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Allantoinases from bacterial, plant and animal sources I. Purification and enzymic properties
4N2
III()CHIMICAET BI()PItYSICA .\(;T.\
m~a 05445 A L L A N T O I N A S E S FROM BACTERIAL, PLANT AND ANIMAL S()URCES 1. P U R I F I C A T I O N A N D ENZYMIC P R O P E R T I E S
G. D. VOGEl.S, F. TRIJBELS AND A. UFFINK Department of Biochemistry, University of Ni#negen, Nijrnegen (The Netherla~zds) (Received February ioth, ~960)
SUMMARY
I. Allantoinases (allantoin amidohydrolase, EC 3.5.2.5) from Streptococcus allantoicus, Arthrobacter allantoicus, Escherichia coli, Pseudomonas fluorescens, Pseudomonas acidovorans, frog liver, goldfish liver, Phaseolus hysteri~us and Glycim" hi@ida were studied. 2. The enzymes were purified 2.5- to 37-fold by (NH4)oSO 4 precipitation and DEAE-cellulose chromatography. 3. The p H optimum curve, Michaelis constant, activation energy, stability and specificity were determined and compared. 4. The enzymes from S. allantoicus, A. allantoicus and E. coli were aspecific; the others degraded (--)-allantoin 4-22 times faster than (--)-allantoin. 5. The allantoinases from Ph. hysterinus and G. hi@ida were activated by acid pretreatment below p H 5.4-
INTRODUCTION Allantoinase (allantoin amidohydrolase, EC 3.5.2.5) is widely distributed among animals :-3, higher plants 4, algae 5,6 and microorganismsL Its occurrence was first demonstrated in soybeans s. At this moment some of the properties are known of the enzyme from soybeansg, 10, mnng beans::, various basidiomycetes TM, bakers' yeast and Candida utilis 1°, Streptococcus allantoicus and Arthrobacger allantoicusL Certain differences in properties between tile allantoinases from different sources have been noted. Allantoinases from soybeans :° and Phaseolus hysterinus 13 are inhibited almost completely by reducing substances, whereas the bacterial enzymes are activated by these compounds. Differences have also been reported ff~r the effect of manganous and other bivalent cations. As opposed to other allantoinases:°,:4,15, the enzymes from S. allantoicus and A, allantoicus are not specific for one of the optical isomers of allantoinL A systematic comparative study of the properties of the allantoinases from
Biochim. Bioph3,s. Acta, 122 (1966) 482-496
ALLANTOINASES. I
483
various sources was therefore considered to be desirable. The present communication deals with the purification and enzymic properties of allantoinases from S. allantoicus,
A. allantoicus, Escherichia coli, Pseudomonas acidovorans, Pseudomonas fluoreseens, frog liver, goldfish liver, Glycine hispida and Ph. hysterinus. In a following communication TM the influences of bivalent cations and of reducing substances will be reported. EXPERIMENTAL
Materials Allantoin was purchased from Fluka AG. Methylolallantoin was prepared according to YANO et al. ~7, 3-methylallantoin according to BILTZ AND ROBLTM, I-acetylallantoin according to BILTZ AND LOEWE 19, 5-aminohydantoin according to BILTZ AND GIESLER20 and BILTZ AND HANISCH21, sodium allantoate as described previously 7. The microorganisms were kindly supplied by Professor T. O. WIK~N, Laboratory of Microbiology at Delft, and were cultivated as described earlier 7. Seeds of Ph. hysterinus Dur. and G. hispida L. were a gift of Professor H. F. LINSKENS, Laboratory of Botany, University of Nijmegen. Other plant materials were obtained from a local market. The frogs tested were Rana escuIenta, the goldfish Carassius
auratus. Cell-free extracts of microorganisms were prepared as described previously 22. Plant materials were ground in a seed grinder, and the resulting meal was extracted with 0.05 M Tris-HC1 buffer (pH 7.6) containing o.17 #mole EDTA per ml. The extraction was performed in a Virtis homogenizer (4° ooo rev./min) at 3 ° for a few minutes. The resulting suspension was centrifuged at o ° successively at IO ooo X g for 15 min and at IOO ooo × g for 45 rain. The animal organs were treated in a Virtis homogenizer in the same manner, disintegrated with a MSE 5oo-W ultrasonic disintegrator, and a IOO ooo × g supernatant was prepared at o °.
Methods Analytical procedures. Allantoate, ureidoglycolate and glyoxylate were measured by differential glyoxylate analysis 14. Absorption was not read immediately as described by LEE AND ROUSH 1°, but after about 15 rain. After this period the analyses were quite reproducible and suitable for large series of determinations. Protein was determined according to LOWRY et al. 23. The amount of nucleic acids was calculated from the absorbances at 260 m# and 280 m#. Polarimetric experiments were performed in a Io-cm cell at 3 °° in a Perkin-Etmer polarimeter with sodium vapor lamp. Enzyme purification. Tile clear IOO ooo × g supernatants were fractionated by addition of a saturated (NH4)2SO a solution (pH 7.6) at o °. The precipitated fractions were collected by centrifugation at IO ooo × g for 15 min at o °, and dissolved in 0.05 M Tris-HC1 buffer (pH 7.6) containing o.17 #mole EDTA per ml. The active fraction (Table IV) was dialyzed against the same buffer for 16 h at 3 ° and applied to a DEAEcellulose column (20 cm × 1.8 cm) equilibrated with the buffer. The enzyme was eluted step-wise by increasing amounts of NaC1 dissolved in the same buffer. The active fractions were collected, dialyzed as above and stored at --20 °. Standard conditions. The standard allantoinase test was performed at 3°° in a mixture containing, per ml, 40 #moles allantoin and 18o #moles diethanolamine-HC1 Biochim. Biophys. Acta, 122 (1966) 482-496
4,~4
~i. l). \'(}(;I~LS, I:. "II,HJBI.:I.S . . \ N I ) \ .
[I,I:INI<
buffer (pH 7.7). Enzymes activated by manganous ions ~)r reduced glutathione ~'; we,-t: assayed in the presence of o. r or io #moles/ml of these substances, respectively. One unit of enzyme activity was defined as the amount which will catalyze the transformation of I/zmole ()f substrate per rain. Specific activity is expressed in units per mg protein. Velocities of the enzyme reaction are given in m/2tn(}les of allantoi~ converted per ml per rain. Buffer molarities refer t() the concentrations of the buffering substances. TABLE
[
MICROORGANISMS
GROWING
UNDER
AEROBIC
OR
ANAEROBIC
CONDITIONS
IN
MEDIA
CONTAINING
A L L A N T O I N AS T H E S O L E O R G A N I C C O M P O U N D
Growth was tested in a synthetic allantoin mediumL
Aerobic conditions
A~merobic co*zditions
P. fluorescens P. acidovorans P. aeruginosa Pen. notatum Pen. cilreo-w:ride
S. allantoicus* A. allantoicus E. coli E. coli v a r . a c i d i l a c t i c i t 3. .[reundii
* This bacterium
also needs some amino acids in the medimn.
RESULTS
Occurrence of the enzyme As sources for bacterial allantoinases five species were chosen from the ten microorganisms, which had previously ~ been found to use allantoin as the main or sole source of carbon, nitrogen and energy for growth (Table I). Three bacteria tested in this report (S. allantoicus, A. allantoicus and E. coli) grow well only under anaerobic conditions, two others (P. acidovorans and P.fluorescens) only under aerobic conditions. The latter organisms have different pathways of allantoate degradation 14,24. The specific allantoinase activities in the crude cell-free extracts of tile bacteria w~ried between I and 2, except in P.fluorescens with a specific activity about 0.3. Therefore, LASKOWSKI1 was right in supposing that S. allantoicus would be an excellent source of allantoinase. The specific activity was 200 and 60 times higher than that of purified allantoinases from bakers' yeasO ° and mung beans n. In the yeast species the enzyme was reported to be constitutive 1°, whereas in the bacteria tested here, in Candida utilis ~° and in a Pseudomonas species 25 the enzyme is adaptive. The allantoinase activities in Pseudomonas aeruginosa and in the Penicillium species were low. In frog and goldfish allantoinase activity was high in liver, low in kidney and heart (Table II). In frog allantoicase (allantoate amidinohydrolase, EC 3-5.3-4) was highest in the liver and ureidoglycolase (ureidoglycolate amidinohydrolase) highest in the kidney. In goldfish allantoicase or allantoate amidohydrolase ~2 activities could not be detected. "Fable I I I presents the allantoinase activities in some plant seeds. Seeds of soybeans (G. hi@ida) and Ph. hysterinus were used as enzyme sources in this study, as previous studies13,28,2~ had demonstrated that the amount of allantoinase was Biochim. Biophys. Acta, 122 (1966) 4 8 2 - 4 9 6
ALLANTOINASES. I
485
T A B L E II SPECIFIC ALLANTOINASE, FROG AND GOLDFISH
ALLANTOICASE
AND
UREIDOGLYCOLASE
ACTIVITIES
IN SOME
ORGANS
OF
Allantoicase activity was tested at 30 ° in a mixture containing, per ml, 23/*moles allantoate, ioo/zmoles triethanolamine-HCl buffer (pH 7.4) and an adequate amount of the ioo ooo × g s u p e r n a t a n t of the materials. The degradation was measured by the differential glyoxylate analysis (method D) 14. Ureidoglycolase activity was tested at 3 °0 in a mixture containing, per ml, 23 t,moles ( + )-ureidoglycolate, lOO/,moles triethanolamine-HC1 buffer (pH 7-4) and an adequate a m o u n t of the ioo ooo × g s u p e r n a t a n t of the materials. Glyoxylate formed was measured according to method C (ref. 14).
Organism
Frog liver Frog kidney Frog heart Goldfish liver Goldfish kidney Goldfish heart
Specific activities A llantoinase
A llantoicase
Ureidoglycolase
0.55 0.04 o 0.o 7 o o
o.2i 0.05 o o o o
0.04 0.22 o 0.26 0.05 o
constant in both plants during germination of the seeds and the first stages of growth. The same is true for lupin seeds and seedlings. Purification and stability of allantoinases A l l a n t o i n a s e s i n c r u d e cell-free e x t r a c t s o f t h e b a c t e r i a , a n i m a l l i v e r s a n d p l a n t s e e d s w e r e p u r i f i e d b y (NH4)~SO 4 p r e c i p i t a t i o n a n d D E A E - c e l l u l o s e c h r o m a t o g r a p h y . T h e s a t u r a t i o n r a n g e o f (NH4)2SO * i n w h i c h t h e a l l a n t o i n a s e s p r e c i p i t a t e d a n d t h e c o n c e n t r a t i o n r a n g e o f NaC1 i n w h i c h t h e e n z y m e w a s e l u t e d f r o m t h e c o l u m n a r e g i v e n i n T a b l e I V . N o s t a n d a r d p u r i f i c a t i o n p r o c e d u r e c a n b e g i v e n for a l l a n t o i n a s e s , as i n 4 o f 9 e x a m p l e s (E. coli, P. acidovorans a n d t h e a n i m a l livers) t h e e n z y m e p r e c i p i t a t e d i n a b o u t h a l f - s a t u r a t e d (NH4)2SO 4 s o l u t i o n , w h e r e a s i n t h e o t h e r s a h i g h e r c o n c e n t r a tion was needed. The allantoinases from animal and plant origins emerged from the c o l u m n a t d i s t i n c t l y l o w e r NaC1 c o n c e n t r a t i o n s t h a n t h e b a c t e r i a l e n z y m e s . F u r t h e r m o r e t h e a l l a n t o i n a s e s d i f f e r e d as t o t h e i r s t a b i l i t y d u r i n g t h e p u r i f i c a t i o n p r o c e d u r e . TABLE lit ALLANTOINASE ACTIVITY IN PLANT SEEDS
A llantoinase activity (units per zoo g) Pea
I
Barley Gherkin Lupin
Ph. hysterinus G. hispida Mung bean*
7 5 5 35o 47 ° 6o
* Calculated from the data given by •AGAI AND FUNAHASH111.
t~iochim. Biophys. Mcta, 122 (1966) 482-496
(;. I). \'()GI£1.S, 1.. "IRI.II;EI.S e\NI) .\. U l q ; I N t ,
4~() "I'A BLtg I V PU RIFICATION
S. A. E. P. P.
allantoic~s allautoicus coli acidovorans Iguorescens
Frog liver Goldfish liver
Pk. hvsterinus (;. hi@ida
OV . \ I A ~ A N T 0 1 N AN I';N
5,'pec~fic activity qf starting material
Precipitation hv (NH4)2HO 4 ~'al~rali(m range ("o)
Elution j r o m Specll/~c DE,.:IE-celhdose aclivitv l'21ulion range q/ (31 .\: aC l ) purified material
t)uri/ication factor
Recovery (Oo)
2.J t.2 ~.35
57 1oo 57 1oo o 33
o.2o 0.35 o.2o o . 2 8 o.22 0.35 o.2, 0.35 o. t5 0.35 o. io o . t 5 o.o5 o. io o. t5 0.25 o o. 1o
5.5 4.5 4.5 37 5-5 2. 5 3" 15 I-'
37 ~o t5 83 75
~.5 o.2~) o.55 0.07 0.029 °.°37
0 57 57 t o o o..to o 57 45 ioo 45 t~5
ll.(, 5. t ~,.o
55 1-55 t.43 o.2" 0.45 o.45
* M e a s u r e d a f t e r (NH~),aS( h p r e c i p i t a t i o n . T h e a c t i v i t y pletely after D E A E - c e l l u l o s e c h r o m a t o g r a p h y a n d dialysis.
had
disappeared
2" 7 °* (1o 7°
almost com-
No or only small loss of activity occurred during the purification of the enzymes from Pseudomonas species and higher plants. Also, the enzymes from soybeans ~° and mung beans n appeared rather stable. Allantoinases from E. coli, Penicillium ,notatum and animal livers were easily inactivated, mainly during DEAE-cellulose chromatography and dialysis. As a result of the purification procedure the allantoinases were purified 2.5- to 37-fold. The amount of nucleic acids in the bacterial extracts diminished from above 2o% to below 2%. No allantoate degrading enzymes were present in the purified allantoinases, except in the case of A. allantoicus; this fact, however, did not affect the analytical measurements, as the products formed from allantoate react positively in the differential glyoxylate analysis according to method B 14. NAGAI AND F U N A H A S H I n purified allantoinase from mung beans by calcium phosphate-gel treatment, followed by (NH4) 2SO4 and acetone fractionation ; the specific activity increased from o.oo5 to o.3, and only 32% of the original activity was lost. LEE AND ROUSH~° enhanced the specific activity of bakers' yeast allantoinase (o.oo4 units/mg protein) 6-fold by a similar procedure to that given here ; 42 % of the original activity remained. Under the same conditions the enzyme from Candida utilis was easily inactivated 1°. Thus it appears that allantoinases from higher plants and Pseudomonas species are relatively stable, whereas the enzymes from other bacteria, fungi, yeasts and basidiomycetes ~ are rather unstable.
pH @lima Ro 9 found an optimal allantoinase activity between pH 6.6 and 7.3 for the enzyme from soybeans, LEE AND ROUSH1° in the range from p H 7 8 for the enzyme from bakers' yeast and VInLERST 5 at p H 7.3 for allantoinase from algae. NAGAI AND FUNAHASHIn obtained a p H optimum between 7.5-8.5 for mung bean allantoinase, this pH optimum curve was measured partly in phosphate buffer. In view of the inBiochim. Biophys. ,4cta, 122 (1966) 4 8 2 - 4 9 6
487
ALLANTOINASES. I
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~250~
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Figs. Ia a n d lb. p H o p t i m u m c u r v e s of a l l a n t o i n a s e s f r o m eight different origins. A t 3 °0 a m i x t u r e w a s i n c u b a t e d c o n t a i n i n g , per rnl, 4o/zmoles allantoin, i 6 o p m o l e s buffer a n d purified a l l a n t o i n a s e : 2 5 # g (d. allantoicus, C u r v e i); i i tzg (E. coli, C u r v e 2); 4o/zg (S. aUantoicus, C u r v e 3) ; 1.2 ~g (P. acidovorans, C u r v e 4) ; 5.3/~g (frog liver, C u r v e s 5 a n d 5 a) ; 4 ° / ~ g (goldfish liver, C u r v e s 6 a n d 6a) ; 290 # g (Ph. hysterinus, Curves 7 a n d 7 a) a n d 53 ° / z g (soybeans, Curves 8 a n d 8a). Moreover, o.I /*mole MnSO 4 a n d io # m o l e s G S H were p r e s e n t in t h e first t h r e e e x a m p l e s . Tris HC1 buffers were u s e d in t h e r a n g e p H 6.9-8.1, d i e t h a n o l a m i n e - H C 1 buffers in t h e r a n g e p H 7.6-9.0, N a 2 C O s - N a H C O s buffers in t h e r a n g e p H 8.4~9. 4 a n d acetic a c i d - s o d i u m a c e t a t e buffers in t h e r a n g e p H 5.7-6.1. I n f o u r cases (Curves 5a, 6a, 7 a a n d 8a) p h o s p h a t e buffers were u s e d in t h e r a n g e p H 6.5-7.5. A l l a n t o a t e f o r m e d w a s m e a s u r e d b y t h e differential g l y o x y l a t e analysis.
hibiting effect of phosphate ions, to be discussed elsewhere 16, the lower range of their p H optimum curve would seem to be invalid. p H optimum curves of eight allantoinases are shown in Figs. I a and lb. The curve found for P. fluorescens was the same as that for P. acidovorans. The p H optim u m ranges of the bacterial allantoinases are narrow; the aChvlhes of the enzymes from higher plants and animal livers are less p H dependent. Optimal activity of allantoinase was observed between p H 7.5 and 8.5. Although p H activity curves with phosphate buffers are given for some preparations, the p H optima were derived only from experiments without phosphate present. ~ m values
Table V presents the Kra values of a series of aUantoinases. The values v a r y from 5" IO-a M (S. allantoicus and animal livers) to about 4 °. lO -3 M (Pseudomonas species and most plants). These values refer to ( + - - ) - a l l a n t o i n as substrate. As only the three first mentioned allantoinases are aspecific, it is more exact to consider ( ~-)Biochim. Biophys. Acta, 122 (1966) 4 5 2 - , 9 6
4~N
~,. IL Vt)(;I;LS, 1,. TI(IJIH~I.S . \ N I ) . \ .
Ut.'I;INI(
"1'.\ }~LI~ V ]£m \ ' A L / ! E S ANI) A C T I V A T I O N
I';NI-RIIIES ~)F ALLANTOINASP;-%
1(~,, v a l u e s w e r e ( l e t e r m i n e d b y m e a s u r i n g t h e v e l o c i t i e s a t d i f f e r e n t s u b s t r a t e c o n ( e n t r a t i o n s a n d p](~tting t h e results a c c o r d i n g t~) t h e m e t h o d o f t~[OFSTEE 2s. T]le i n c u b a t i o n m i x t u r e s c o n t a i n e d . p e r ml, i 7 5 1 m u ) l c s d i e t h a n o l a m i n e HCI b u f f e r ( p l l 7.7), p u r i i i e d a l l a n t o i n a s e , a n d a l l a n t c m l v a r y i n g f r o m (~ 3 9 / t m o l e s . In t ) r e p a r a t i o n s f r o m N. allantoicus, 1'2. coil a n d ..I. a l l a n l , i c u s , o. i / t m o h , MnS()t and 1o/tmoles (;SIt were also present. The mixtures were incubated at 3° . Allantoatc f i ) r m e d was d e t e r m i n e d b y t h e difl\~rential g l y o x y l a t e a n a l y s i s . A c t i v a t i o n energies were d e t e r m i n e d b y m e a s u r i n g t h e v e l o c i t i e s a t d i f f e r e n t t e m p e r a t u r e s in m i x t u r e s containing, p e r ml, 1 9 o / , m o l e s d i e t h a n o l a m i n e tt(71 buffer ( p l t 7.7), f )urified a l l a n t o i n a s e , a n d 4.2 p m o t e s a l l a n t o i n . In e x p e r i m e n t s w i t h N. altantoicus, l J. coli a n d ,q. allanloicus o.t t , m o l e M n S ( ) t a n d l o / , l n o l e s ( ; S H w e r e a l s o p r e s e n t , a n d in e x p e r i m e n t s w i t h P h . h v s t e r i n u s a n d s ~ y l ) e a n s o. ~ /ma(He M n S ( ) ~ A r r h e n i u s p l o t s x~ere m a d e , a n d t h e a c t i v a t i o n energies w e r e c a l c u l a t e d . OrgarHsm
Nm ( M × zo")
. t cli~,alion c~tergy (cal/mole)
5,'. allanloicus =t. allantoicus
4.9 14
t ~ Soo 9 o50 8 35 ° I I 75 ° i t 9o0 S ~)oo
l'2. coil
22
P. acidovorans P . fluorescens F r o g liver
45 35 6 S. 4 .t(i 14
Goldfish liver P h . hysterim~s
G. hi@ida Bakers' yeast*
3o
M u n g b¢2an **
4°
9 ooo ~) t o o
* V a l u e f r o m L E E AND [{OUSI{ 10. ** V & l u e f r o l n N A G A I AND F U N A H A S H 1 1 1 .
allantoin as the substrate for the others. Then, two groups of Km values can be distinguished: ( 1 8 ± 4 ) . I o a M for A. allantoicus, E. coli, the Pseudomonas species, bakers' yeast and plants, except soybeans, and ( 5 ~ 2 ) " 1 o -3 M for S. allantoicus, the animal livers and soybeans. For allantoinase from S. allantoicus the same Km was found if Mn 2+ was omitted from the medium or replaced by Zn 2+. Moreover, this value did not change if the diethanolamine-HC1 buffer was replaced by a phosphate buffer (pH 7.5).
Activation energies The activation energy was determined by comparison of the allantoinase activity at different incubation temperatures (Table V). Three activation energies amounted to 11.8 kcal/mole (S. allantoicus and the Pseudomonas species); in five other preparations this value was about 8. 9 kcal/mole. These values were measured in the temperature range from o-27 °, where the Arrhenius plots showed straight lines (Fig. 2) ; above 27 ° deviations occurred from a straight line plot. Similar results were found for other enzymes e.g. urease ~9 (urea amidohydrolase, EC 3.5.1.5) and glycogen phosphorylasO ° (~-i,4-glucan: orthophosphate glucosyltransferase, EC 2.4.1.1 ). An explanation for these results cannot be given at present; the deflection is probably not due to a denaturation effect, as the enzyme, especially from plants, is stable at temperatures up to 55 °. Biochim. ~)iophys. /tcta, 1,22 (1966) 482-,t96
ALLANTOINASES. I
489
>
~2 r~
320
.~40
360
'
!.Io 5 T
Fig. 2. D e t e r m i n a t i o n of t h e activation energies of allantoinases from Ph. hysterinus (i), P. fluorescens (2) and frog liver (3). I n c u b a t i o n and analysis were performed as given in Table V.
I _
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Fig. 3- D e g r a d a t i o n of ( + - - ) - a l l a n t o i n b y allantoinase f r o m P. acidovorans, change in optical rotation and d e t e r m i n a t i o n of the specific optical r o t a t i o n of (--)-allantoin. The i n c u b a t i o n m i x t u r e at 3 °o contained, per ml, 46/~moles ( + - - ) - a l l a n t o i n , zoo/*moles diethanolamine-HC1 buffer (pH 7.7) and 15. 5/zg purified allantoinase. Allantoate formed was m e a s u r e d b y t h e differential glyoxylate analysis.
Biochim. Biophys. Acta, 122 (1966) 482-496
4()0
t;. IL V()(',I~I.S, 1,'. I'RIJBI'A~S ANI/ .\. I'FFINI,~
optical specificity I n previous studies a st)ecificity fl~r (F)-allantoin was reported fl)r tilt: e n z w n c , from soybcansl°,la, la, Ph. hysteri'mts 13, P. acidovorans 14 and fish liver al. The enzvm~,~ from S. allantoicus and A. alla~ztoicus were not specific f~)r one of the optical Jsolner~ of allantoin 7. Results obtained with allantoinase from P. acidovorans demonstrated that this enzyme degraded (+)-allantoin nmch faster than (--)-allantoin (Fig. 3). The apparent specific rotation [a]~° was calculated from the a m o u n t of allantoate formed and th/~ observed optical rotation. By extrapolation of these values a specific rotation [c~i~/°of 8 o ~ i ° for ( -)-allantoin was estimated, which is lower than the value of - 9 2~ found by FOSSE, THOMAS AND DE (IRAEVE ~5, but in good agreement with the results of I,EE AND ROUSH ~°. On the basis of this value the initial velocities ti)r the degradation of (i-~) - and ( --)-allantoin were calculated from the analytical and polarimetric data. Figs. 4 a and 4b show the results for frog liver and Ph. hysterinus. Three allantoinases (S. allantoicus, A. allantoicus and E. coli) are aspecific (Table VI); the degradation rates for the two optical isomers differed less than I ~,; and never was there an excess of more than o.5% of one of the isomers during degradation. With the enzyme from all other sources the ( + ) - f o r m was degraded faster than the ( - ) - f o r m , but the latter was always degraded to(). In our hands none of the allantoinases was absolutely stereospecific. LEE AND ROUSH 1°, while obtaining results similar to ours fi>r bakers' yeast, concluded from their experiments t h a t soybean allantoinase was completely stereospecific. However, "FH(~51as AND DE (IRAEVE 32 had previously demonstrated that the enzyme from soybeans degraded (--)-allantoin 2.6-fold faster than (--)-allantoin. T A B L E VI SPECIFICITY ALLANTOIN
OF AND
ALLANTOINASES
FOR
THE
OPTICAL
ISOMERS
OF
ALLANTOIN
ANt)
FOR
METHYI.OL
-
5-AMINOHYDANTO1N
The e x p e r i m e n t s were performed at 3°o in m i x t u r e s containing, per ml, 39/tmoles ( I )-allantoin, nlethylolallantoin or 5 - a m i n o h y d a n t o i n (for P. acidovorans 46pmoles), I75 ttmoles diethanolamine ItCI buffer (pH 7.7) (for P. acidovora~s 200 t~moles) and purified allantoinase: 68tlg (5". allantoicus) ; 02/zg ( A . allantoiczts); 50big (E. coli); I5.5t~g (P. acidovora~s) ; 3o/~g (P. ftuoresce~s) ; 15o fig (frog liver) ; 8 0 o / , g (Ph. h3,sterinus ) or 46o tzg (soybeans). In the e x p e r i m e n t s with ~4. alla~ztoiczes and E. coli, moreover, 12/*moles thioglycolate were present and in those with S. allantoicus, A. allantoicus, E. coli, Ph. hysterinus and s o y b e a n s o . i / , m o l e MnSO 4. The degradation of the s u b s t r a t e was followed by m e a s u r i n g the p r o d u c t by the differential glyoxylate analysis (method B). Two e x a m p l e s of the calculation of the degradation ratio ( + )-allantoin to ( )-allan-toin are given in Figs. 4 a and 4b.
Orga~dsm
Degradation rates
( ~ J-form~( J-form
S. A. E. P. P.
allantoicus allantoicus coli acidovorans fluorescens
Frog liver
Ph. hysterinus G. hispida
Degradation rates*
(%)
Methylolallantoi~z
5-,4 mi nohydantoin
1 I
45 25
6-4 4
1
6
2i. 5 6. 3 4-9 13.,5 4-4
8 3° 3° 37.5 5°
o o o o-7 0.9 0.5
* Given in per cent of the degradation rate obtained with ( ~
Biochim. Bioph),s. Acta, 122 (196t,) 482-496
)-allantoin.
ALLANTOINASES. I
49I
~2s
1 c+ -)-allantom"
~ 20
', [ ~- 4
C+-)-allantom"
jx
/
x
, ,_ 20 °
oO15 U
< lo L
54
il
,Y/
' J
zx~/(÷~-allantoin
o ~
0 0
30
60
90
120 150 180 0 I~cu]0atio~ time (rain)
60
120 180 240 I n c u b a t i o n time (rain)
Figs. 4 a a n d 4 b. D e g r a d a t i o n of ( + - - ) - a l l a n t o i n a n d c h a n g e in optical r o t a t i o n b y a l l a n t o i n a s e s f r o m Ph. hysterinus (Fig. 4 a) a n d frog liver (Fig. 4b). T h e e x p e r i m e n t s were p e r f o r m e d a t 3 °° in m i x t u r e s c o n t a i n i n g , per ml, 3 9 . o / , m o l e s allantoin, 1 7 5 / , m o l e s d i e t h a n o l a m i n e - H C 1 buffer (pH 7.7) a n d purified a l l a n t o i n a s e (o.86 m g f r o m Ph. hysterinus or o.15 m g f r o m frog liver). I n t h e f o r m e r case o. I/~mole M n S O 4 w a s p r e s e n t . F r o m t h e d e g r a d a t i o n c u r v e of ( + - - ) - a l l a n t o i n a n d t h e c h a n g e in optical r o t a t i o n t h e d e g r a d a t i o n s of ( + ) - a n d ( - - ) - a l l a n t o i n were calculated.
Two groups of allantoinases can be distinguished on the basis of optical specificity. The first group is formed by the aspecific enzymes from S. allantoicus, A. allantoicus and E. coli, bacteria that grow in allantoin media only under anaerobic conditions. In the second group the enzymes from higher plants, several microorganisms (Pseudomonas species, bakers' yeast 1°) and animal livers (frog, goldfish, Raja clavata al) can be classified that degrade (+)-allantoin 5-20 times faster than (--)-allantoin.
Degradation of allantoin derivatives 3-Methylallantoin and i-acetylallantoin were not degraded by any of the nine enzymes tested. 1,3-Diacetylallantoin and 5-acetylaminohydantoin were not attacked by allantoinases from S. allantoicus and A. alIantoicusL Methylolallantoin and 5aminohydantoin were broken down at velocities presented in Table VI and Figs. 5 a
//1
~50
o~
~4o u t~
20 ¸
/ /x
10.
lO 20 30 40 0 Incubatior~ t i m e (rain)
2()
40 6'0 8'0 100 I n c u b a t i o r l t i m e (rain)
Figs. 5a a n d 5b. D e g r a d a t i o n of a l l a n t o i n (i), m e t h y l o l M l a n t o i n (2) a n d 5 - a m i n o h y d a n t o i n (3) b y a l l a n t o i n a s e s f r o m A. allantoicus (Fig. 5 a) a n d E. coli (Fig. 5b). D e g r a d a t i o n w a s t e s t e d u n d e r c o n d i t i o n s g i v e n in T a b l e VI.
Biochim. Biophys. Acta, 122 (1966) 482-496
4{)2
~;. 1). \'{)(;El.S, F. "II~IJBIiI.S .\NI~ .\. I'FFINi,;
and 51). All allantoin derivatives were tested under the conditions given in Table V I. Methylolatlantoin was degraded at 25-5o~!i~ of the rate for allantoin; ,mlv I~,r allantoinases from E. coil and 1'. acidovoraus was the rate lower (8o:o). The positi(m of the methylol group in the allantoin molecule is unknown; since substituents in the hydantoin moiety prevent degradation by allantoinase, the most probable positi(m of the m e t h y M group is the 8- or 6-position in the molecule. Allantoinases from 5. alla~2loicHs and A. allantoicus could use 5-aminohydantoin as substrate, although slowh'. The other allantoinases degraded this compound at a very l.w rate or not at all. Allantoinases activated by Mn '+ (ref. 16) degraded methylolallantoin and 5aminohydantoin faster in the presence of these ions. The enhancement of activity was for all substrates in the same order of magnitude.
Heat stability The effect of 5 min heat pretreatment on the activity of allantoinases is shown in Fig. 6. Most of the enzymes were largely inactivated by heating between 5o ° and 6o °. The enzymes from soybeans and Ph. hysterinus were much more stable, though not as stable as reported by Ro 9 who found only little loss of activity after heating for 30 rain at 7°o and loss of half the activity at 8o ° for 30 min. The stability . f
~1oo~.~.~.~ , 6O
"
20 x
Vxx,~ V 0 0
. 40"
.
& .
. 5'0°
60 °
i 70 °
60 ° Temperc~ture
Fig. O. I n a c t i v a t i o n b y h e a t p r e t r e a t m e n t o f a l l a n t o i n a s e s f r o m E. coli (k~), P. acidovorans (W), f r o g l i v e r (A), S. allantoicus ( 0 ) , A. allantoicus (0), P. fluoresrens ((~), g o l d f i s h l i v e r ( × ) , Ph. hvsterinus ([7) a n d G. hi@ida (11). A n a l i q u o t o f e n z y m e w a s h e a t e d for 5 m i n a t t h e i n d i c a t e d t e m p e r a t u r e s a t p H 7-5- T h e r e s u l t i n g a c t i v i t y w a s t e s t e d u n d e r s t a n d a r d c o n d i t i o n s a n d c o m pared with untreated material.
Biochim. Biophys. Acta, 122 (t066) 4 8 2 - 4 9 6
ALLANTOINASES. I
493
allantoinase from bakers' yeast 1° was similar to that found for most allantoinases. This enzyme was stable up to 4°° and was inactivated above 55 ° when held at pH 7 for 20 rain,
Inactivation by urea After pretreatment in 5 M urea for IO rain at 20 °, enzyme activity was tested in mixtures containing I or 5 M urea. The activities measured were compared with the activity of untreated material in the absence of urea (Table vii). In the presence of 5 M urea the allantoinase activities were very low; after dilution to I M urea, the activities were about half those of the untreated material. TABLE
VII
EFFECT OF PRETREATMENT OF ALLANTOINASES IN 5 M UREA A n a l i q u o t o f t h e e n z y m e s o l u t i o n w a s m i x e d w i t h a n e q u a l v o l u m e o f i o M u r e a ( p H 8.o) a t 2o °. A f t e r IO m i n o n e s a m p l e w a s i n c u b a t e d a t 3 °0 i n a m i x t u r e c o n t a i n i n g , p e r m l , 2 0 / ~ m o l e s a l l a n t o i n , i o o / ~ m o l e s d i e t h a n o l a m i n e - H C 1 b u f f e r ( p H 8.0) a n d s u c h a n a m o u n t of t h e t r e a t e d e n z y m e t h a t t h e f i n a l c o n c e n t r a t i o n o f u r e a w a s I M, A n o t h e r s a m p l e w a s t e s t e d i n a s i m i l a r m a n n e r i n an incubation mixture containing 5 M urea. The velocities measured were compared with the r e s u l t s o f a t e s t in w h i c h u r e a w a s o m i t t e d . M n 2+ ( o . i # m o l e ] m l ) w a s p r e s e n t i n t h e t e s t s w i t h S. allantoicus, A. allantoicus, E. coli, Ph. hysterinus a n d s o y b e a n s ; G S H ( 3 / ~ m o l e s / m l ) i n t h e t e s t s w i t h E. coli a n d A . allantoicus.
Organism
Activity ( % ) 5 M Urea I M Urea
S. A. E. P. P.
allantoicus allantoicus coli acidovorans fluorescens
Frog liver Goldfish liver
Ph. hysterinus G. hispida
io 16 2
o o I io 6 6
54 75 18 33 61 42 49 64 55
Stability in acidic solutions The effect of incubation with o.I M sodium acetate-acetic acid buffers at pH values between 5-4 and 4.0 is presented in Fig. 7. Most allantoinases were inactivated readily under these conditions. The enzyme from E. coli was inactivated partly even at a pH of about 7, but a small activity remained at pH 4.0. Allantoinases from Ph. hysterinus and soybeans were activated 1.3- to 1.6-fold below pH 5.4. The effect of temperature and preincubation time is given in Fig. 8. The activating effect of acid pretreatment on these allantoinases is perhaps due to a mechanism similar to that in the case of allantoate amidohydrolases from S, allantoicus, A. allantoicus, E. coli and P. acidovoransT, 2~. The activity of these enzymes is enhanced IO-IOO times by acid pretreatment at o ° for 30 sec. Preliminary results obtained with aUantoinases from Pen. notatum, wheat and gherkin indicate that these enzymes are inactivated by acid pretreatment. Biochim. Biophys. Acta, 122 (1966) 4 8 2 - 4 9 6
41)4
(;.
IL
V()GIC, I . S ,
I,'. F I , : I J I { I Z L S
..\NI)A.
UI;I;IN[,,
$ 175 >,
/
x" " ~ " ×---....x....x
150
\/'~'~ I00
75 G.
50
(•)
\\\
l~a.(*) A.=~o)
\k\ V X,~
25
,
,
em"~ I
j
5 4 pH d u r i n g p r e t r e a t m e n t
Fig. 7. S t a b i l i t y of a l l a n t o i n a s e s in acidic media. To aliquots of t h e e n z y m e solution 3 v o l u m e s of o.i M s o d i u m a c e t a t e - a c e t i c acid buffer w i t h different p H v a l u e s were added. T h e p H of t h e mixt u r e s is i n d i c a t e d in t h e figure. After 6 m i n at 3 °0 a s a m p l e of t h e m i x t u r e was a d d e d to a n allantoin solution. I n t h e i n c u b a t i o n m i x t u r e s a t 3 °° t h e r e were present, per mI, 2oo/*moles d i e t h a n o l a m i n e HC1 buffer (pH 7.7) a n d 4 2/tlnoles allantoin. F u r t h e r m o r e , io/~lnoles G S H were p r e s e n t in t h e t e s t s with ,4. allantoicus a n d E. coli, a n d o . i / , m o l e s MnSO 4 in t h e s a m e t e s t s a n d in t h e experim e n t s w i t h S. allanloicus, Ph. h.vsterinus a n d s o y b e a n s . T h e a m o u n t s of purified a l l a n t o i n a s e s (in/~g) p r e s e n t per ml i n c u b a t i o n m i x t u r e , a n d t h e velocities m e a s u r e d , w h e n i n s t e a d of a c e t a t e buffer w a t e r was used, are given in this order w i t h i n p a r e n t h e s e s : S. allanloicus (S.a., 15.6, lO5), ~4. allanloicus (A.a., 8, 23), E. coli (E.c., 5 o, I24), P. acidovorans (P.a., 66, 29), P. fluorescens (P.f., I1, 4.5), frog liver (F., 12.5, 7.8), goldfish liver (G., 57 o, 5.I), Ph. hvsterinus (P.h. 21o, 75) a n d s o y b e a n s (G.h., 380, 99). T h e velocities m e a s u r e d after p r e t r e a t m e n t a t t h e indicated p H are e x p r e s s e d in % of t h e velocity m e a s u r e d with t h e u n t r e a t e d material.
DISCUSSION
Allantoinase is the only enzyme known to attack allantoin, perhaps with the exception of "allantoin oxidase", which was postulated by FRANKE3a for Alternaria tenuis, but this needs confirmation. In this study a comparison was made of some properties of allantoinases from S. allantoicus, A. allantoicus, E. coli, P. acidovorans, P. fluorescens, frog liver, goldfish liver, Ph. hysterinus and soybeans. Furthermore, the results were compared with previous studies of the enzymes from bakers' yeast, Candida utilis, Pen. notatum, basidiomycetes, Raja clavata and mung beans. The behaviors during a purification procedure, involving (NH4)~SO 4 precipitation and DEAE-cellulose chromatography, the pH optimum curve, Kin, activation energy and the stability on storage, heating, urea treatment and acid pretreatment were compared. The specificity for ( + ) - and (--)-allantoin and allantoin derivatives was also studied. The allantoinases differed from each other in each of the properties mentioned. Four groups of enzyme sources were used in this study: animal livers, Biochi,m. Biophys. ,4eta, t22 (1966) 482 496
ALLANTOINASES. I
495
160 v
< 140
120
7"
pH during pretrealrnent
Fig. 8. A c i d a c t i v a t i o n o f a l l a n t o i n a s e f r o m Ph. hysterinus. T h e e x p e r i m e n t w a s p e r f o r m e d as d e s c r i b e d in Fig. 7. T h e p r e t r e a t m e n t w i t h a c e t a t e buffer w a s p e r f o r m e d e i t h e r d u r i n g 6 rain a t 3 °0 (I), 6 m i n at o ° (2) or h a l f a m i n u t e a t o ° (3)'
plant seeds, Pseudomonas species and three bacteria that can use allantoin as sole organic substrate for growth under anaerobic conditions. Some differences in the enzyme properties followed this classification, namely pH optimum curve and stability on storage, heating and acid pretreatment. Other differences showed no correlation with this classification, namely behavior during purification, Kin, activation energy and specificity for allantoin derivatives. The enzymes from bacteria that degrade allantoin under anaerobic conditions were not stereospecific. Since allantoin can racemize 15, the compound may be present in the optically inactive form in decaying biological material. S. allantoicus, A. allantoicus, and Escherichia species are probably the scavengers of this compound, which is present in large quantities in nature as an excretion product of animals and as a compound involved in the nitrogen storage and transport in a large number of plant families. REFERENCES i 2 3 4 5 6 7 8 9 io II
M. LASKOWSKI,Enzymes, I (1951) 946. L. GOLDS'rEIN AND R. P. FOSTER, Comp. Biochem. Physiol., 14 (1965) 567. I. GOODBOD'¢, J. Exptl. Biol., 42 (1965) 299. M. V. TRACEY, in K. PAECH AND M. V. TRACEY, Moderne Methoden der Pflanzenanalyse, Vol. 4. Springer, Berlin, 1955, p. 119. S. VILLERET, Compt. Rend., 241 (1955) 90. S. VILLERET, Compt. Rend., 246 (1958) 1452. G. D. VOGELS, Thesis, I n s t i t u t e o f T e c h n o l o g y , Delft, 1963. R. F o s s e AND A. BRUNEL, Compt. Rend., 188 (1929) 426. K. Ro, J. Biochem. Tokyo, 14 (1932) 405 ` K. W. LEE AND A. H. ROUSH, Arch. Biochem. Biophys., lO8 (1964) 46o. Y. NAGAI AND S. FUNAHASHI, Agr. Biol. Chem. Tokyo, 25 (1961) 265.
Biochim. Biophys. Acta, 122 (1966) 482-496
490 I2 13 14 15 16 17 I8 19 2o 2I 22 23 x4 25 26 27 28 29 3° 31 32 33
~;. IL \:~(il,;l.S, 1~. IRIJt3F.I.5 AND A. UFI:INIx
A. ]~RUNEL, Thesis, l i n i x e r s i t v of P a r i s , 1931~. CHR. VAN DER DRIFT AND (;. :1). VOGELS, .qcla ]JOla~¢. ~\:eer[., 15 (:900) -'(J~. F. TRIJBELS AND (;. 1). VOGELS, Biochim. Biophys. ,qcta, i t 3 (19()()) 2(~2. R. FOSSE, P . - E . THOMAS AND 1'. DE (;RAI~:VI.:, Conzpl. l¢~nd., i 9 8 (1034) ogq. G. I). VOGELS a~,'I) ('HR. VAN DER I)RIFT, Bi,wkim. Biopk3's..q~/a, ,2" (I9o()) 497. t,2. YANo, T. IKEr)A, S. TSVI~tTA, C.'I'ANL lq. NAKAJ1MA AND Y. MoJ~I,C/2em. qb~tr.,45 (~(J491 759 o. H. BIL'rZ AND 1{. I{OBL, I/of., 54 ( r 9 2 I ) 2448. H. BILTZ AND L. LOEWE, .[. Prakt. Chem., 14[ (1934) 284. H. BILTZ AND E . GIESLVR, 13er., 40 (1913) 3410H. BILTZ AND H . ItANlSCH, .[. Prakt. Che~n., ~I2 (1920) 138. G. D. VOGELS, Biochim. I3iopkx,s. dcta, i [ 3 (1900) 277. O. H . I . O W R Y , N. J. I~OSEBROUGH, \ . 1.. FARR AND R. J. I{ANDALL, J . l~iol. Chem., lq 3 (~t~5I) 265. F. TRIJBELS AND G. I). \'Oc;t':LS, Biochim. Biophys. dcla, t18 ([900) 387 . L. L. CAMPBELL, JR., J . Bacteriol., 08 (I954) 598. IC ECNEVlN AND A. BRUNEL, CompL Rend., 2o 5 (1937) 294. 1C t~;CHFVlN AND A. B~UNEL, Compl. l?e~d., 2o8 (~939) 826. B. H. J. HOI~'STEE, Ncience, I ~, (1952) 329 . G. B. KISTIAKOWSKY AND 1C LUMR¥, .]..qm. Chem. Sot., 71 (1949) 2oo0. D. J. (;RAVin:s, R. \V. SI':ALOCK AND J. H. WANC, Biochemisl~9,, 4 (1965) 29 °. R. FOSSE, I'.-E. THOS~AS AND I'. IIr: GRArVE, Compl. Rend., 198 (1934) 1374P . - E . THOMAS AND P. DE GIaAEVE, CompL Rend., 108 (1934) 22o5. W. FRANKE, Z. Vilamin Hormo~ I:ermen(forsch., 5 (1953) 279.
Biovhim. Biophys. Acta, i22 (L966) 4 8 z - 4 9 0
III()CHIMICAET BI()PItYSICA .\(;T.\
m~a 05445 A L L A N T O I N A S E S FROM BACTERIAL, PLANT AND ANIMAL S()URCES 1. P U R I F I C A T I O N A N D ENZYMIC P R O P E R T I E S
G. D. VOGEl.S, F. TRIJBELS AND A. UFFINK Department of Biochemistry, University of Ni#negen, Nijrnegen (The Netherla~zds) (Received February ioth, ~960)
SUMMARY
I. Allantoinases (allantoin amidohydrolase, EC 3.5.2.5) from Streptococcus allantoicus, Arthrobacter allantoicus, Escherichia coli, Pseudomonas fluorescens, Pseudomonas acidovorans, frog liver, goldfish liver, Phaseolus hysteri~us and Glycim" hi@ida were studied. 2. The enzymes were purified 2.5- to 37-fold by (NH4)oSO 4 precipitation and DEAE-cellulose chromatography. 3. The p H optimum curve, Michaelis constant, activation energy, stability and specificity were determined and compared. 4. The enzymes from S. allantoicus, A. allantoicus and E. coli were aspecific; the others degraded (--)-allantoin 4-22 times faster than (--)-allantoin. 5. The allantoinases from Ph. hysterinus and G. hi@ida were activated by acid pretreatment below p H 5.4-
INTRODUCTION Allantoinase (allantoin amidohydrolase, EC 3.5.2.5) is widely distributed among animals :-3, higher plants 4, algae 5,6 and microorganismsL Its occurrence was first demonstrated in soybeans s. At this moment some of the properties are known of the enzyme from soybeansg, 10, mnng beans::, various basidiomycetes TM, bakers' yeast and Candida utilis 1°, Streptococcus allantoicus and Arthrobacger allantoicusL Certain differences in properties between tile allantoinases from different sources have been noted. Allantoinases from soybeans :° and Phaseolus hysterinus 13 are inhibited almost completely by reducing substances, whereas the bacterial enzymes are activated by these compounds. Differences have also been reported ff~r the effect of manganous and other bivalent cations. As opposed to other allantoinases:°,:4,15, the enzymes from S. allantoicus and A, allantoicus are not specific for one of the optical isomers of allantoinL A systematic comparative study of the properties of the allantoinases from
Biochim. Bioph3,s. Acta, 122 (1966) 482-496
ALLANTOINASES. I
483
various sources was therefore considered to be desirable. The present communication deals with the purification and enzymic properties of allantoinases from S. allantoicus,
A. allantoicus, Escherichia coli, Pseudomonas acidovorans, Pseudomonas fluoreseens, frog liver, goldfish liver, Glycine hispida and Ph. hysterinus. In a following communication TM the influences of bivalent cations and of reducing substances will be reported. EXPERIMENTAL
Materials Allantoin was purchased from Fluka AG. Methylolallantoin was prepared according to YANO et al. ~7, 3-methylallantoin according to BILTZ AND ROBLTM, I-acetylallantoin according to BILTZ AND LOEWE 19, 5-aminohydantoin according to BILTZ AND GIESLER20 and BILTZ AND HANISCH21, sodium allantoate as described previously 7. The microorganisms were kindly supplied by Professor T. O. WIK~N, Laboratory of Microbiology at Delft, and were cultivated as described earlier 7. Seeds of Ph. hysterinus Dur. and G. hispida L. were a gift of Professor H. F. LINSKENS, Laboratory of Botany, University of Nijmegen. Other plant materials were obtained from a local market. The frogs tested were Rana escuIenta, the goldfish Carassius
auratus. Cell-free extracts of microorganisms were prepared as described previously 22. Plant materials were ground in a seed grinder, and the resulting meal was extracted with 0.05 M Tris-HC1 buffer (pH 7.6) containing o.17 #mole EDTA per ml. The extraction was performed in a Virtis homogenizer (4° ooo rev./min) at 3 ° for a few minutes. The resulting suspension was centrifuged at o ° successively at IO ooo X g for 15 min and at IOO ooo × g for 45 rain. The animal organs were treated in a Virtis homogenizer in the same manner, disintegrated with a MSE 5oo-W ultrasonic disintegrator, and a IOO ooo × g supernatant was prepared at o °.
Methods Analytical procedures. Allantoate, ureidoglycolate and glyoxylate were measured by differential glyoxylate analysis 14. Absorption was not read immediately as described by LEE AND ROUSH 1°, but after about 15 rain. After this period the analyses were quite reproducible and suitable for large series of determinations. Protein was determined according to LOWRY et al. 23. The amount of nucleic acids was calculated from the absorbances at 260 m# and 280 m#. Polarimetric experiments were performed in a Io-cm cell at 3 °° in a Perkin-Etmer polarimeter with sodium vapor lamp. Enzyme purification. Tile clear IOO ooo × g supernatants were fractionated by addition of a saturated (NH4)2SO a solution (pH 7.6) at o °. The precipitated fractions were collected by centrifugation at IO ooo × g for 15 min at o °, and dissolved in 0.05 M Tris-HC1 buffer (pH 7.6) containing o.17 #mole EDTA per ml. The active fraction (Table IV) was dialyzed against the same buffer for 16 h at 3 ° and applied to a DEAEcellulose column (20 cm × 1.8 cm) equilibrated with the buffer. The enzyme was eluted step-wise by increasing amounts of NaC1 dissolved in the same buffer. The active fractions were collected, dialyzed as above and stored at --20 °. Standard conditions. The standard allantoinase test was performed at 3°° in a mixture containing, per ml, 40 #moles allantoin and 18o #moles diethanolamine-HC1 Biochim. Biophys. Acta, 122 (1966) 482-496
4,~4
~i. l). \'(}(;I~LS, I:. "II,HJBI.:I.S . . \ N I ) \ .
[I,I:INI<
buffer (pH 7.7). Enzymes activated by manganous ions ~)r reduced glutathione ~'; we,-t: assayed in the presence of o. r or io #moles/ml of these substances, respectively. One unit of enzyme activity was defined as the amount which will catalyze the transformation of I/zmole ()f substrate per rain. Specific activity is expressed in units per mg protein. Velocities of the enzyme reaction are given in m/2tn(}les of allantoi~ converted per ml per rain. Buffer molarities refer t() the concentrations of the buffering substances. TABLE
[
MICROORGANISMS
GROWING
UNDER
AEROBIC
OR
ANAEROBIC
CONDITIONS
IN
MEDIA
CONTAINING
A L L A N T O I N AS T H E S O L E O R G A N I C C O M P O U N D
Growth was tested in a synthetic allantoin mediumL
Aerobic conditions
A~merobic co*zditions
P. fluorescens P. acidovorans P. aeruginosa Pen. notatum Pen. cilreo-w:ride
S. allantoicus* A. allantoicus E. coli E. coli v a r . a c i d i l a c t i c i t 3. .[reundii
* This bacterium
also needs some amino acids in the medimn.
RESULTS
Occurrence of the enzyme As sources for bacterial allantoinases five species were chosen from the ten microorganisms, which had previously ~ been found to use allantoin as the main or sole source of carbon, nitrogen and energy for growth (Table I). Three bacteria tested in this report (S. allantoicus, A. allantoicus and E. coli) grow well only under anaerobic conditions, two others (P. acidovorans and P.fluorescens) only under aerobic conditions. The latter organisms have different pathways of allantoate degradation 14,24. The specific allantoinase activities in the crude cell-free extracts of tile bacteria w~ried between I and 2, except in P.fluorescens with a specific activity about 0.3. Therefore, LASKOWSKI1 was right in supposing that S. allantoicus would be an excellent source of allantoinase. The specific activity was 200 and 60 times higher than that of purified allantoinases from bakers' yeasO ° and mung beans n. In the yeast species the enzyme was reported to be constitutive 1°, whereas in the bacteria tested here, in Candida utilis ~° and in a Pseudomonas species 25 the enzyme is adaptive. The allantoinase activities in Pseudomonas aeruginosa and in the Penicillium species were low. In frog and goldfish allantoinase activity was high in liver, low in kidney and heart (Table II). In frog allantoicase (allantoate amidinohydrolase, EC 3-5.3-4) was highest in the liver and ureidoglycolase (ureidoglycolate amidinohydrolase) highest in the kidney. In goldfish allantoicase or allantoate amidohydrolase ~2 activities could not be detected. "Fable I I I presents the allantoinase activities in some plant seeds. Seeds of soybeans (G. hi@ida) and Ph. hysterinus were used as enzyme sources in this study, as previous studies13,28,2~ had demonstrated that the amount of allantoinase was Biochim. Biophys. Acta, 122 (1966) 4 8 2 - 4 9 6
ALLANTOINASES. I
485
T A B L E II SPECIFIC ALLANTOINASE, FROG AND GOLDFISH
ALLANTOICASE
AND
UREIDOGLYCOLASE
ACTIVITIES
IN SOME
ORGANS
OF
Allantoicase activity was tested at 30 ° in a mixture containing, per ml, 23/*moles allantoate, ioo/zmoles triethanolamine-HCl buffer (pH 7.4) and an adequate amount of the ioo ooo × g s u p e r n a t a n t of the materials. The degradation was measured by the differential glyoxylate analysis (method D) 14. Ureidoglycolase activity was tested at 3 °0 in a mixture containing, per ml, 23 t,moles ( + )-ureidoglycolate, lOO/,moles triethanolamine-HC1 buffer (pH 7-4) and an adequate a m o u n t of the ioo ooo × g s u p e r n a t a n t of the materials. Glyoxylate formed was measured according to method C (ref. 14).
Organism
Frog liver Frog kidney Frog heart Goldfish liver Goldfish kidney Goldfish heart
Specific activities A llantoinase
A llantoicase
Ureidoglycolase
0.55 0.04 o 0.o 7 o o
o.2i 0.05 o o o o
0.04 0.22 o 0.26 0.05 o
constant in both plants during germination of the seeds and the first stages of growth. The same is true for lupin seeds and seedlings. Purification and stability of allantoinases A l l a n t o i n a s e s i n c r u d e cell-free e x t r a c t s o f t h e b a c t e r i a , a n i m a l l i v e r s a n d p l a n t s e e d s w e r e p u r i f i e d b y (NH4)~SO 4 p r e c i p i t a t i o n a n d D E A E - c e l l u l o s e c h r o m a t o g r a p h y . T h e s a t u r a t i o n r a n g e o f (NH4)2SO * i n w h i c h t h e a l l a n t o i n a s e s p r e c i p i t a t e d a n d t h e c o n c e n t r a t i o n r a n g e o f NaC1 i n w h i c h t h e e n z y m e w a s e l u t e d f r o m t h e c o l u m n a r e g i v e n i n T a b l e I V . N o s t a n d a r d p u r i f i c a t i o n p r o c e d u r e c a n b e g i v e n for a l l a n t o i n a s e s , as i n 4 o f 9 e x a m p l e s (E. coli, P. acidovorans a n d t h e a n i m a l livers) t h e e n z y m e p r e c i p i t a t e d i n a b o u t h a l f - s a t u r a t e d (NH4)2SO 4 s o l u t i o n , w h e r e a s i n t h e o t h e r s a h i g h e r c o n c e n t r a tion was needed. The allantoinases from animal and plant origins emerged from the c o l u m n a t d i s t i n c t l y l o w e r NaC1 c o n c e n t r a t i o n s t h a n t h e b a c t e r i a l e n z y m e s . F u r t h e r m o r e t h e a l l a n t o i n a s e s d i f f e r e d as t o t h e i r s t a b i l i t y d u r i n g t h e p u r i f i c a t i o n p r o c e d u r e . TABLE lit ALLANTOINASE ACTIVITY IN PLANT SEEDS
A llantoinase activity (units per zoo g) Pea
I
Barley Gherkin Lupin
Ph. hysterinus G. hispida Mung bean*
7 5 5 35o 47 ° 6o
* Calculated from the data given by •AGAI AND FUNAHASH111.
t~iochim. Biophys. Mcta, 122 (1966) 482-496
(;. I). \'()GI£1.S, 1.. "IRI.II;EI.S e\NI) .\. U l q ; I N t ,
4~() "I'A BLtg I V PU RIFICATION
S. A. E. P. P.
allantoic~s allautoicus coli acidovorans Iguorescens
Frog liver Goldfish liver
Pk. hvsterinus (;. hi@ida
OV . \ I A ~ A N T 0 1 N AN I';N
5,'pec~fic activity qf starting material
Precipitation hv (NH4)2HO 4 ~'al~rali(m range ("o)
Elution j r o m Specll/~c DE,.:IE-celhdose aclivitv l'21ulion range q/ (31 .\: aC l ) purified material
t)uri/ication factor
Recovery (Oo)
2.J t.2 ~.35
57 1oo 57 1oo o 33
o.2o 0.35 o.2o o . 2 8 o.22 0.35 o.2, 0.35 o. t5 0.35 o. io o . t 5 o.o5 o. io o. t5 0.25 o o. 1o
5.5 4.5 4.5 37 5-5 2. 5 3" 15 I-'
37 ~o t5 83 75
~.5 o.2~) o.55 0.07 0.029 °.°37
0 57 57 t o o o..to o 57 45 ioo 45 t~5
ll.(, 5. t ~,.o
55 1-55 t.43 o.2" 0.45 o.45
* M e a s u r e d a f t e r (NH~),aS( h p r e c i p i t a t i o n . T h e a c t i v i t y pletely after D E A E - c e l l u l o s e c h r o m a t o g r a p h y a n d dialysis.
had
disappeared
2" 7 °* (1o 7°
almost com-
No or only small loss of activity occurred during the purification of the enzymes from Pseudomonas species and higher plants. Also, the enzymes from soybeans ~° and mung beans n appeared rather stable. Allantoinases from E. coli, Penicillium ,notatum and animal livers were easily inactivated, mainly during DEAE-cellulose chromatography and dialysis. As a result of the purification procedure the allantoinases were purified 2.5- to 37-fold. The amount of nucleic acids in the bacterial extracts diminished from above 2o% to below 2%. No allantoate degrading enzymes were present in the purified allantoinases, except in the case of A. allantoicus; this fact, however, did not affect the analytical measurements, as the products formed from allantoate react positively in the differential glyoxylate analysis according to method B 14. NAGAI AND F U N A H A S H I n purified allantoinase from mung beans by calcium phosphate-gel treatment, followed by (NH4) 2SO4 and acetone fractionation ; the specific activity increased from o.oo5 to o.3, and only 32% of the original activity was lost. LEE AND ROUSH~° enhanced the specific activity of bakers' yeast allantoinase (o.oo4 units/mg protein) 6-fold by a similar procedure to that given here ; 42 % of the original activity remained. Under the same conditions the enzyme from Candida utilis was easily inactivated 1°. Thus it appears that allantoinases from higher plants and Pseudomonas species are relatively stable, whereas the enzymes from other bacteria, fungi, yeasts and basidiomycetes ~ are rather unstable.
pH @lima Ro 9 found an optimal allantoinase activity between pH 6.6 and 7.3 for the enzyme from soybeans, LEE AND ROUSH1° in the range from p H 7 8 for the enzyme from bakers' yeast and VInLERST 5 at p H 7.3 for allantoinase from algae. NAGAI AND FUNAHASHIn obtained a p H optimum between 7.5-8.5 for mung bean allantoinase, this pH optimum curve was measured partly in phosphate buffer. In view of the inBiochim. Biophys. ,4cta, 122 (1966) 4 8 2 - 4 9 6
487
ALLANTOINASES. I
"~
1
~moo
~250~
"B
"-6
o
6
6--
~5o
a
E
~'800 I I
I 600
/,
150q
×'i
;×
I I i
\
30
-i I I
400
I I00 1
,,
// /,/
I
i
~"
'
o
200
/ I
OI 6
,, *,
2
%, •
%
,, ,,
/
Sa
:/
,
x, /'
-t
I I !
/ 4',N
I i&
I
"i
I I
;
//
/ 7
6
9
6 pH
7
6a ,'95a
6
9
pH
Figs. Ia a n d lb. p H o p t i m u m c u r v e s of a l l a n t o i n a s e s f r o m eight different origins. A t 3 °0 a m i x t u r e w a s i n c u b a t e d c o n t a i n i n g , per rnl, 4o/zmoles allantoin, i 6 o p m o l e s buffer a n d purified a l l a n t o i n a s e : 2 5 # g (d. allantoicus, C u r v e i); i i tzg (E. coli, C u r v e 2); 4o/zg (S. aUantoicus, C u r v e 3) ; 1.2 ~g (P. acidovorans, C u r v e 4) ; 5.3/~g (frog liver, C u r v e s 5 a n d 5 a) ; 4 ° / ~ g (goldfish liver, C u r v e s 6 a n d 6a) ; 290 # g (Ph. hysterinus, Curves 7 a n d 7 a) a n d 53 ° / z g (soybeans, Curves 8 a n d 8a). Moreover, o.I /*mole MnSO 4 a n d io # m o l e s G S H were p r e s e n t in t h e first t h r e e e x a m p l e s . Tris HC1 buffers were u s e d in t h e r a n g e p H 6.9-8.1, d i e t h a n o l a m i n e - H C 1 buffers in t h e r a n g e p H 7.6-9.0, N a 2 C O s - N a H C O s buffers in t h e r a n g e p H 8.4~9. 4 a n d acetic a c i d - s o d i u m a c e t a t e buffers in t h e r a n g e p H 5.7-6.1. I n f o u r cases (Curves 5a, 6a, 7 a a n d 8a) p h o s p h a t e buffers were u s e d in t h e r a n g e p H 6.5-7.5. A l l a n t o a t e f o r m e d w a s m e a s u r e d b y t h e differential g l y o x y l a t e analysis.
hibiting effect of phosphate ions, to be discussed elsewhere 16, the lower range of their p H optimum curve would seem to be invalid. p H optimum curves of eight allantoinases are shown in Figs. I a and lb. The curve found for P. fluorescens was the same as that for P. acidovorans. The p H optim u m ranges of the bacterial allantoinases are narrow; the aChvlhes of the enzymes from higher plants and animal livers are less p H dependent. Optimal activity of allantoinase was observed between p H 7.5 and 8.5. Although p H activity curves with phosphate buffers are given for some preparations, the p H optima were derived only from experiments without phosphate present. ~ m values
Table V presents the Kra values of a series of aUantoinases. The values v a r y from 5" IO-a M (S. allantoicus and animal livers) to about 4 °. lO -3 M (Pseudomonas species and most plants). These values refer to ( + - - ) - a l l a n t o i n as substrate. As only the three first mentioned allantoinases are aspecific, it is more exact to consider ( ~-)Biochim. Biophys. Acta, 122 (1966) 4 5 2 - , 9 6
4~N
~,. IL Vt)(;I;LS, 1,. TI(IJIH~I.S . \ N I ) . \ .
Ut.'I;INI(
"1'.\ }~LI~ V ]£m \ ' A L / ! E S ANI) A C T I V A T I O N
I';NI-RIIIES ~)F ALLANTOINASP;-%
1(~,, v a l u e s w e r e ( l e t e r m i n e d b y m e a s u r i n g t h e v e l o c i t i e s a t d i f f e r e n t s u b s t r a t e c o n ( e n t r a t i o n s a n d p](~tting t h e results a c c o r d i n g t~) t h e m e t h o d o f t~[OFSTEE 2s. T]le i n c u b a t i o n m i x t u r e s c o n t a i n e d . p e r ml, i 7 5 1 m u ) l c s d i e t h a n o l a m i n e HCI b u f f e r ( p l l 7.7), p u r i i i e d a l l a n t o i n a s e , a n d a l l a n t c m l v a r y i n g f r o m (~ 3 9 / t m o l e s . In t ) r e p a r a t i o n s f r o m N. allantoicus, 1'2. coil a n d ..I. a l l a n l , i c u s , o. i / t m o h , MnS()t and 1o/tmoles (;SIt were also present. The mixtures were incubated at 3° . Allantoatc f i ) r m e d was d e t e r m i n e d b y t h e difl\~rential g l y o x y l a t e a n a l y s i s . A c t i v a t i o n energies were d e t e r m i n e d b y m e a s u r i n g t h e v e l o c i t i e s a t d i f f e r e n t t e m p e r a t u r e s in m i x t u r e s containing, p e r ml, 1 9 o / , m o l e s d i e t h a n o l a m i n e tt(71 buffer ( p l t 7.7), f )urified a l l a n t o i n a s e , a n d 4.2 p m o t e s a l l a n t o i n . In e x p e r i m e n t s w i t h N. altantoicus, l J. coli a n d ,q. allanloicus o.t t , m o l e M n S ( ) t a n d l o / , l n o l e s ( ; S H w e r e a l s o p r e s e n t , a n d in e x p e r i m e n t s w i t h P h . h v s t e r i n u s a n d s ~ y l ) e a n s o. ~ /ma(He M n S ( ) ~ A r r h e n i u s p l o t s x~ere m a d e , a n d t h e a c t i v a t i o n energies w e r e c a l c u l a t e d . OrgarHsm
Nm ( M × zo")
. t cli~,alion c~tergy (cal/mole)
5,'. allanloicus =t. allantoicus
4.9 14
t ~ Soo 9 o50 8 35 ° I I 75 ° i t 9o0 S ~)oo
l'2. coil
22
P. acidovorans P . fluorescens F r o g liver
45 35 6 S. 4 .t(i 14
Goldfish liver P h . hysterim~s
G. hi@ida Bakers' yeast*
3o
M u n g b¢2an **
4°
9 ooo ~) t o o
* V a l u e f r o m L E E AND [{OUSI{ 10. ** V & l u e f r o l n N A G A I AND F U N A H A S H 1 1 1 .
allantoin as the substrate for the others. Then, two groups of Km values can be distinguished: ( 1 8 ± 4 ) . I o a M for A. allantoicus, E. coli, the Pseudomonas species, bakers' yeast and plants, except soybeans, and ( 5 ~ 2 ) " 1 o -3 M for S. allantoicus, the animal livers and soybeans. For allantoinase from S. allantoicus the same Km was found if Mn 2+ was omitted from the medium or replaced by Zn 2+. Moreover, this value did not change if the diethanolamine-HC1 buffer was replaced by a phosphate buffer (pH 7.5).
Activation energies The activation energy was determined by comparison of the allantoinase activity at different incubation temperatures (Table V). Three activation energies amounted to 11.8 kcal/mole (S. allantoicus and the Pseudomonas species); in five other preparations this value was about 8. 9 kcal/mole. These values were measured in the temperature range from o-27 °, where the Arrhenius plots showed straight lines (Fig. 2) ; above 27 ° deviations occurred from a straight line plot. Similar results were found for other enzymes e.g. urease ~9 (urea amidohydrolase, EC 3.5.1.5) and glycogen phosphorylasO ° (~-i,4-glucan: orthophosphate glucosyltransferase, EC 2.4.1.1 ). An explanation for these results cannot be given at present; the deflection is probably not due to a denaturation effect, as the enzyme, especially from plants, is stable at temperatures up to 55 °. Biochim. ~)iophys. /tcta, 1,22 (1966) 482-,t96
ALLANTOINASES. I
489
>
~2 r~
320
.~40
360
'
!.Io 5 T
Fig. 2. D e t e r m i n a t i o n of t h e activation energies of allantoinases from Ph. hysterinus (i), P. fluorescens (2) and frog liver (3). I n c u b a t i o n and analysis were performed as given in Table V.
I _
L
E
40 i"
tJ o
."
20~ I
z
(.R
t
I I
,7 30
/
t
I
I
I-
i
l F 15
I t
'
i
I I
/
20
×
t t t
L 10 I
×
/
//
! oo
I
x/
I r
!
o
I
/× I0
,
e.~.
I
-~ -70 ° b
~ -60
/
o
-50"
I I
, •
L I I I I
O
O Incu]:,,=tlon time (n',An)
Fig. 3- D e g r a d a t i o n of ( + - - ) - a l l a n t o i n b y allantoinase f r o m P. acidovorans, change in optical rotation and d e t e r m i n a t i o n of the specific optical r o t a t i o n of (--)-allantoin. The i n c u b a t i o n m i x t u r e at 3 °o contained, per ml, 46/~moles ( + - - ) - a l l a n t o i n , zoo/*moles diethanolamine-HC1 buffer (pH 7.7) and 15. 5/zg purified allantoinase. Allantoate formed was m e a s u r e d b y t h e differential glyoxylate analysis.
Biochim. Biophys. Acta, 122 (1966) 482-496
4()0
t;. IL V()(',I~I.S, 1,'. I'RIJBI'A~S ANI/ .\. I'FFINI,~
optical specificity I n previous studies a st)ecificity fl~r (F)-allantoin was reported fl)r tilt: e n z w n c , from soybcansl°,la, la, Ph. hysteri'mts 13, P. acidovorans 14 and fish liver al. The enzvm~,~ from S. allantoicus and A. alla~ztoicus were not specific f~)r one of the optical Jsolner~ of allantoin 7. Results obtained with allantoinase from P. acidovorans demonstrated that this enzyme degraded (+)-allantoin nmch faster than (--)-allantoin (Fig. 3). The apparent specific rotation [a]~° was calculated from the a m o u n t of allantoate formed and th/~ observed optical rotation. By extrapolation of these values a specific rotation [c~i~/°of 8 o ~ i ° for ( -)-allantoin was estimated, which is lower than the value of - 9 2~ found by FOSSE, THOMAS AND DE (IRAEVE ~5, but in good agreement with the results of I,EE AND ROUSH ~°. On the basis of this value the initial velocities ti)r the degradation of (i-~) - and ( --)-allantoin were calculated from the analytical and polarimetric data. Figs. 4 a and 4b show the results for frog liver and Ph. hysterinus. Three allantoinases (S. allantoicus, A. allantoicus and E. coli) are aspecific (Table VI); the degradation rates for the two optical isomers differed less than I ~,; and never was there an excess of more than o.5% of one of the isomers during degradation. With the enzyme from all other sources the ( + ) - f o r m was degraded faster than the ( - ) - f o r m , but the latter was always degraded to(). In our hands none of the allantoinases was absolutely stereospecific. LEE AND ROUSH 1°, while obtaining results similar to ours fi>r bakers' yeast, concluded from their experiments t h a t soybean allantoinase was completely stereospecific. However, "FH(~51as AND DE (IRAEVE 32 had previously demonstrated that the enzyme from soybeans degraded (--)-allantoin 2.6-fold faster than (--)-allantoin. T A B L E VI SPECIFICITY ALLANTOIN
OF AND
ALLANTOINASES
FOR
THE
OPTICAL
ISOMERS
OF
ALLANTOIN
ANt)
FOR
METHYI.OL
-
5-AMINOHYDANTO1N
The e x p e r i m e n t s were performed at 3°o in m i x t u r e s containing, per ml, 39/tmoles ( I )-allantoin, nlethylolallantoin or 5 - a m i n o h y d a n t o i n (for P. acidovorans 46pmoles), I75 ttmoles diethanolamine ItCI buffer (pH 7.7) (for P. acidovora~s 200 t~moles) and purified allantoinase: 68tlg (5". allantoicus) ; 02/zg ( A . allantoiczts); 50big (E. coli); I5.5t~g (P. acidovora~s) ; 3o/~g (P. ftuoresce~s) ; 15o fig (frog liver) ; 8 0 o / , g (Ph. h3,sterinus ) or 46o tzg (soybeans). In the e x p e r i m e n t s with ~4. alla~ztoiczes and E. coli, moreover, 12/*moles thioglycolate were present and in those with S. allantoicus, A. allantoicus, E. coli, Ph. hysterinus and s o y b e a n s o . i / , m o l e MnSO 4. The degradation of the s u b s t r a t e was followed by m e a s u r i n g the p r o d u c t by the differential glyoxylate analysis (method B). Two e x a m p l e s of the calculation of the degradation ratio ( + )-allantoin to ( )-allan-toin are given in Figs. 4 a and 4b.
Orga~dsm
Degradation rates
( ~ J-form~( J-form
S. A. E. P. P.
allantoicus allantoicus coli acidovorans fluorescens
Frog liver
Ph. hysterinus G. hispida
Degradation rates*
(%)
Methylolallantoi~z
5-,4 mi nohydantoin
1 I
45 25
6-4 4
1
6
2i. 5 6. 3 4-9 13.,5 4-4
8 3° 3° 37.5 5°
o o o o-7 0.9 0.5
* Given in per cent of the degradation rate obtained with ( ~
Biochim. Bioph),s. Acta, 122 (196t,) 482-496
)-allantoin.
ALLANTOINASES. I
49I
~2s
1 c+ -)-allantom"
~ 20
', [ ~- 4
C+-)-allantom"
jx
/
x
, ,_ 20 °
oO15 U
< lo L
54
il
,Y/
' J
zx~/(÷~-allantoin
o ~
0 0
30
60
90
120 150 180 0 I~cu]0atio~ time (rain)
60
120 180 240 I n c u b a t i o n time (rain)
Figs. 4 a a n d 4 b. D e g r a d a t i o n of ( + - - ) - a l l a n t o i n a n d c h a n g e in optical r o t a t i o n b y a l l a n t o i n a s e s f r o m Ph. hysterinus (Fig. 4 a) a n d frog liver (Fig. 4b). T h e e x p e r i m e n t s were p e r f o r m e d a t 3 °° in m i x t u r e s c o n t a i n i n g , per ml, 3 9 . o / , m o l e s allantoin, 1 7 5 / , m o l e s d i e t h a n o l a m i n e - H C 1 buffer (pH 7.7) a n d purified a l l a n t o i n a s e (o.86 m g f r o m Ph. hysterinus or o.15 m g f r o m frog liver). I n t h e f o r m e r case o. I/~mole M n S O 4 w a s p r e s e n t . F r o m t h e d e g r a d a t i o n c u r v e of ( + - - ) - a l l a n t o i n a n d t h e c h a n g e in optical r o t a t i o n t h e d e g r a d a t i o n s of ( + ) - a n d ( - - ) - a l l a n t o i n were calculated.
Two groups of allantoinases can be distinguished on the basis of optical specificity. The first group is formed by the aspecific enzymes from S. allantoicus, A. allantoicus and E. coli, bacteria that grow in allantoin media only under anaerobic conditions. In the second group the enzymes from higher plants, several microorganisms (Pseudomonas species, bakers' yeast 1°) and animal livers (frog, goldfish, Raja clavata al) can be classified that degrade (+)-allantoin 5-20 times faster than (--)-allantoin.
Degradation of allantoin derivatives 3-Methylallantoin and i-acetylallantoin were not degraded by any of the nine enzymes tested. 1,3-Diacetylallantoin and 5-acetylaminohydantoin were not attacked by allantoinases from S. allantoicus and A. alIantoicusL Methylolallantoin and 5aminohydantoin were broken down at velocities presented in Table VI and Figs. 5 a
//1
~50
o~
~4o u t~
20 ¸
/ /x
10.
lO 20 30 40 0 Incubatior~ t i m e (rain)
2()
40 6'0 8'0 100 I n c u b a t i o r l t i m e (rain)
Figs. 5a a n d 5b. D e g r a d a t i o n of a l l a n t o i n (i), m e t h y l o l M l a n t o i n (2) a n d 5 - a m i n o h y d a n t o i n (3) b y a l l a n t o i n a s e s f r o m A. allantoicus (Fig. 5 a) a n d E. coli (Fig. 5b). D e g r a d a t i o n w a s t e s t e d u n d e r c o n d i t i o n s g i v e n in T a b l e VI.
Biochim. Biophys. Acta, 122 (1966) 482-496
4{)2
~;. 1). \'{)(;El.S, F. "II~IJBIiI.S .\NI~ .\. I'FFINi,;
and 51). All allantoin derivatives were tested under the conditions given in Table V I. Methylolatlantoin was degraded at 25-5o~!i~ of the rate for allantoin; ,mlv I~,r allantoinases from E. coil and 1'. acidovoraus was the rate lower (8o:o). The positi(m of the methylol group in the allantoin molecule is unknown; since substituents in the hydantoin moiety prevent degradation by allantoinase, the most probable positi(m of the m e t h y M group is the 8- or 6-position in the molecule. Allantoinases from 5. alla~2loicHs and A. allantoicus could use 5-aminohydantoin as substrate, although slowh'. The other allantoinases degraded this compound at a very l.w rate or not at all. Allantoinases activated by Mn '+ (ref. 16) degraded methylolallantoin and 5aminohydantoin faster in the presence of these ions. The enhancement of activity was for all substrates in the same order of magnitude.
Heat stability The effect of 5 min heat pretreatment on the activity of allantoinases is shown in Fig. 6. Most of the enzymes were largely inactivated by heating between 5o ° and 6o °. The enzymes from soybeans and Ph. hysterinus were much more stable, though not as stable as reported by Ro 9 who found only little loss of activity after heating for 30 rain at 7°o and loss of half the activity at 8o ° for 30 min. The stability . f
~1oo~.~.~.~ , 6O
"
20 x
Vxx,~ V 0 0
. 40"
.
& .
. 5'0°
60 °
i 70 °
60 ° Temperc~ture
Fig. O. I n a c t i v a t i o n b y h e a t p r e t r e a t m e n t o f a l l a n t o i n a s e s f r o m E. coli (k~), P. acidovorans (W), f r o g l i v e r (A), S. allantoicus ( 0 ) , A. allantoicus (0), P. fluoresrens ((~), g o l d f i s h l i v e r ( × ) , Ph. hvsterinus ([7) a n d G. hi@ida (11). A n a l i q u o t o f e n z y m e w a s h e a t e d for 5 m i n a t t h e i n d i c a t e d t e m p e r a t u r e s a t p H 7-5- T h e r e s u l t i n g a c t i v i t y w a s t e s t e d u n d e r s t a n d a r d c o n d i t i o n s a n d c o m pared with untreated material.
Biochim. Biophys. Acta, 122 (t066) 4 8 2 - 4 9 6
ALLANTOINASES. I
493
allantoinase from bakers' yeast 1° was similar to that found for most allantoinases. This enzyme was stable up to 4°° and was inactivated above 55 ° when held at pH 7 for 20 rain,
Inactivation by urea After pretreatment in 5 M urea for IO rain at 20 °, enzyme activity was tested in mixtures containing I or 5 M urea. The activities measured were compared with the activity of untreated material in the absence of urea (Table vii). In the presence of 5 M urea the allantoinase activities were very low; after dilution to I M urea, the activities were about half those of the untreated material. TABLE
VII
EFFECT OF PRETREATMENT OF ALLANTOINASES IN 5 M UREA A n a l i q u o t o f t h e e n z y m e s o l u t i o n w a s m i x e d w i t h a n e q u a l v o l u m e o f i o M u r e a ( p H 8.o) a t 2o °. A f t e r IO m i n o n e s a m p l e w a s i n c u b a t e d a t 3 °0 i n a m i x t u r e c o n t a i n i n g , p e r m l , 2 0 / ~ m o l e s a l l a n t o i n , i o o / ~ m o l e s d i e t h a n o l a m i n e - H C 1 b u f f e r ( p H 8.0) a n d s u c h a n a m o u n t of t h e t r e a t e d e n z y m e t h a t t h e f i n a l c o n c e n t r a t i o n o f u r e a w a s I M, A n o t h e r s a m p l e w a s t e s t e d i n a s i m i l a r m a n n e r i n an incubation mixture containing 5 M urea. The velocities measured were compared with the r e s u l t s o f a t e s t in w h i c h u r e a w a s o m i t t e d . M n 2+ ( o . i # m o l e ] m l ) w a s p r e s e n t i n t h e t e s t s w i t h S. allantoicus, A. allantoicus, E. coli, Ph. hysterinus a n d s o y b e a n s ; G S H ( 3 / ~ m o l e s / m l ) i n t h e t e s t s w i t h E. coli a n d A . allantoicus.
Organism
Activity ( % ) 5 M Urea I M Urea
S. A. E. P. P.
allantoicus allantoicus coli acidovorans fluorescens
Frog liver Goldfish liver
Ph. hysterinus G. hispida
io 16 2
o o I io 6 6
54 75 18 33 61 42 49 64 55
Stability in acidic solutions The effect of incubation with o.I M sodium acetate-acetic acid buffers at pH values between 5-4 and 4.0 is presented in Fig. 7. Most allantoinases were inactivated readily under these conditions. The enzyme from E. coli was inactivated partly even at a pH of about 7, but a small activity remained at pH 4.0. Allantoinases from Ph. hysterinus and soybeans were activated 1.3- to 1.6-fold below pH 5.4. The effect of temperature and preincubation time is given in Fig. 8. The activating effect of acid pretreatment on these allantoinases is perhaps due to a mechanism similar to that in the case of allantoate amidohydrolases from S, allantoicus, A. allantoicus, E. coli and P. acidovoransT, 2~. The activity of these enzymes is enhanced IO-IOO times by acid pretreatment at o ° for 30 sec. Preliminary results obtained with aUantoinases from Pen. notatum, wheat and gherkin indicate that these enzymes are inactivated by acid pretreatment. Biochim. Biophys. Acta, 122 (1966) 4 8 2 - 4 9 6
41)4
(;.
IL
V()GIC, I . S ,
I,'. F I , : I J I { I Z L S
..\NI)A.
UI;I;IN[,,
$ 175 >,
/
x" " ~ " ×---....x....x
150
\/'~'~ I00
75 G.
50
(•)
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l~a.(*) A.=~o)
\k\ V X,~
25
,
,
em"~ I
j
5 4 pH d u r i n g p r e t r e a t m e n t
Fig. 7. S t a b i l i t y of a l l a n t o i n a s e s in acidic media. To aliquots of t h e e n z y m e solution 3 v o l u m e s of o.i M s o d i u m a c e t a t e - a c e t i c acid buffer w i t h different p H v a l u e s were added. T h e p H of t h e mixt u r e s is i n d i c a t e d in t h e figure. After 6 m i n at 3 °0 a s a m p l e of t h e m i x t u r e was a d d e d to a n allantoin solution. I n t h e i n c u b a t i o n m i x t u r e s a t 3 °° t h e r e were present, per mI, 2oo/*moles d i e t h a n o l a m i n e HC1 buffer (pH 7.7) a n d 4 2/tlnoles allantoin. F u r t h e r m o r e , io/~lnoles G S H were p r e s e n t in t h e t e s t s with ,4. allantoicus a n d E. coli, a n d o . i / , m o l e s MnSO 4 in t h e s a m e t e s t s a n d in t h e experim e n t s w i t h S. allanloicus, Ph. h.vsterinus a n d s o y b e a n s . T h e a m o u n t s of purified a l l a n t o i n a s e s (in/~g) p r e s e n t per ml i n c u b a t i o n m i x t u r e , a n d t h e velocities m e a s u r e d , w h e n i n s t e a d of a c e t a t e buffer w a t e r was used, are given in this order w i t h i n p a r e n t h e s e s : S. allanloicus (S.a., 15.6, lO5), ~4. allanloicus (A.a., 8, 23), E. coli (E.c., 5 o, I24), P. acidovorans (P.a., 66, 29), P. fluorescens (P.f., I1, 4.5), frog liver (F., 12.5, 7.8), goldfish liver (G., 57 o, 5.I), Ph. hvsterinus (P.h. 21o, 75) a n d s o y b e a n s (G.h., 380, 99). T h e velocities m e a s u r e d after p r e t r e a t m e n t a t t h e indicated p H are e x p r e s s e d in % of t h e velocity m e a s u r e d with t h e u n t r e a t e d material.
DISCUSSION
Allantoinase is the only enzyme known to attack allantoin, perhaps with the exception of "allantoin oxidase", which was postulated by FRANKE3a for Alternaria tenuis, but this needs confirmation. In this study a comparison was made of some properties of allantoinases from S. allantoicus, A. allantoicus, E. coli, P. acidovorans, P. fluorescens, frog liver, goldfish liver, Ph. hysterinus and soybeans. Furthermore, the results were compared with previous studies of the enzymes from bakers' yeast, Candida utilis, Pen. notatum, basidiomycetes, Raja clavata and mung beans. The behaviors during a purification procedure, involving (NH4)~SO 4 precipitation and DEAE-cellulose chromatography, the pH optimum curve, Kin, activation energy and the stability on storage, heating, urea treatment and acid pretreatment were compared. The specificity for ( + ) - and (--)-allantoin and allantoin derivatives was also studied. The allantoinases differed from each other in each of the properties mentioned. Four groups of enzyme sources were used in this study: animal livers, Biochi,m. Biophys. ,4eta, t22 (1966) 482 496
ALLANTOINASES. I
495
160 v
< 140
120
7"
pH during pretrealrnent
Fig. 8. A c i d a c t i v a t i o n o f a l l a n t o i n a s e f r o m Ph. hysterinus. T h e e x p e r i m e n t w a s p e r f o r m e d as d e s c r i b e d in Fig. 7. T h e p r e t r e a t m e n t w i t h a c e t a t e buffer w a s p e r f o r m e d e i t h e r d u r i n g 6 rain a t 3 °0 (I), 6 m i n at o ° (2) or h a l f a m i n u t e a t o ° (3)'
plant seeds, Pseudomonas species and three bacteria that can use allantoin as sole organic substrate for growth under anaerobic conditions. Some differences in the enzyme properties followed this classification, namely pH optimum curve and stability on storage, heating and acid pretreatment. Other differences showed no correlation with this classification, namely behavior during purification, Kin, activation energy and specificity for allantoin derivatives. The enzymes from bacteria that degrade allantoin under anaerobic conditions were not stereospecific. Since allantoin can racemize 15, the compound may be present in the optically inactive form in decaying biological material. S. allantoicus, A. allantoicus, and Escherichia species are probably the scavengers of this compound, which is present in large quantities in nature as an excretion product of animals and as a compound involved in the nitrogen storage and transport in a large number of plant families. REFERENCES i 2 3 4 5 6 7 8 9 io II
M. LASKOWSKI,Enzymes, I (1951) 946. L. GOLDS'rEIN AND R. P. FOSTER, Comp. Biochem. Physiol., 14 (1965) 567. I. GOODBOD'¢, J. Exptl. Biol., 42 (1965) 299. M. V. TRACEY, in K. PAECH AND M. V. TRACEY, Moderne Methoden der Pflanzenanalyse, Vol. 4. Springer, Berlin, 1955, p. 119. S. VILLERET, Compt. Rend., 241 (1955) 90. S. VILLERET, Compt. Rend., 246 (1958) 1452. G. D. VOGELS, Thesis, I n s t i t u t e o f T e c h n o l o g y , Delft, 1963. R. F o s s e AND A. BRUNEL, Compt. Rend., 188 (1929) 426. K. Ro, J. Biochem. Tokyo, 14 (1932) 405 ` K. W. LEE AND A. H. ROUSH, Arch. Biochem. Biophys., lO8 (1964) 46o. Y. NAGAI AND S. FUNAHASHI, Agr. Biol. Chem. Tokyo, 25 (1961) 265.
Biochim. Biophys. Acta, 122 (1966) 482-496
490 I2 13 14 15 16 17 I8 19 2o 2I 22 23 x4 25 26 27 28 29 3° 31 32 33
~;. IL \:~(il,;l.S, 1~. IRIJt3F.I.5 AND A. UFI:INIx
A. ]~RUNEL, Thesis, l i n i x e r s i t v of P a r i s , 1931~. CHR. VAN DER DRIFT AND (;. :1). VOGELS, .qcla ]JOla~¢. ~\:eer[., 15 (:900) -'(J~. F. TRIJBELS AND (;. 1). VOGELS, Biochim. Biophys. ,qcta, i t 3 (19()()) 2(~2. R. FOSSE, P . - E . THOMAS AND 1'. DE (;RAI~:VI.:, Conzpl. l¢~nd., i 9 8 (1034) ogq. G. I). VOGELS a~,'I) ('HR. VAN DER I)RIFT, Bi,wkim. Biopk3's..q~/a, ,2" (I9o()) 497. t,2. YANo, T. IKEr)A, S. TSVI~tTA, C.'I'ANL lq. NAKAJ1MA AND Y. MoJ~I,C/2em. qb~tr.,45 (~(J491 759 o. H. BIL'rZ AND 1{. I{OBL, I/of., 54 ( r 9 2 I ) 2448. H. BILTZ AND L. LOEWE, .[. Prakt. Chem., 14[ (1934) 284. H. BILTZ AND E . GIESLVR, 13er., 40 (1913) 3410H. BILTZ AND H . ItANlSCH, .[. Prakt. Che~n., ~I2 (1920) 138. G. D. VOGELS, Biochim. I3iopkx,s. dcta, i [ 3 (1900) 277. O. H . I . O W R Y , N. J. I~OSEBROUGH, \ . 1.. FARR AND R. J. I{ANDALL, J . l~iol. Chem., lq 3 (~t~5I) 265. F. TRIJBELS AND G. I). \'Oc;t':LS, Biochim. Biophys. dcla, t18 ([900) 387 . L. L. CAMPBELL, JR., J . Bacteriol., 08 (I954) 598. IC ECNEVlN AND A. BRUNEL, CompL Rend., 2o 5 (1937) 294. 1C t~;CHFVlN AND A. B~UNEL, Compl. l?e~d., 2o8 (~939) 826. B. H. J. HOI~'STEE, Ncience, I ~, (1952) 329 . G. B. KISTIAKOWSKY AND 1C LUMR¥, .]..qm. Chem. Sot., 71 (1949) 2oo0. D. J. (;RAVin:s, R. \V. SI':ALOCK AND J. H. WANC, Biochemisl~9,, 4 (1965) 29 °. R. FOSSE, I'.-E. THOS~AS AND I'. IIr: GRArVE, Compl. Rend., 198 (1934) 1374P . - E . THOMAS AND P. DE GIaAEVE, CompL Rend., 108 (1934) 22o5. W. FRANKE, Z. Vilamin Hormo~ I:ermen(forsch., 5 (1953) 279.
Biovhim. Biophys. Acta, i22 (L966) 4 8 z - 4 9 0