n t o a t e were equal in t h e w a v e l e n g t h region 215 to 24o nm, it seems u n l i k e l y t h a t the i n h i b i t o r i n d u c e d a gross change in t h e p r o t e i n conformation. I n the region of side chain c h r o m o p h o r e s of t h e enzyme, the CD is resolved into several positive b a n d s at 258, 275, 282, 288 a n d 294 nm. The m a g n i t u d e s of the b a n d s at 282, 288 a n d 294 n m were r e d u c e d to a b o u t h a l f of the original value in the presence of IO 4 M N - c a r b a m o y l - ( R ) - a s p a r a g i n e , whereas t h e b a n d at 258 n m increased a b o u t 2-fold (Fig. 3)U n d e r the conditions a p p l i e d a b o u t 4 o % of the e n z y m e will be b o u n d to the inhibitor.

Biochim. Biophys. Acta, 251 (1971) 393-406

E.J. 'S-GRAVENMADEet al.

402

This result strongly indicates that the Cotton bands at 294, 288, 282 and perhaps also the one at 275 nm are absent in the enzyme-inhibitor complex. Hydantoate exerted a similar effect and the Cotton effect for the enzyme material at about 280 nm were greatly decreased in the presence of this inhibitor (Fig. I). In the case of N-carbamoyl(R)-asparagine a correction was made for the intrinsic rotation of this compound. Performing the same experiment with N-carbamoyt-(S)-asparagine (lO -4 M) we observed no change of the CD and optical rotation in the region 25 ° to 45o nm. The interaction between allantoicase (E) and inhibitors (I) can be analyzed by measuring the change of the levorotation at 4oo nm. Assuming that this change for any given molar ratio of the inhibitor to allantoicase is directly proportional to the fraction of allantoicase which is associated with the inhibitor molecule, the following equation is obtained: k

i

zla

[EI]

K~

i

[E]t[I] + EE]t

in which IE]t = IEI + EEl] and k is a constant. A linear relationship has been obtained between Aa calculated and Aa observed.

ORD and CD of metal-free enzyme and some metalloallantoicases ORD and CD measurements were performed with the metal-free enzyme and with the enzyme, in which Mn 2+ was replaced by Cd 2+, Cu 2+ and Co2+. Since only small amounts of these enzymes were available, reliable ORD and CD spectra could be measured only in the wavelength region from about 200 to 240 nm. The differences in the ORD spectra were small, but the CD spectra showed differences in the location of the minima of the curves (Fig. 8). 0 (de, :.10 -1) *0.2

~0.2

~\\

i\ 2",.3,~

-0.4

~' ' x

F

]I /

"N:[-" /

- 0.6

-08

200

220

240

X(nm)

F i g . 8. C i r c u l a r d i c h r o i s m o f m e t a l - f r e e e n z y m e a n d s o m e m e t a l l o a l l a n t o i c a s e s . C u r v e i r e f e r s t o m e t a l - f l e e e n z y m e , C u r v e 2 r e f e r s t o C d 2+- a n d M n * + - a l l a n t o i c a s e a n d C u r v e s 3 a n d 4 r e f e r t o Co 2+- a n d C u 2 + - a l l a n t o i e a s e , r e s p e c t i v e l y . T h e c o n c e n t r a t i o n o f t h e m e t a l - f r e e e n z y m e a n d t h e m e t a l l o a l l a n t o i c a s e s w a s a b o u t 5oltg/ml. T h e o p t i c a l p a t h w a y w a s IO.O r a m . All m e a s u r e m e n t s w e r e p e r f o r m e d w i t h e n z y m e s o l u t i o n s i n o . o I M T r i s - H C 1 b u f f e r , p H 7-5.

The metal-free enzyme exhibited negative CD bands at about 208 and 222 nm due to (¢r°-~-) and (nl-7c) amide transitions, respectively. These ellipticity bands and the distinct notch at 2~5 nm suggested the presence of a-helical structure. From the reference values of poly-a,L-glutamic acid (Table I) it appeared that the metal-free enzyme contained about 35% a-helix. The Co2+-enzyme also exhibited a notch at 215 nm, but less distinct than the one that is characteristic for the a-helical structure Biochim. Biophys. Acta, 251 (1971) 3 9 3 - 4 o 6

CONFORMATION OF ALLANTOICASE

403

in s y n t h e t i c p o l y a m i n o acids 2~. T h e Cu2+-enzyme exposed a b a n d at 222 n m an d only a v e r y small shoulder at a b o u t 21o nm. T h e Cd 2+- a n d Mn2+-enzyme showed a m i n i m u m at a b o u t 219 nm, which indicates t he presence of still a n o t h e r ordered s t r u c t u r e t h a n the a-helix. Mn 2+- and Cd2+-allantoicases show t h e highest e n z y m i c a c t i v i t y , followed by t h e Cu 2+- a n d Co2+-enzymes, whereas t h e metal-free e n z y m e is w i t h o u t a c t i v i t y 7. T h e a m o u n t of a-helical s t r u c t u r e seems to increase in t h e same order. Q u a n t i t a t i v e m e a s u r e m e n t s of th e a m o u n t of a-helical s t r u c t u r e in t h e various metalloallantoicases could not be p e r f o r m e d because t h e c o n c e n t r a t i o n s of t h e e n z y m e were not k n o w n exactly.

Infrared absorption of o.9-S and xo.8-S allantoicase I n f r a r e d s p e c t r a of o.9-S and IO.8-S allantoicase were m e a s u r e d in ~H~O and in solid state. Th e a m id e I b a n d is c h a r a c t e r i z e d b y a peak near I632 cm -1 an d a w e a k shoulder at I65o cm -1. T h e first b a n d indicates th e presence offl structure, t h e second b a n d the presence of a-helix s t r u c t u r e 25. I n t e r p r e t a t i o n of t h e absorption in t h e amide I I region (15oo-155o cm -1) in 2H20 solutions is m o r e difficult, because of the overl a p p i n g of ionized c a r b o x y l groups in t h e solution. MIYAZAWA26 proposed the use of t he a m i d e V b a n d for b e t t e r diagnosis especially when various c o n f o r m a t i o n s coexist, as is t r u e for allantoicase. T h e a m id e V b a n d of allantoicase is ch ar act er i zed b y w e a k peaks at 61o and 698 cm -1, a n d a shoulder at 650 cm -1. These characteristics are i n d i c a t i v e ~v of t h e presence of disordered structure, fl s t r u c t u r e and a-helix s t r u c t u r e in allantoicase. This result is in accordance with t h e results o b t a i n e d in studies on t he O R D an d CD. Analysis of the ORD of o.9-S and io.8-S allantoicase T h e e s t i m a t i o n of the a m o u n t of a-helical c o n f o r m a t i o n of o.9-S an d IO.8-S allantoicase was p e r f o r m e d b y m e a n s of three different methods. T h e results are r e p r e s e n t e d in Table II. In all instances poly-a,L-glutamic acid was used as a reference TABLE II R O T A T O R Y CONSTANTS OF o . 9 - S AND I o . 8 - S

ALLANTOICASE IN A Q U E O U S S O L U T I O N

d llantoicase

pH bo* % (from b0) A*{a,p) (193)

A*(a,o}2~5 H'193

H*~25 [O']'220 %

0.9 S

0.9 S

7.6 -- 123 25 + 545 -- 445 35 19 --5700 25

4.6 -- 17o 32 + 677 -- 542 39 25 --7800 31

Poly-a,L-glutamic acid Io.8 S 7.6 --

4.3 630 IOO**

7500 3°

+

7.1 5° O ~**

+ 2900 -2050

75° -- 60

--32000 ioo

+31oo o

* b0 M O F F I T T - - Y A N G 28 rotatory parameter ; A (a,e)(193)and M(a,o)22~S H E C H T E R AND B L O U T 29"31 rotatory parameters; H193 and H,25 helical contents calculated from the parameters at 193 nm and 225 nm, respectively; [0']22o corrected mean residual specific ellipticity at 220 nm. ** Assumed to be lOO% helical. *** Assumed to be 0% helical.

Biochim. Biophys. Acta. 25I (I971) 393-406

404

E.J. 'S-GRAVENMADEet al.

compound and it was assumed that a linear relationship exists between the features measured and the a-helix content. Application of the method of MOFFITT-YANG2s and the two-term Drude equation by SHECHTERAND BLOUT29-31 did not yield linear plots for allantoicase due to the presence of the extrinsic and aromatic Cotton effects. Therefore, we measured the CD spectra of these chromophores and calculated their contribution to the rotatory dispersion by application of the Kronig-Kramers' transformation. The rotatory values obtained were substracted from the original rotatory dispersion spectra and in this way, linear plots were obtained. No linear relation was found between H193 and H225 in a plot according to SHECHTER AND BLOUT29-al. This proves that structures other than a-helix and random coil are present in the enzyme, which is in accordance with the infrared and spectropolarimetric data. DISCUSSION

The relationship between the secondary, tertiary and quaternary structure of allantoicase and the ability of the enzyme molecules to function catalytically was one of the subjects under investigation. For this reason the amino acid composition, infrared spectra, ORD and CD spectra, and the behavior in the ultracentrifuge were studied. From ultracentrifugation studies it appeared that at least two enzymically active types of allantoicase are present with sedimentation coefficients o. 9 S and lO.8 S. Both types exhibited about the same specific activity towards allantoate, the same infrared spectra in eH,,O and in solid state, and their ORD and CD spectra differed only slightly. These observations suggest a very close relationship between the two forms and they could be converted into each other under certain conditions. The characteristic ORD, CD and infrared features of o.9-S and IO.8-S allantoicase indicate that//structure, a-helix and random coil conformations were present in both. Correlation of ORD data and infrared characteristics did not allow identification of a particular type of fi structure (parallel, antiparallel or cross-//conformation). An estimate of the amount of//structure must await an assessment of the contribution to overall rotatory properties of the protein by: (I) the rotatory characteristics of amino acid residues, (2) of structures other than t h e / / s t r u c t u r e and the a-helix and (3) the effects of aggregation, environmental factors and specific differences between //structures. The characteristics of the ORD (trough at 23o nm and peak at 2o5 nm) suggest that the Mn2+-enzyme behaves as a form I-fl polypeptide according to the classification of lCASMANAND POTTER32. The acidic amino acids are twice as abundant in allantoicase than the basic ones. The molecular weight of o.9-S allantoicase calculated from the weights of the various components is about i i ooo, which is in good agreement with that obtained from sedimentation studies (II ooo -t= 3oo) and on basis of the metal content. The molecular weight of IO.8-S allantoicase was found to be 154 ooo 4- 8ooo. Preincubation for 15 min at pH 4.6 resulted in an apparent enhancement of the amount of a-helical structure, measured at pH 7.6, from the original 25% to about 32% after preincubation (Table II). This treatment reduced the enzymic activity to about half the original value. It appeared that I/~mole metal ion is present per about I I ooo #g protein. The latter value is the weight of a o.9-S particle and it seems justified to assume that one metal ion is bound to one o.9-S particle. Biochim. Biophys. Acta, 251 (1971) 393 4°6

CONFORMATION OF ALLANTOICASE

405

Comparison of the a-helix contents of some metalloallantoicases indicated that the enzymes with low helix contents exhibited high enzymic activities. The amount of a-helix present in metal-free allantoicase was estimated to be 350/0. Allantoicase showed a multiple Cotton effect at wavelengths between 250 and 300 nm. This Cotton effect was strongly influenced by the addition of inhibitors, e.g. hydantoate and N-carbamoyl-(R)-asparagine. The detailed information obtained for the latter substance showed that 4 or 5 Cotton bands decreased strongly and were perhaps absent in the enzyme-inhibitor complex, whereas one band located at 258 nm increased. These data suggest that the inhibitors strongly influenced the environment of some aromatic groups of the enzyme. The gross structure of the enzyme appeared not to be markedly affected by these inhibitors. The binding of Mn 2+ to the enzyme appeared to be accompanied by a relatively small decrease of the a-helix content. More information on the mode of Mn 2+ binding was revealed from a study of the absorption, ORD and CD spectra at wavelengths above 400 nm. Two effects of these spectra were studied in more detail: addition of inhibitors and lowering the of pH. Lowering of the pH of an allantoiease solution from 7.6 to 4.6 resulted in a shift of the maximum of the absorption band at 416 nm (e416 n m : 8000 mole 1.cm 1) to 394 n m (E394 n m : 9°00 mole -l"cm-1) • Simultaneously a slight decrease of the aromatic absorption bands occurred. In contrast to the absorption band at 416 nm, the band at 394 nm exposed no CD spectrum ([O']41s n m = + 14o degrees, cm 2- dmole-1). The shift is reversible and the original band at 416 nm reappeared when the pH was adjusted again to 7.6. The absorption coefficients of both bands are indicative of charge-transfer reactions 33-3a. Mn 2+ appeared to be bound to the enzyme both at pH 7.6 and 4.6, but a distinct alteration in the mode of binding occurs. This alteration is associated with loss of enzymic activityL However, both forms of the Mn2+-enzyme complex seem to bind the substrates with equal effectiveness, since the Km value of the substrates do not alter in the pH region from 4.6 to 7.6 (ref. 6). The addition of competitive inhibitors resulted in a slight decrease of the absorption and CD bands at 416 nm and 418 nm, respectively. The positive Cotton effect with a maximum at 4 1 8 n m diminished about lO% upon addition of N-carbamoyl-(R)-asparagine or hydantoate. The K, values of the inhibitors determined from the change of the optical rotation in the presence of inhibitors agree well with those resulting from enzymic studies s. The results are consistent with the idea that the binding of competitive inhibitors causes alterations in the state of ligands in the active centre of the enzyme Mn 2+ complex and that simultaneously the environment of certain aromatic groups is strongly affected by placement of these chromophoric residues out of an asymmetric environment. These groups may play an essential role in the binding of substrates and inhibitors by the complex. REFERENCES i 2 3 4

F. TRYBELS, T h e s i s , N i j m e g e n , 1 9 6 7 . G. D. VOGELS, T h e s i s , D e l f t , 1 9 6 3 . G. D . VOGELS AND C. VAN DER DRIFT, Rec. Tray. Chim., 88 (1969) 9 5 1 . E. J . 's-GRAVENMADE, G. D . VOGELS AND C. VAN DER DRIFT, Biochim. Biophys. Acta, 198 (197o) 569.

Biochim. Biophys. Acta, 251 (1971) 3 9 3 - 4 0 6

406 5 6 7 8 9 io II 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 3° 31 32 33 34 35

E.J. 'S-GRAVENMADEet al.

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Biochim. Biophys. Acta, 251 (197 I) 393-406