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Allantoinases from bacterial, plant and animal sources II. Effect of bivalent cations and reducing substances on the enzymic activity
BIOCHIMICA ET BIOPHYSICA ACTA
497
BBA 65446 A L L A N T O I N A S E S FROM B A C T E R I A L , P L A N T AND ANIMAL SOURCES II. E F F E C T OF B I V A L E N T CATIONS AND R E D U C I N G SUBSTANCES ON T H E ENZYMIC A C T I V I T Y
G. D. V O G E L S AND C H R . V A N D E R D R I F T
Department of Biochemistry, University of Nijmegen, Nijmegen (The Netherlands) (Received F e b r u a r y i o t h , 1966)
SUMMARY
I. The effect of manganous and other bivalent cations and of reducing substances on the activity of nine allantoinases (aUantoin amidohydrolase, EC 3.5.2.5) was studied. 2. At least four groups of allantoinases could be distinguished. The first group includes the enzymes from Streptococcus allantoicus, Arthrobacter allantoicus and Escherichia coli; the second group includes the enzymes from two Pseudomonas species; the third, the enzymes from frog and goldfish liver; and the fourth, the enzymes from Phaseolus hysterinus Dur. and Glycine hispida L. 3. The enzymes in the first group were aspecific for ( + ) - or (--)-allantoin, in contrast to the other allantoinases, and were activated by Mn 2+ and reducing substances. The allantoinases in the second group were inhibited b y Mn 2+ and insensitive to reducing substances. The third group includes enzymes which were inhibited b y both additions and the enzymes of the fourth group were activated b y Mn 2+ and inhibited b y cysteine. 4. The effects of Mn *+ and of reducing substances on allantoinases of the first and fourth groups influenced each other. 5. The inhibiting effect of Cd 2+ on the activity of allantoinases from the first group was largely prevented by previous addition of GSH. 6. In phosphate buffers the activity of allantoinases, except for those in the second group, was inhibited. This effect was partly due to removal of Mn 2+ from the enzyme. 7. The structural differences of the active sites in allantoinases of the four groups are discussed.
INTRODUCTION
Allantoinases (allantoin amidohydrolase, EC 3.5.2.5) from Streptococcus allantoicus and Arthrobacter allantoicus are activated b y Mn ~+ and reducing substances 1. Biochim. Biophys. Acta, 122 (1966) 497-5o9
4(),N
(i. ]3. Vi)(;I,;I.S A N I ) ( . V A N 1)1~1,~ I)RIF'I
No activating effect of Mn s4 or other bivalent cations was found fi)r allant(~inases from mung beans 2 and baker~' yeast 3. The enzymes from bakers' yeast, soybeans :~ and Phaseolus hystefinus 4 are strongly inhibited on addition of cysteine. In order to obtain more information on the observed differences, the effects of bivalent cations and r~ ducing substances on the activities of atlantoinases from nine origins were studied and are reported in the present paper. The properties and purification of the enzymes haw~ been reported previously ~. EXPERIMENTAL
Growth of bacteria, preparation of cell-free extracts and purification of the enzymes were performed as previously described 5. The degradation of allantoin was measured by the differential glyoxylate analysis (method B) after incubation under standard conditions ~. RESULTS
Effect of bivalent cations on allantoinases NAGAI AND FUNAHASHI2 tested the effect of Mg s+, Mn 2+, Ca 2+, Cos+, Ni s+, Zn s+, Cu s+, Fe 3+, EDTA, iodoacetate and KCN (all at lO -3 M) on purified allantoinase from mung beans. Cu 2+ inhibited the reaction strongly, Mn 2+ diminished the activity by 5% and the other additions were without effect. LEE AND ROUSH3 reported that ions inhibited bakers' yeast allantoinase only slightly, and stated that no direct evidence was found for a co-factor for this enzyme. Nevertheless, yeast allantoinase lost 65 % activity on dialysis, in contrast with soybean allantoinase. Allantoinases from S. allantoicus and A. allantoicus were inhibited by Hg s+, Cu 2+, Zn ~+, Fe 2+, Co2+ and EDTA (lO -3 and lO -4 M) 1. No change in activity was caused by Mg s+, Ca 2+, Sr 2+, Ba 2+, Ni s+ and Fe 3+, but Mn ~+ enhanced the enzymic activity in crude extracts 1.3- and 2.o-fold, respectively. Table I presents the effects of Mn 2+, Zn ~+, Co 2+, Cd 2+ and EDTA on purified allantoinases from S. allantoicus, A. allantoicus, Escherichia coli, Pseudomonas acidovorans, Pseudomonas fluorescens, liver of frogs (Rana esculenta) and goldfishes (Carassius auratus), Phaseolus hysterinus Dur. and soybeans (Glycine hi@ida L.). Zn 2+, Co2+ and Cd 2+ inhibited all allantoinases, except those from higher plants which were slightly activated. Allantoinases from Pseudomonas species were strongly inhibited by these ions, the enzymes from animal livers, S. allantoicus, A. allantoicus and E. coli were less inhibited. Pseudomonas allantoinases were inhibited strongly by Mn s+, enzymes from animal livers only IO%, and enzymes from higher plants were activated about 2-fold. Not all plant allantoinases were activated by Mn s+ however, since preliminary results with the enzymes from wheat and gherkin showed inhibition by I O - ~ M Mn 2+. The effect of Mn 2+, Cd s+, Cos+, Zn 2+ and EDTA on the allantoinase from S. allantoicus is shown in Figs. Ia and lb. These additions were tested at various concentrations in the presence or absence of 7 • lO -5 M MnSO 4. Zn s+, Co2+ and EDTA lowered the activity to about one-third of the original activity in the presence of MnSO4, whereas in the absence of Mn ~+ only a slight inhibiting effect was observed. Since EDTA, Zn 2+ and Co~+ inhibited the reaction to the same extent and were inhibitors Biochim. Biophys. Acta, 1 2 2 ( 1 9 6 6 ) 4 9 7 - 5 0 9
~o
I
~o &
t~
7~
I ON THE ACTIVITIES OF ALLANTOINASES
allantoicus allantoicus coli acidovorans fluorescens
20 22 I 9 4 45 25 ° 560
17
Purified allantoinase (#g/ml)
15o 117 116 54 12 7.6 6. 3 54 41
v*
8 o 90 85 185 195
---47 15 90 9o I72 229
M ----
5 "I0-8
2 . 5 "I 0 - ~
M
5 "10-3
36 15 I2 o o 8 o 75 98
M
Z n SO 4
MnSO,
A clivities *(%)
* v (m/,moles allantoin converted per ml per min) was measured p o u n d s is g i v e n i n °/o o f t h i s v a l u e .
Ph. hyslerinus G. hi@ida
Frog liver Goldfish liver
S. A. E. P. P.
Organism
2.5 "I0-~
35 32 16 o o 54 51 116 IiO
M
5 "10-3
2.5 "I0-4
93 IOI 94 51 44 1°3 86 115 115
M
43 22 13 o o 13 o IOI lO 7
M
5 "I0-8
CdS04 2-5 "10-4
96 lO6 IOI o o 4° 4 123 I2O
M
35 7 6 lO8 Io 4 IOO 1°5 IOI lO 5
M
5 "I 0 - 8
ED TA 2.5 "10-1
35 8 9 98 ioo IOO IOI IOO lO 3
M
in t h e a b s e n c e o f a d d i t i o n s , a n d t h e a c t i v i t y i n t h e p r e s e n c e o f t h e s e c o m -
53 54 83 o 4 42 16 III 12o
M
CoSO 4
A l l a n t o i n a s e a c t i v i t i e s w e r e d e t e r m i n e d a t 3 °0 i n m i x t u r e s c o n t a i n i n g , p e r m l , 3 3 / , m o l e s a l l a n t o i n , 1 6 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 7-7) a n d t h e i n d i c a t e d a m o u n t s o f p u r i f i e d a l l a n t o i n a s e a n d c a t i o n s o r E D T A . I n t h e e x p e r i m e n t s w i t h S. allantoicus, A. aUantoicus a n d E. coli, m o r e o v e r , 0 . 0 7 / , m o l e M n S O 4 a n d 8. 7 / , m o l e s G S H w e r e p r e s e n t p e r m l .
EFFECT OF i n 2+, Z n 2+, Co 2+, C d 2+ AND E D T A
TABLE
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o
>
5oo
(;. 1). \'()(;I!;LS A N I ) ( .
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A
V.\N
DEI,~ I)I.HFF
1
>
160
120
60
x
4
•
£
\
40 5
B
A 0
,
10-4
10-3
J
10-a 10-'1
C o n c e n t r c ~ t i o n (M)
10-3
10-a
C o n c e n t z c r t i o n (M)
F i g s . i a a n d i b . E f f e c t s o f M n 24 (I), Cd 2¢ (2), Co "°" (3) a n d Z n 2+ (4) a n d E I ) T A (5) in d i f f e r e n t c o n c e n t r a t i o n s o n t h e a l l a n t o i n a s e a c t i v i t y o f S. allantoicus in t h e p r e s e n c e (Fig. l a ) a n d a b s e n c e (Fig. i b ) o f 7" I ° - 5 M MnSO4. T e s t s w e r e p e r f o r m e d i n t h e s a m e m a n n e r as g i v e n in T a b l e 1, e x c e p t that in the second experiment MnSO 4 and GSH were omitted from the nledinm. The activities a r e e x p r e s s e d in ~/~ o f t h e a c t i v i t y w i t h o u t a d d i t i o n s .
only in the presence of the activator Mn 2+, it is probable that the inhibition was due to replacement of Mn 2+ from the enzyme molecule. The inhibiting effect of Cd 29 seems to be of a different character, since Cd 2+ at higher concentrations inhibited the enzyme strongly in the absence of Mn 2+. Mn 2+ protected the enzyme against the inhibiting effect of Cd 2+. Manganous ions were not essential for enzymic activity of S. allantoicus allantoinase, but enhanced the activity about 2-fold. In the absence of these ions the enzymic activity was not inhibited by EDTA and i,io-phenanthroline. The results obtained with allantoinases from A. allangoicus and E. coli were similar to these x~dth S. allantoicus; the activities in the absence of Mn ~+ and GSH were about 10% and 20%, respectively, of those in the presence of these compounds.
Effect of reducing substances and K C N BACHRACH6 reported an inhibition of allantoin degradation by cell suspensions of unidentified Pseudomonas species in the presence of KCN : 5" IO-3 M KCN caused 40% inhibition, 5 .IO 2 M lOO% inhibition. No effect on the activity of mung bean allantoinase was caused by lO -3 M KCN (ref. 2). Yeast allantoinase was strongly inhibited (90%) by lO -2 M KCN (ref. 3). The enzymes from yeast, soybeans and Ph. hysterinus 4 were strongly inhibited by cysteine and GSH, but soybean allantoinase Biochim. Biophvs. ,4eta, 122 (1960) 497 509
ALLANTOINASES.
II
5Ol
lost no activity upon 3 days dialysis against o.oi M KCN according to LEE AND ROUSHs. Allantoinases from S. allantoicus and A. allantoicus were activated by cysteine and GSH (ref. I). The effect of cysteine, thioglycolate, GSH and KCN on nine allantoinases is shown in Table II. The allantoinases of one group (S. allantoicus, A. allantoicus and E. coli) were activated by reducing substances and KCN, which can act as a reducing agent to open disulfide links in enzymes. The activity of allantoinases from Pseudomonas species was not influenced by these additions; the enzymes from animals and higher plants were inhibited. Iodoacetate tested under the conditions given in Table II did not inhibit any of the allantoinases. According to LEE A N D R O U S H 3, yeast allantoinase was inhibited only at concentrations near lO -2 M, whereas the activity of the enzyme from mung beans 2 was not influenced. LEE AND ROUSH3 stated that yeast allantoinase did not appear to have sulfhydryl groups essential for activity.
Interaction of reducing substances and manganous ions It appears that at least four groups of allantoinases can be distinguished on the basis of the effects of Mn ~+ and reducing substances. The enzymes from S. allantoicus, A. allantoicus and E. coli are activated both by Mn 2+ and reducing substances. Allantoinases from Pseudomonas species are inhibited by Mn 2+ and are insensitive to reducing substances. Allantoinases from animal livers are inhibited by both additions, whereas those from soybeans and Ph. hysterinus are activated by Mn ~+ and inhibited by reducing compounds. The inhibition of animal allantoinases by cysteine was not influenced by the TABLE
II
EFFECT OF REDUCING SUBSTANCES AND K C N ON THE ENZYMIC ACTIVITIES OF ALLANTOINASES A l l a n t o i n a s e a c t i v i t i e s w e r e d e t e r m i n e d a t 3 °0 i n m i x t u r e s c o n t a i n i n g , p e r m l , 3 9 / z m o l e s a l l a n t o i n , 200/,moles diethanolamine-HC1 b u f f e r ( p H 7.7) a n d t h e i n d i c a t e d a m o u n t s o f p u r i f i e d a l l a n t o i n a s e a n d o f r e d u c i n g s u b s t a n c e s . I n t h e e x p e r i m e n t s w i t h S. allantoicus, A. allantoicus a n d E. coli o . i # m o l e M n S O 4 w a s a d d e d t o g e t h e r w i t h t h e r e d u c i n g s u b s t a n c e s a n d a l l a n t o i n , v w a s m e a s u r e d i n t h e a b s e n c e o f r e d u c i n g s u b s t a n c e s a n d K C N . T h e o t h e r v a l u e s a r e e x p r e s s e d in % of this value.
Organism
Purified aUantoinase (l~g/ml)
v*
Activities (%) Cysteine Io -3 ~VI
7['hioglycolate
Glutathione Io -2 M
• o -~ M
S. A. E. P. V.
aUantoicus allantoicus eoli acidovorans fluorescens
Frog liver Goldfish liver
Ph. hysterinus G. hispida
72 24 38 i.i io 4.5 52 29o 640
409 14 19 60 12 7.6 7-3 62 47.7
181 114o ilOO ioo lO6 25 33 o 7
* v in m/2moles allantoin converted
18o 650 95 ° ioo 116 60 85 66 68
148 55 ° 91o ioo 115 ioo ioo 66 86
KCN 2.5 "to-3
2.5"zo-~
l~l
M
ioo 31o 13o 95 ioo 39 3° 83 90
95 -ioo ioo ioo 76 77 87 95
per ml per min.
B iochim. Biophys. Acta, 122 (1966) 4 9 7 - 5 0 9
502
(,. D. V()GEI~S ANI) ('. V.-\N I)FA{ I)RII:I
TA BLE I I 1 ACTIVATION AND INHIBITION OF SOME ALLANTOINASES BY MANGANOUS IONS AND RI,21)UCING
SUBSTANCES Allantoinase activities w e r e m e a s u r e d a t 3 o ' in m i x t u r e s containing, t)er m l , in t h e lirst t h r e e experiments, t6o/~moles diethanolamine H ( ' I b u f f e r ( p H 7.7), 3 3 / m m l e s allantoin a n d t h e ind i c a t e d a m o u n t s of purified allantoinase, which w e r e a d d e d t o the o t h e r w i s e c o m p l e t e m i x t u r e . M n S O 4 a n d G S I t were present in a m o u n t s of 1.25 and 8. 7 / , m o l e s , r e s p e c t i v e l y . I n t h e tests w i t h a l l a n t o i n a s e s f r o m higher p l a n t s there were present, per ml, 2 o o / , m o l e s d i e t h a n o l a m i n e HC1 buffer ( p H 7-7), 3 9 / ~ m o l e s a l l a n t o i n and the indicated a m o u n t s of purified a l l a n t o i n a s e a d d e d to t h e o t h e r w i s e c o m p l e t e m i x t u r e . M n S ( ) a and cysteine were present in a m o u n t s o f o . 0 8 an(l io/~moles, respectively. Orgauism
Purified allan/oinase
v"
Activities" (V/.o) q M$~2+ ~ (~SH
S. allanloicus A. allanloicus E. coli Ph. hysterinus G. hi@ida
34 2o.6 8.9 290 425
+ ~Vin2+ +
q C3'steine
+ M n 2~ + cvslei~ze
o
t23 i79
GSH
(l*glml) 71 r>.0 3.8 54 41.4
153 485 19o 214 262
119 124 105
228 133o 74 ° o
" v in m # m o l e s a l l a n t o i n c o n v e r t e d per ml per min. The activities m e a s u r e d in the presence of Mn 2+ and reducing s u b s t a n c e s are given in °/o of this value.
addition of Mn 2+. A distinct interaction of the reducing substances and Mn 2+ was observed with allantoinases from higher plants, S. allantoicus, A. allantoicus and E. coli (Table III). W h e n both substances were added simultaneously, the observed activity was higher than would be expected from results with the additions tested separately. Plant allantoinases were inhibited very strongly by cysteine; the inhibiting effect was abolished largely by the addition of Mn 2+ (Figs. 2a and 2b). The allantoinases from Ph. hysterinus and soybeans were inhibited almost completely by 2. Io -a M cysteine in the absence of Mn ~+. In the presence of Mn 2+ this inhibition was only 4o% and 3O9/o, even in the presence of i o 2 M cysteine. The s a m e effect on cysteine-inhibited allantoinases was found when Mn 2+ was added 4o min after the start of the experiment. Soybean allantoinase was not reactivated c o m p l e t e l y under these conditions. This result was probably due to a decreasing sensitivity of the enz y m e to the activating effect of Mn 2+ on preincubation with allantoin (Fig. 3). Preincubation with Mn ~+ in the absence of allantoin resulted, however, in a more active enzyme.
Inhibition of allantoinase from S. allantoicus by Cd 2+. Effect of GSH and Mn 2+ It appears from the results presented in Fig. Ib that the inhibiting effect of Cd 2+ on S. allantoicus allantoinase involves another m e c h a n i s m than the inhibition by Zn 2+, Co 2+ and E D T A ; the latter c o m p o u n d s remove added Mn ~+ from the e n z y m e molecule. Cd z~ inhibits the reaction also in the absence of Mn ~+. In order to s t u d y the m e c h a n i s m of the inhibition by Cd 2+, this c o m p o u n d was added to allantoinase from S. allantoicus in the presence of the activators Mn 2+ and GSH (Table IV). The order of addition was varied. Cd 2+ added separately or in combination with Mn 2+ inhibited the reaction strongly (Expts. 4, 7 and 8). In combination Biochim. Biophys. Acta, 122 (1966) ,t97 5 0 9
ALLANTOINASES. II
503
"~ 120. ~
Glyeine hiapid~
Phcraeolua hysterinus
-
\
~oo
60 0.08Nmole~ MnVat t: 0 mila
0 . 0 8 ~ m o ] e s Mn2'at t,O rain x
60
o
0.08#mo]e~ MnZ*at t = 4 0 m i n 0.08~mo]es Mna* at t = 4 0 m i n
40
\
20
no M n a ' a d d e d A
0
t
o
2
t
t
~
t
i
~
i
lO o 4 6 Cy~teine ( ~ m o l e $ / m l )
uo] i
~ t
i
'
~
t
t
t
B
4 6 8 lO Cyst~ixae ( p m o I e ~ / m ] )
Figs. 2a a n d 2b. I n h i b i t i o n of a l l a n t o i n a s e s f r o m Ph. hysterinus (Fig. 2a) a n d s o y b e a n s (Fig. 2b) b y different a m o u n t s of cysteine, a n d c o u n t e r a c t i o n of t h i s inhibition b y M n 2+. T h e i n c u b a t i o n m i x t u r e s a t 3 °° contained, per ml, 200/*moles d i e t h a n o l a m i n e - H C 1 buffer (pH 7.7), 39/*moles allantoin, t h e i n d i c a t e d a m o u n t s of c y s t e i n e a n d 0.29 or 0.42 m g of purified a l l a n t o i n a s e f r o m Ph. hysterinus or s o y b e a n s , respectively. T w o series of i n c u b a t i o n m i x t u r e s were t e s t e d ; to one series 0.08 /*mole M n S O 4 was a d d e d t o g e t h e r w i t h t h e a l l a n t o i n a s e s a t t h e s t a r t of t h e experim e n t . I n t h e second series t h e v e l o c i t y was followed for 4 ° m i n in t h e absence of M n *+. T h e n , o . o 8 / , m o l e M n S O , w a s added, a n d t h e a c t i v i t y was m e a s u r e d d u r i n g a p r o l o n g e d i n c u b a t i o n t i m e of 4 ° min.
with GSH, Cd 2+ inhibited only if added before GSH (Expts. lO-12, 14 and 25) ; GSH protected the enzyme against the inhibiting effect ofCd ~+ (Expts. 9, 13, 15, 16 and 24). Allantoin protected the enzyme against the inhibiting effect of Cd ~+ (Expts. 19 and 23), but counteracted the activating effect of Mn 2+ too (Expts. 18 and 20). The order of the addition of Mn 2+ and Cd 2+ (Expts. 7 and 8) or of Mn z+ and GSH (Expts. 5 and 6) did not change the resulting allantoinase activity considerably. The activating effect of Mn 2+ and GSH together was larger, however, than would be expected from the results with these compounds added separately.
Inhibition by phosphate buffers LEE AND ROUSHs first reported an inhibition of allantoinase from bakers' yeast by o.I M phosphate buffer, but they did not state the pH of the buffer. In a preceding communication ~ the effect of phosphate buffers with different pH values was given. Allantoinases from Ph. hysterinus, soybeans, goldfish liver and frog liver were increasingly inhibited upon lowering the pH. At pH 7-5 the enzymes from frog and goldfish liver were inhibited 50% and 60%, respectively, whereas the plant enzymes Biochim. Biophys. Acta, 122 (1966) 497-509
t;. 1). V()(iEI.S AND (:. VAN DI{R I ) R I I ; I
.~O4 TAI3I.I'; IV ACTIVATION
O F ,%'.
THE
OF
ORI)EI/
alla}llolclt,g
ALL.\NTt)INASE
I / Y ,~tll 2 ! . \ N i l
( ;~1 [ ; INHIBIT[tIN
13V ( ( l 2 ~ . I':FFKt"I" t ) I
AI)DIT[ONS
T() o. 5 m l cell-free e x t r a c t of .S'. alla~lloiclis, c o n t a i n i n g [ t o / m l o l e s d i e t h a n o l a m i n c t:1(21 buflk?r ( p t l 7.7) a n d 98 [*g p r o t e i n , t h c r c x~cre a d d e d in t h e o r d e r g i v e n /)elow: (a) o . - ml o f a solutiol~ c o n t a i n i n g I 3 / m m l e s G S H a n d 5 3 1 m m l e s d i e t h a n o l a m i n e H(-1 troffer ( p H 7-7); (b) o . r ml con t a i n i n g 1 . 9 / l m o l e MnS()~; (c) <).J m l c o n t a i n i n g t.9 l, m o l c C(1S()~ a n d ((1) r m l c o n t a i n i n g 5 o . 6 / m ~ o l e s a l l a n t o i n a n d 23 ° / t m o l e s d i e t h a n o l a m i n e ftC1 1)ufl\w (t)H 7.7}. "fhe linal v o l u m e w a s [.0 m l . W h e n o n e o f t h e a d d i t i o n s w a s o m i t t e d it w a s r e p l a c e d b y t h e s a m e v o l u m e o f w a t e r . All i n c u l ) a t i o n s w e r e p e r f o r m e d a t 3 ° ' . I n E x p t s . I 10 t h e r e w a s a n i n t e r v a l o f 5 inin b e t w e e n eac]l a d d i t i o n , a n d t h e v e l o c i t y w a s m e a s u r e d lo, 2o a n d 3 ° r a i n a f t e r a d d i t i o n o f t h e a l l a n t o i n s o l u t i o n . I n t h e E x p t s . r 7 25 t h e r e w a s also a n i n t e r v a l o f 5 r a i n a f t e r t h e a d d i t i o n o f t h e first c o m p o u n d t o t h e e x t r a c t . T h e n , a l l a n t o i n w a s a d d e d a n d t h e v e l o c i t y (%) w a s m e a s u r e d 5 a n d io rain a f t e r t h i s a d d i t i o n . A s e c o n d c o m p o u n d w a s a d d e d ~5 r a i n a f t e r t h e a d d i t i o n o f a l l a n t o i n , a n d t h e v e l o c i t y {%) w a s m e a s u r e d 5, 15 a n d 25 rain a f t e r t h i s a d d i t i o n , v is e x p r e s s e d in m / t m o l e s a l l a n t o i n c o n v e r t e d p e r r a i n p e r ml.
l:.xpl.
Order ~f addition (~)
I 2 3 4 .5 () 7 8 9 o ii 12 13 4 15 i() 17 i~ IQ 2o 2~ 22 23 24 25
Extract Extract Extract Extract Extract Extract Extract Extract Extract Extract Extract Extract Extract Extract Extract Extract Extract Extract Extract Extract Extract Extract Extract Extract Extract
(2) . . . . --. . . . . . ..... ---Cd 2 ~ Cd 2 ~M n 2~ Mn 2~ GSH GSH -
GSH M n 2+ Cd 2+ M n 2~ GSH Cd 2+
c' (3)
-
GStt M n 2' ( ' d 2+ Mn 2 GSH Cd 2 Mn 2~ GSH GSH Cd 2 Cd 24 M n 2+ Allantoin Allantoin Allantoin Allantoin Allantoin Atlantoin Allantoin Allantoin Allantoin
(4)
(5)
-GSH M n 2~ C(I 2 ~ M n 2+ GSH M n 2~ (;(t 2, Cd 2 ~ GSH GSH M n 2~ (;d 2~ GSH M n 2+ Cd 2+
Allantoin Allantoin Allantoin Allantoin Allantoin Allantoin Allantoin Allantoin Allantoin Allantoin Allantoin Allantoin Allantoin Allantoin Allantoin Allantoin
vl
Addition al I 15
va
45 45 45
GSH Mn2+ Cd2+
52
Mn 2 ~
~)7
GSH
4
Mn2+
4
66 52 5
Cd 2+ C(I 2~ GSH
47 59 8
45 49 ()6
0 92 93 2
12 52 8 2 lO ~ 19 22 i 12
114 5° 51 36 05 81
were not inhibited. Allantoinases from Pseudomonas species were not inhibited by phosphate buffers between pH 7.8 and 6. 7. At pH 7.7 only the enzymes from S. allantoicus, A. allantoicus, E. coli and animal livers were inhibited by phosphate buffers (Fig. 4). In the experiments with bacterial allantoinases Mn 2+ was present in the incubation mixture. The effect of this ion on the inhibiting effect of phosphate buffers is shown in Fig. 5. S. allantoicus allantoinase was inhibited only slightly in the absence, but rather strongly in the presence, of Mn 2+. In the presence of Mn =+ the activity was lowered to the same value as that found in its absence. Therefore, the inhibition by phosphate buffers was probably due to removal of Mn 2+ from allantoinase by combination with H2P04-. 13iochim. t3iophys. A eta, 122 (i960} 497 5o9
ALLANTOINASES.
II
505
60
40
0
10
1'5
2'0 15 3'0 35 PrebacuBct~io~a {line (mha)
Fig. 3. Effect of p r e i n c u b a t i o n w i t h Mn 2+ (I) o r allantoin (2) on the enzymic activity of aliantoinase f r o m soybeans. P r e i n c u b a t i o n with Mn z+ was performed in a m i x t u r e containing, per ml, 18o/,moles diethanolamine-HC1 buffer (pH 7.7), o . i / , m o l e MnSO 4 and 3.3 mg of purified allantoinase f r o m soybeans. At the indicated time intervals an aliquot (o.i ml) of the m i x t u r e was added to I ml of an allantoin solution in diethanolamine buffer containing a second a m o u n t of Mn 2+. The resulting incubation m i x t u r e s at 3 °0 contained, per ml, 45/~moles allantoin, o.i/~mole MnSO4, 220 #moles diethanolamine-HC1 buffer (pH 7.7) and 0. 3 m g of the purified enzyme. Prei n c u b a t i o n with allantoin was performed in a m i x t u r e containing, per ml, 45/~moles allantoin, 220/,moles diethanolamine-HC1 buffer (pH 7.7) and 0. 3 mg of the purified enzyme. The allantoin conversion was m e a s u r e d in this m i x t u r e and at the indicated time intervals MnSO 4 was added (o.oi ml to I ml p r e i n c u b a t i o n mixture). The final concentration of Mn 2+ was IO -~ M. The velocity of t h e allantoinase reaction was m e a s u r e d again, and the values obtained are presented in the figure. P o i n t s A a n d B indicate the velocities m e a s u r e d w h e n Mn 2+ was added t o g e t h e r w i t h allantoin at the start, or w h e n these ions were o m i t t e d f r o m the medium, respectively.
Experiments with allantoinases from A. allantoicus and E. coli gave rise to the same conclusion. In contrast with these allantoinases, the enzymes from higher plants were inhibited strongly b y phosphate buffer even in the absence of Mn 2+ (Fig. 5); 75% inhibition was measured in o.18 M phosphate buffer (pH 6.9). Allantoinases from frog and goldfish liver were also strongly inhibited in the absence of Mn ~+. Since E D T A in absence of added Mn ~+ does not change the activities of higher plant and animal allantoinases (Table I), it is difficult to explain the phosphate inhibition of these allantoinases on the basis of removal of essential bivalent cations. DISCUSSION
In a preceding communication 5 some enzymic properties of nine allantoinases were studied: p H optimum curve, Kin, activation energy and stability on storage, Biochim. Biophys. Acta, 122 (1966) 497-5o9
50()
(;. I). VOI;I,]LS AND C. VAN I)t,;1,~ 1)RII:']
o•100 >,
[]
42
~d < 80
|
60
A
cl
40
20
o. o
01o
[ Phosphate ]
Fig. 4. Inhibition of allantoinases f r o m S. allantoicus (0), A. allantoicus (×), E. coli (A) and frog liver ([B) b y phosphate buffers (pH 7-7) with increasing molarities. Allantoin solutions in Tris-HC1 buffer (pH 7-7) and in K H ~ P O 4 - N a 2 H P O 4 buffer (pH 7.7) were mixed to obtain the indicated molarity of phosphate buffer. There were present, per ml incubation mixture, 42/*moles allantoin, o.I /,mole MnSO4, 5.5/,moles G S H and 25 ° / * m o l e s buffer (conlposed of Tris and phospilate buffer). MnSO 4 and GSH were absent in the e x p e r i m e n t s with frog liver allantoinase. The a m o u n t of purified allantoinase per ml w a s 21/*g (S. allantoicus), 26/*g (A. aUantoicus), 51/*g (E. coli) and 13. 5 / , g (frog liver). The velocities measured in the absence of phosphate buffers were 177 , 91.5, 153 and 12.2 m/*moles allantoin converted per rain per ml, respectively.
heating, urea treatment and acid pretreatment. Differences between the enzymes were found, but no satisfactory classification of the enzymes w-as possible on this basis. Nor did the specificity of the enzymes for allantoin derivatives and ( + ) - and (--)-allantoin allow a classification. A classification into four groups of allantoinases could be made on the basis of the influence of bivalent cations and reducing substances (Table V). The first group includes the enzymes from S. allantoicus, A. allantoicus and E. coll. These organisms can use allantoin as the main source of carbon, nitrogen and energy for growth, but only under anaerobic conditions. The allantoinases are not optically specific, in contrast with the other allantoinases, and are activated by Mn 2+ and reducing substances. The second group is formed by the allantoinases from two Pseudomonas species which grow in media containing a]lantoin as the sole organic substrate under aerobic conditions only. The enzymes are inhibited by Mn 2+ and several other bivalent cations, whereas reducing substances have no effect on the activity. In the third group two allantoinases from animal livers are classified. These allantoinases are inhibited slightBiochim. Biophys. Acta, 122 (~960) 497-509
ALLANTOINASES.
ii
507
TABLE V SOME PROPERTIES
OF ALLANTOINASES
Group Organism
optical specificity*
A cid pretreatment*
Effect of some additions M n ~+
Zn 2+
Reducing substances
Phosphate buffer (pH 7)
S. allantoicus Aspecific ,4. allantoicus E. coli
Activation
None
Activation
Slight or no inhibition
Inactivation
P. acido-
Specific
Strong inhibition
Strong inhibition
None
None
Inactivation
III
F r o g liver Specifc Goldfish liver
Inhibition
Strong inhibition
Inhibition
Strong inhibition
Inactivation
IV
Ph. hyste-
Activation
Slight activation
Strong inhibition
Inhibition
Activation
I
II
vorans
P. fluorescens
Specific
rinus
G. hi@ida * T a k e n f r o m ref. 5.
6O
] y,\
!
• 200
"~ %v
t
"r
I-
o
I I
"~60 40
"- " . \ 20
,,
5o
31 o
o.65
o/o
o.i~
I
0.20
0
[Ph o~p]~,:tte]
Fig. 5. I n h i b i t i n g effect of p h o s p h a t e buffer (pH 6.9) on t h e a l l a n t o i n a s e activities of S. allantoicus (i), Ph. hysterinus (2) a n d s o y b e a n s (3) in t h e presence (closed symbols) a n d a b s e n c e (open s y m bols) of M n 2+. A l l a n t o i n s o l u t i o n s in Tris-HC1 buffer (pH 6.9) a n d K H 2 P O 4 - N a 2 H P O 4 buffer (pH 6.9) were m i x e d to o b t a i n t h e i n d i c a t e d molarities of p h o s p h a t e buffer. T h e i n c u b a t i o n m i x t u r e s a t 3 o° c o n t a i n e d , per ml, 1 8 2 / , m o l e s buffer ( m i x t u r e s of Tris-HC1 a n d p h o s p h a t e buffer), 46/~moles a l l a n t o i n a n d i i 4 / ~ g (cell-free e x t r a c t of S. allantoicus), 3 o o p g (soybeans) or 34o/~g (Ph. hysterinus) protein. I n one series o f e x p e r i m e n t s (closed s y m b o l s ) , m o r e o v e r , o.i # m o l e M n 2+ w a s 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 .
Biochim. Biophys. Acta, 122 (1966) 497-599
50~
~i. l). V()(iEI_S AN1) ( . V.kN I)1:.1,: I)I,~I1;I
ly by Mn 2 ~, and strongly t)y other bivalent cations. Reducing substancos also) inhibit these enzymes. The fourth group is fi~rmcd by enzymes from higher plants. These allantoinases are activated bv Mn" ~ and several other bivalent cations, but are strongly inhibited by reducing substances. This classification, however, does not seeln to be complete. Preliminary results with allantoinase from Pe~zicillium notatum indicated that this enzyme was inhibited by IO 4 M MnSO a (24~)o inhibition) and actiwtted by IO " M GSH (24~}.;) activation). Baker's yeast allantoinase was strongly inhibited by cysteine but seems not to be activated by Mn 2+ (ref. 3}- Allantoinases from wheat and gherkin were not activated by acid pretreatment, while Mn"~ inhibited these enzymes. Manganous ions activate allanff)inases of groups I and IV. These ions seem not to be essential for the activity of these enzymes, since addition of chelating compounds did not lower the activity or lowered it only slightly. Mn 2÷ inhibited the activity of allantoinases from groups I1 and I[I. Similar results were obtained with reducing substances. Cysteine, GSH or thioglycolate activated the enzymes from group I. Allantoinases from groups I I I and IV were inhibited. In the latter group the inhibition could be counteracted by Mn"~. SII
S
F/
l~oducing •-
/ +
E
SIII)St~MIC(?S i S ] t - M n ''+
¢
SI1 I Mn 2~ '
+ G
Mn 2 ~ - - -
/1 Reducing E ' -->Mn~+--"\ ! substances S
SIt / IL \ SIt
S c h e m e i. F o u r possible c o n f i g u r a t i o n s of a l l a n t o i n a s e s .
The most likely explanation for these differences among the allantoinases is to accept four structural configurations with different activities for all these enzymes (Scheme I). In group i all four configurations are active, the Mn2+-bound reduced form being most active. In group II only the two Mn2+-free configurations are active and the activities of both forms are equal. In group I I I the Mn2+-free oxidized form 0 / active and is most active, the Mn2+-bound oxidized configuration a little less (IO/o) the reduced forms are much less active. In group IV the Mni+-bound oxidized con. figuration has the highest activity; the Mn2+-bound reduced and the Mn2+-free oxidized form have a lower activity (about 5o%), and the Mn2
5o 9
ALLANTOINASES. I I
counteracts the activating effect of Mn 2+ on allantoinases from soybeans and S. allantoicus. Furthermore, the inhibiting effect of Cd 2+ on the latter enzyme is abolished almost completely upon preincubation of the enzyme with allantoin. Some effects observed by KANAMORI AND WIXOM 7 a n d SATYANARAYANA AND RADHAKRISHNAN8 with dihydroxyacid dehydratase (2,3-dihydroxyacid hydro-lyase, EC 4.2.1.9) from Phaseolus radiatus and spinach leaves can be explained by assuming a similar interconversion of active configurations. Mg ~+-, Mn 2+- and Co2+-bound enzymes were 2- 3 times more active than the cation-free configuration. Cysteine, in contrast with GSH, inhibited the reaction. The inhibiting effect of cysteine was largely reversed b y the addition of Mg 2+. MYERS9 observed that the same enzyme from E. coli required cysteine for activity and was, furthermore, activated by Mg 2+ and Mn2+; GSH could replace cysteine with this enzyme. It would be interesting to determine whether any other enzyme has several active configurations of the active site, as postulated here for the allantoinases. REFERENCES i 2 3 4 5 6 7 8 9
G. D. VOGELS, Thesis, I n s t i t u t e of Technology, Delft, 1963. Y. NAGAI AND S. FUNAHASHI, Agr. Biol. Chem. Tokyo, 25 (I96I) 265. K. W. LEE AND A. H. ROUSH, Arch. Biochem. Biophys., lO8 (1964) 460. CHR. VAN hER DRIFT AND G. D. VOGELS, Acta Bo/an. Neerl., 15 (1966) 209. G. D. VOGELS, F. TRIJBELS AND A. UFFINK, Biochim. Biophys. Acta, 122 (I966) 482. U. ]~ACHRACH, J. Sen. Microbiol., 17 (1957) I. M. KANAMORI AND R. L. WIXOM, J. Biol. Chem., 238 (1963) 998. T. SATYANARAYANA AND A. ~N]-.RADHAKRISHNAN, Biochim. Biophys. Acta, 92 (1964) 367 . J. W. MYERS, J. Biol. Chem., 236 (1961) 1414.
Biochim. Biophys. Acta, 122 (1966) 497-5o9
497
BBA 65446 A L L A N T O I N A S E S FROM B A C T E R I A L , P L A N T AND ANIMAL SOURCES II. E F F E C T OF B I V A L E N T CATIONS AND R E D U C I N G SUBSTANCES ON T H E ENZYMIC A C T I V I T Y
G. D. V O G E L S AND C H R . V A N D E R D R I F T
Department of Biochemistry, University of Nijmegen, Nijmegen (The Netherlands) (Received F e b r u a r y i o t h , 1966)
SUMMARY
I. The effect of manganous and other bivalent cations and of reducing substances on the activity of nine allantoinases (aUantoin amidohydrolase, EC 3.5.2.5) was studied. 2. At least four groups of allantoinases could be distinguished. The first group includes the enzymes from Streptococcus allantoicus, Arthrobacter allantoicus and Escherichia coli; the second group includes the enzymes from two Pseudomonas species; the third, the enzymes from frog and goldfish liver; and the fourth, the enzymes from Phaseolus hysterinus Dur. and Glycine hispida L. 3. The enzymes in the first group were aspecific for ( + ) - or (--)-allantoin, in contrast to the other allantoinases, and were activated by Mn 2+ and reducing substances. The allantoinases in the second group were inhibited b y Mn 2+ and insensitive to reducing substances. The third group includes enzymes which were inhibited b y both additions and the enzymes of the fourth group were activated b y Mn 2+ and inhibited b y cysteine. 4. The effects of Mn *+ and of reducing substances on allantoinases of the first and fourth groups influenced each other. 5. The inhibiting effect of Cd 2+ on the activity of allantoinases from the first group was largely prevented by previous addition of GSH. 6. In phosphate buffers the activity of allantoinases, except for those in the second group, was inhibited. This effect was partly due to removal of Mn 2+ from the enzyme. 7. The structural differences of the active sites in allantoinases of the four groups are discussed.
INTRODUCTION
Allantoinases (allantoin amidohydrolase, EC 3.5.2.5) from Streptococcus allantoicus and Arthrobacter allantoicus are activated b y Mn ~+ and reducing substances 1. Biochim. Biophys. Acta, 122 (1966) 497-5o9
4(),N
(i. ]3. Vi)(;I,;I.S A N I ) ( . V A N 1)1~1,~ I)RIF'I
No activating effect of Mn s4 or other bivalent cations was found fi)r allant(~inases from mung beans 2 and baker~' yeast 3. The enzymes from bakers' yeast, soybeans :~ and Phaseolus hystefinus 4 are strongly inhibited on addition of cysteine. In order to obtain more information on the observed differences, the effects of bivalent cations and r~ ducing substances on the activities of atlantoinases from nine origins were studied and are reported in the present paper. The properties and purification of the enzymes haw~ been reported previously ~. EXPERIMENTAL
Growth of bacteria, preparation of cell-free extracts and purification of the enzymes were performed as previously described 5. The degradation of allantoin was measured by the differential glyoxylate analysis (method B) after incubation under standard conditions ~. RESULTS
Effect of bivalent cations on allantoinases NAGAI AND FUNAHASHI2 tested the effect of Mg s+, Mn 2+, Ca 2+, Cos+, Ni s+, Zn s+, Cu s+, Fe 3+, EDTA, iodoacetate and KCN (all at lO -3 M) on purified allantoinase from mung beans. Cu 2+ inhibited the reaction strongly, Mn 2+ diminished the activity by 5% and the other additions were without effect. LEE AND ROUSH3 reported that ions inhibited bakers' yeast allantoinase only slightly, and stated that no direct evidence was found for a co-factor for this enzyme. Nevertheless, yeast allantoinase lost 65 % activity on dialysis, in contrast with soybean allantoinase. Allantoinases from S. allantoicus and A. allantoicus were inhibited by Hg s+, Cu 2+, Zn ~+, Fe 2+, Co2+ and EDTA (lO -3 and lO -4 M) 1. No change in activity was caused by Mg s+, Ca 2+, Sr 2+, Ba 2+, Ni s+ and Fe 3+, but Mn ~+ enhanced the enzymic activity in crude extracts 1.3- and 2.o-fold, respectively. Table I presents the effects of Mn 2+, Zn ~+, Co 2+, Cd 2+ and EDTA on purified allantoinases from S. allantoicus, A. allantoicus, Escherichia coli, Pseudomonas acidovorans, Pseudomonas fluorescens, liver of frogs (Rana esculenta) and goldfishes (Carassius auratus), Phaseolus hysterinus Dur. and soybeans (Glycine hi@ida L.). Zn 2+, Co2+ and Cd 2+ inhibited all allantoinases, except those from higher plants which were slightly activated. Allantoinases from Pseudomonas species were strongly inhibited by these ions, the enzymes from animal livers, S. allantoicus, A. allantoicus and E. coli were less inhibited. Pseudomonas allantoinases were inhibited strongly by Mn s+, enzymes from animal livers only IO%, and enzymes from higher plants were activated about 2-fold. Not all plant allantoinases were activated by Mn s+ however, since preliminary results with the enzymes from wheat and gherkin showed inhibition by I O - ~ M Mn 2+. The effect of Mn 2+, Cd s+, Cos+, Zn 2+ and EDTA on the allantoinase from S. allantoicus is shown in Figs. Ia and lb. These additions were tested at various concentrations in the presence or absence of 7 • lO -5 M MnSO 4. Zn s+, Co2+ and EDTA lowered the activity to about one-third of the original activity in the presence of MnSO4, whereas in the absence of Mn ~+ only a slight inhibiting effect was observed. Since EDTA, Zn 2+ and Co~+ inhibited the reaction to the same extent and were inhibitors Biochim. Biophys. Acta, 1 2 2 ( 1 9 6 6 ) 4 9 7 - 5 0 9
~o
I
~o &
t~
7~
I ON THE ACTIVITIES OF ALLANTOINASES
allantoicus allantoicus coli acidovorans fluorescens
20 22 I 9 4 45 25 ° 560
17
Purified allantoinase (#g/ml)
15o 117 116 54 12 7.6 6. 3 54 41
v*
8 o 90 85 185 195
---47 15 90 9o I72 229
M ----
5 "I0-8
2 . 5 "I 0 - ~
M
5 "10-3
36 15 I2 o o 8 o 75 98
M
Z n SO 4
MnSO,
A clivities *(%)
* v (m/,moles allantoin converted per ml per min) was measured p o u n d s is g i v e n i n °/o o f t h i s v a l u e .
Ph. hyslerinus G. hi@ida
Frog liver Goldfish liver
S. A. E. P. P.
Organism
2.5 "I0-~
35 32 16 o o 54 51 116 IiO
M
5 "10-3
2.5 "I0-4
93 IOI 94 51 44 1°3 86 115 115
M
43 22 13 o o 13 o IOI lO 7
M
5 "I0-8
CdS04 2-5 "10-4
96 lO6 IOI o o 4° 4 123 I2O
M
35 7 6 lO8 Io 4 IOO 1°5 IOI lO 5
M
5 "I 0 - 8
ED TA 2.5 "10-1
35 8 9 98 ioo IOO IOI IOO lO 3
M
in t h e a b s e n c e o f a d d i t i o n s , a n d t h e a c t i v i t y i n t h e p r e s e n c e o f t h e s e c o m -
53 54 83 o 4 42 16 III 12o
M
CoSO 4
A l l a n t o i n a s e a c t i v i t i e s w e r e d e t e r m i n e d a t 3 °0 i n m i x t u r e s c o n t a i n i n g , p e r m l , 3 3 / , m o l e s a l l a n t o i n , 1 6 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 7-7) a n d t h e i n d i c a t e d a m o u n t s o f p u r i f i e d a l l a n t o i n a s e a n d c a t i o n s o r E D T A . I n t h e e x p e r i m e n t s w i t h S. allantoicus, A. aUantoicus a n d E. coli, m o r e o v e r , 0 . 0 7 / , m o l e M n S O 4 a n d 8. 7 / , m o l e s G S H w e r e p r e s e n t p e r m l .
EFFECT OF i n 2+, Z n 2+, Co 2+, C d 2+ AND E D T A
TABLE
%D
oo
>
o
>
5oo
(;. 1). \'()(;I!;LS A N I ) ( .
£00
A
V.\N
DEI,~ I)I.HFF
1
>
160
120
60
x
4
•
£
\
40 5
B
A 0
,
10-4
10-3
J
10-a 10-'1
C o n c e n t r c ~ t i o n (M)
10-3
10-a
C o n c e n t z c r t i o n (M)
F i g s . i a a n d i b . E f f e c t s o f M n 24 (I), Cd 2¢ (2), Co "°" (3) a n d Z n 2+ (4) a n d E I ) T A (5) in d i f f e r e n t c o n c e n t r a t i o n s o n t h e a l l a n t o i n a s e a c t i v i t y o f S. allantoicus in t h e p r e s e n c e (Fig. l a ) a n d a b s e n c e (Fig. i b ) o f 7" I ° - 5 M MnSO4. T e s t s w e r e p e r f o r m e d i n t h e s a m e m a n n e r as g i v e n in T a b l e 1, e x c e p t that in the second experiment MnSO 4 and GSH were omitted from the nledinm. The activities a r e e x p r e s s e d in ~/~ o f t h e a c t i v i t y w i t h o u t a d d i t i o n s .
only in the presence of the activator Mn 2+, it is probable that the inhibition was due to replacement of Mn 2+ from the enzyme molecule. The inhibiting effect of Cd 29 seems to be of a different character, since Cd 2+ at higher concentrations inhibited the enzyme strongly in the absence of Mn 2+. Mn 2+ protected the enzyme against the inhibiting effect of Cd 2+. Manganous ions were not essential for enzymic activity of S. allantoicus allantoinase, but enhanced the activity about 2-fold. In the absence of these ions the enzymic activity was not inhibited by EDTA and i,io-phenanthroline. The results obtained with allantoinases from A. allangoicus and E. coli were similar to these x~dth S. allantoicus; the activities in the absence of Mn ~+ and GSH were about 10% and 20%, respectively, of those in the presence of these compounds.
Effect of reducing substances and K C N BACHRACH6 reported an inhibition of allantoin degradation by cell suspensions of unidentified Pseudomonas species in the presence of KCN : 5" IO-3 M KCN caused 40% inhibition, 5 .IO 2 M lOO% inhibition. No effect on the activity of mung bean allantoinase was caused by lO -3 M KCN (ref. 2). Yeast allantoinase was strongly inhibited (90%) by lO -2 M KCN (ref. 3). The enzymes from yeast, soybeans and Ph. hysterinus 4 were strongly inhibited by cysteine and GSH, but soybean allantoinase Biochim. Biophvs. ,4eta, 122 (1960) 497 509
ALLANTOINASES.
II
5Ol
lost no activity upon 3 days dialysis against o.oi M KCN according to LEE AND ROUSHs. Allantoinases from S. allantoicus and A. allantoicus were activated by cysteine and GSH (ref. I). The effect of cysteine, thioglycolate, GSH and KCN on nine allantoinases is shown in Table II. The allantoinases of one group (S. allantoicus, A. allantoicus and E. coli) were activated by reducing substances and KCN, which can act as a reducing agent to open disulfide links in enzymes. The activity of allantoinases from Pseudomonas species was not influenced by these additions; the enzymes from animals and higher plants were inhibited. Iodoacetate tested under the conditions given in Table II did not inhibit any of the allantoinases. According to LEE A N D R O U S H 3, yeast allantoinase was inhibited only at concentrations near lO -2 M, whereas the activity of the enzyme from mung beans 2 was not influenced. LEE AND ROUSH3 stated that yeast allantoinase did not appear to have sulfhydryl groups essential for activity.
Interaction of reducing substances and manganous ions It appears that at least four groups of allantoinases can be distinguished on the basis of the effects of Mn ~+ and reducing substances. The enzymes from S. allantoicus, A. allantoicus and E. coli are activated both by Mn 2+ and reducing substances. Allantoinases from Pseudomonas species are inhibited by Mn 2+ and are insensitive to reducing substances. Allantoinases from animal livers are inhibited by both additions, whereas those from soybeans and Ph. hysterinus are activated by Mn ~+ and inhibited by reducing compounds. The inhibition of animal allantoinases by cysteine was not influenced by the TABLE
II
EFFECT OF REDUCING SUBSTANCES AND K C N ON THE ENZYMIC ACTIVITIES OF ALLANTOINASES A l l a n t o i n a s e a c t i v i t i e s w e r e d e t e r m i n e d a t 3 °0 i n m i x t u r e s c o n t a i n i n g , p e r m l , 3 9 / z m o l e s a l l a n t o i n , 200/,moles diethanolamine-HC1 b u f f e r ( p H 7.7) a n d t h e i n d i c a t e d a m o u n t s o f p u r i f i e d a l l a n t o i n a s e a n d o f r e d u c i n g s u b s t a n c e s . I n t h e e x p e r i m e n t s w i t h S. allantoicus, A. allantoicus a n d E. coli o . i # m o l e M n S O 4 w a s a d d e d t o g e t h e r w i t h t h e r e d u c i n g s u b s t a n c e s a n d a l l a n t o i n , v w a s m e a s u r e d i n t h e a b s e n c e o f r e d u c i n g s u b s t a n c e s a n d K C N . T h e o t h e r v a l u e s a r e e x p r e s s e d in % of this value.
Organism
Purified aUantoinase (l~g/ml)
v*
Activities (%) Cysteine Io -3 ~VI
7['hioglycolate
Glutathione Io -2 M
• o -~ M
S. A. E. P. V.
aUantoicus allantoicus eoli acidovorans fluorescens
Frog liver Goldfish liver
Ph. hysterinus G. hispida
72 24 38 i.i io 4.5 52 29o 640
409 14 19 60 12 7.6 7-3 62 47.7
181 114o ilOO ioo lO6 25 33 o 7
* v in m/2moles allantoin converted
18o 650 95 ° ioo 116 60 85 66 68
148 55 ° 91o ioo 115 ioo ioo 66 86
KCN 2.5 "to-3
2.5"zo-~
l~l
M
ioo 31o 13o 95 ioo 39 3° 83 90
95 -ioo ioo ioo 76 77 87 95
per ml per min.
B iochim. Biophys. Acta, 122 (1966) 4 9 7 - 5 0 9
502
(,. D. V()GEI~S ANI) ('. V.-\N I)FA{ I)RII:I
TA BLE I I 1 ACTIVATION AND INHIBITION OF SOME ALLANTOINASES BY MANGANOUS IONS AND RI,21)UCING
SUBSTANCES Allantoinase activities w e r e m e a s u r e d a t 3 o ' in m i x t u r e s containing, t)er m l , in t h e lirst t h r e e experiments, t6o/~moles diethanolamine H ( ' I b u f f e r ( p H 7.7), 3 3 / m m l e s allantoin a n d t h e ind i c a t e d a m o u n t s of purified allantoinase, which w e r e a d d e d t o the o t h e r w i s e c o m p l e t e m i x t u r e . M n S O 4 a n d G S I t were present in a m o u n t s of 1.25 and 8. 7 / , m o l e s , r e s p e c t i v e l y . I n t h e tests w i t h a l l a n t o i n a s e s f r o m higher p l a n t s there were present, per ml, 2 o o / , m o l e s d i e t h a n o l a m i n e HC1 buffer ( p H 7-7), 3 9 / ~ m o l e s a l l a n t o i n and the indicated a m o u n t s of purified a l l a n t o i n a s e a d d e d to t h e o t h e r w i s e c o m p l e t e m i x t u r e . M n S ( ) a and cysteine were present in a m o u n t s o f o . 0 8 an(l io/~moles, respectively. Orgauism
Purified allan/oinase
v"
Activities" (V/.o) q M$~2+ ~ (~SH
S. allanloicus A. allanloicus E. coli Ph. hysterinus G. hi@ida
34 2o.6 8.9 290 425
+ ~Vin2+ +
q C3'steine
+ M n 2~ + cvslei~ze
o
t23 i79
GSH
(l*glml) 71 r>.0 3.8 54 41.4
153 485 19o 214 262
119 124 105
228 133o 74 ° o
" v in m # m o l e s a l l a n t o i n c o n v e r t e d per ml per min. The activities m e a s u r e d in the presence of Mn 2+ and reducing s u b s t a n c e s are given in °/o of this value.
addition of Mn 2+. A distinct interaction of the reducing substances and Mn 2+ was observed with allantoinases from higher plants, S. allantoicus, A. allantoicus and E. coli (Table III). W h e n both substances were added simultaneously, the observed activity was higher than would be expected from results with the additions tested separately. Plant allantoinases were inhibited very strongly by cysteine; the inhibiting effect was abolished largely by the addition of Mn 2+ (Figs. 2a and 2b). The allantoinases from Ph. hysterinus and soybeans were inhibited almost completely by 2. Io -a M cysteine in the absence of Mn ~+. In the presence of Mn 2+ this inhibition was only 4o% and 3O9/o, even in the presence of i o 2 M cysteine. The s a m e effect on cysteine-inhibited allantoinases was found when Mn 2+ was added 4o min after the start of the experiment. Soybean allantoinase was not reactivated c o m p l e t e l y under these conditions. This result was probably due to a decreasing sensitivity of the enz y m e to the activating effect of Mn 2+ on preincubation with allantoin (Fig. 3). Preincubation with Mn ~+ in the absence of allantoin resulted, however, in a more active enzyme.
Inhibition of allantoinase from S. allantoicus by Cd 2+. Effect of GSH and Mn 2+ It appears from the results presented in Fig. Ib that the inhibiting effect of Cd 2+ on S. allantoicus allantoinase involves another m e c h a n i s m than the inhibition by Zn 2+, Co 2+ and E D T A ; the latter c o m p o u n d s remove added Mn ~+ from the e n z y m e molecule. Cd z~ inhibits the reaction also in the absence of Mn ~+. In order to s t u d y the m e c h a n i s m of the inhibition by Cd 2+, this c o m p o u n d was added to allantoinase from S. allantoicus in the presence of the activators Mn 2+ and GSH (Table IV). The order of addition was varied. Cd 2+ added separately or in combination with Mn 2+ inhibited the reaction strongly (Expts. 4, 7 and 8). In combination Biochim. Biophys. Acta, 122 (1966) ,t97 5 0 9
ALLANTOINASES. II
503
"~ 120. ~
Glyeine hiapid~
Phcraeolua hysterinus
-
\
~oo
60 0.08Nmole~ MnVat t: 0 mila
0 . 0 8 ~ m o ] e s Mn2'at t,O rain x
60
o
0.08#mo]e~ MnZ*at t = 4 0 m i n 0.08~mo]es Mna* at t = 4 0 m i n
40
\
20
no M n a ' a d d e d A
0
t
o
2
t
t
~
t
i
~
i
lO o 4 6 Cy~teine ( ~ m o l e $ / m l )
uo] i
~ t
i
'
~
t
t
t
B
4 6 8 lO Cyst~ixae ( p m o I e ~ / m ] )
Figs. 2a a n d 2b. I n h i b i t i o n of a l l a n t o i n a s e s f r o m Ph. hysterinus (Fig. 2a) a n d s o y b e a n s (Fig. 2b) b y different a m o u n t s of cysteine, a n d c o u n t e r a c t i o n of t h i s inhibition b y M n 2+. T h e i n c u b a t i o n m i x t u r e s a t 3 °° contained, per ml, 200/*moles d i e t h a n o l a m i n e - H C 1 buffer (pH 7.7), 39/*moles allantoin, t h e i n d i c a t e d a m o u n t s of c y s t e i n e a n d 0.29 or 0.42 m g of purified a l l a n t o i n a s e f r o m Ph. hysterinus or s o y b e a n s , respectively. T w o series of i n c u b a t i o n m i x t u r e s were t e s t e d ; to one series 0.08 /*mole M n S O 4 was a d d e d t o g e t h e r w i t h t h e a l l a n t o i n a s e s a t t h e s t a r t of t h e experim e n t . I n t h e second series t h e v e l o c i t y was followed for 4 ° m i n in t h e absence of M n *+. T h e n , o . o 8 / , m o l e M n S O , w a s added, a n d t h e a c t i v i t y was m e a s u r e d d u r i n g a p r o l o n g e d i n c u b a t i o n t i m e of 4 ° min.
with GSH, Cd 2+ inhibited only if added before GSH (Expts. lO-12, 14 and 25) ; GSH protected the enzyme against the inhibiting effect ofCd ~+ (Expts. 9, 13, 15, 16 and 24). Allantoin protected the enzyme against the inhibiting effect of Cd ~+ (Expts. 19 and 23), but counteracted the activating effect of Mn 2+ too (Expts. 18 and 20). The order of the addition of Mn 2+ and Cd 2+ (Expts. 7 and 8) or of Mn z+ and GSH (Expts. 5 and 6) did not change the resulting allantoinase activity considerably. The activating effect of Mn 2+ and GSH together was larger, however, than would be expected from the results with these compounds added separately.
Inhibition by phosphate buffers LEE AND ROUSHs first reported an inhibition of allantoinase from bakers' yeast by o.I M phosphate buffer, but they did not state the pH of the buffer. In a preceding communication ~ the effect of phosphate buffers with different pH values was given. Allantoinases from Ph. hysterinus, soybeans, goldfish liver and frog liver were increasingly inhibited upon lowering the pH. At pH 7-5 the enzymes from frog and goldfish liver were inhibited 50% and 60%, respectively, whereas the plant enzymes Biochim. Biophys. Acta, 122 (1966) 497-509
t;. 1). V()(iEI.S AND (:. VAN DI{R I ) R I I ; I
.~O4 TAI3I.I'; IV ACTIVATION
O F ,%'.
THE
OF
ORI)EI/
alla}llolclt,g
ALL.\NTt)INASE
I / Y ,~tll 2 ! . \ N i l
( ;~1 [ ; INHIBIT[tIN
13V ( ( l 2 ~ . I':FFKt"I" t ) I
AI)DIT[ONS
T() o. 5 m l cell-free e x t r a c t of .S'. alla~lloiclis, c o n t a i n i n g [ t o / m l o l e s d i e t h a n o l a m i n c t:1(21 buflk?r ( p t l 7.7) a n d 98 [*g p r o t e i n , t h c r c x~cre a d d e d in t h e o r d e r g i v e n /)elow: (a) o . - ml o f a solutiol~ c o n t a i n i n g I 3 / m m l e s G S H a n d 5 3 1 m m l e s d i e t h a n o l a m i n e H(-1 troffer ( p H 7-7); (b) o . r ml con t a i n i n g 1 . 9 / l m o l e MnS()~; (c) <).J m l c o n t a i n i n g t.9 l, m o l c C(1S()~ a n d ((1) r m l c o n t a i n i n g 5 o . 6 / m ~ o l e s a l l a n t o i n a n d 23 ° / t m o l e s d i e t h a n o l a m i n e ftC1 1)ufl\w (t)H 7.7}. "fhe linal v o l u m e w a s [.0 m l . W h e n o n e o f t h e a d d i t i o n s w a s o m i t t e d it w a s r e p l a c e d b y t h e s a m e v o l u m e o f w a t e r . All i n c u l ) a t i o n s w e r e p e r f o r m e d a t 3 ° ' . I n E x p t s . I 10 t h e r e w a s a n i n t e r v a l o f 5 inin b e t w e e n eac]l a d d i t i o n , a n d t h e v e l o c i t y w a s m e a s u r e d lo, 2o a n d 3 ° r a i n a f t e r a d d i t i o n o f t h e a l l a n t o i n s o l u t i o n . I n t h e E x p t s . r 7 25 t h e r e w a s also a n i n t e r v a l o f 5 r a i n a f t e r t h e a d d i t i o n o f t h e first c o m p o u n d t o t h e e x t r a c t . T h e n , a l l a n t o i n w a s a d d e d a n d t h e v e l o c i t y (%) w a s m e a s u r e d 5 a n d io rain a f t e r t h i s a d d i t i o n . A s e c o n d c o m p o u n d w a s a d d e d ~5 r a i n a f t e r t h e a d d i t i o n o f a l l a n t o i n , a n d t h e v e l o c i t y {%) w a s m e a s u r e d 5, 15 a n d 25 rain a f t e r t h i s a d d i t i o n , v is e x p r e s s e d in m / t m o l e s a l l a n t o i n c o n v e r t e d p e r r a i n p e r ml.
l:.xpl.
Order ~f addition (~)
I 2 3 4 .5 () 7 8 9 o ii 12 13 4 15 i() 17 i~ IQ 2o 2~ 22 23 24 25
Extract Extract Extract Extract Extract Extract Extract Extract Extract Extract Extract Extract Extract Extract Extract Extract Extract Extract Extract Extract Extract Extract Extract Extract Extract
(2) . . . . --. . . . . . ..... ---Cd 2 ~ Cd 2 ~M n 2~ Mn 2~ GSH GSH -
GSH M n 2+ Cd 2+ M n 2~ GSH Cd 2+
c' (3)
-
GStt M n 2' ( ' d 2+ Mn 2 GSH Cd 2 Mn 2~ GSH GSH Cd 2 Cd 24 M n 2+ Allantoin Allantoin Allantoin Allantoin Allantoin Atlantoin Allantoin Allantoin Allantoin
(4)
(5)
-GSH M n 2~ C(I 2 ~ M n 2+ GSH M n 2~ (;(t 2, Cd 2 ~ GSH GSH M n 2~ (;d 2~ GSH M n 2+ Cd 2+
Allantoin Allantoin Allantoin Allantoin Allantoin Allantoin Allantoin Allantoin Allantoin Allantoin Allantoin Allantoin Allantoin Allantoin Allantoin Allantoin
vl
Addition al I 15
va
45 45 45
GSH Mn2+ Cd2+
52
Mn 2 ~
~)7
GSH
4
Mn2+
4
66 52 5
Cd 2+ C(I 2~ GSH
47 59 8
45 49 ()6
0 92 93 2
12 52 8 2 lO ~ 19 22 i 12
114 5° 51 36 05 81
were not inhibited. Allantoinases from Pseudomonas species were not inhibited by phosphate buffers between pH 7.8 and 6. 7. At pH 7.7 only the enzymes from S. allantoicus, A. allantoicus, E. coli and animal livers were inhibited by phosphate buffers (Fig. 4). In the experiments with bacterial allantoinases Mn 2+ was present in the incubation mixture. The effect of this ion on the inhibiting effect of phosphate buffers is shown in Fig. 5. S. allantoicus allantoinase was inhibited only slightly in the absence, but rather strongly in the presence, of Mn 2+. In the presence of Mn =+ the activity was lowered to the same value as that found in its absence. Therefore, the inhibition by phosphate buffers was probably due to removal of Mn 2+ from allantoinase by combination with H2P04-. 13iochim. t3iophys. A eta, 122 (i960} 497 5o9
ALLANTOINASES.
II
505
60
40
0
10
1'5
2'0 15 3'0 35 PrebacuBct~io~a {line (mha)
Fig. 3. Effect of p r e i n c u b a t i o n w i t h Mn 2+ (I) o r allantoin (2) on the enzymic activity of aliantoinase f r o m soybeans. P r e i n c u b a t i o n with Mn z+ was performed in a m i x t u r e containing, per ml, 18o/,moles diethanolamine-HC1 buffer (pH 7.7), o . i / , m o l e MnSO 4 and 3.3 mg of purified allantoinase f r o m soybeans. At the indicated time intervals an aliquot (o.i ml) of the m i x t u r e was added to I ml of an allantoin solution in diethanolamine buffer containing a second a m o u n t of Mn 2+. The resulting incubation m i x t u r e s at 3 °0 contained, per ml, 45/~moles allantoin, o.i/~mole MnSO4, 220 #moles diethanolamine-HC1 buffer (pH 7.7) and 0. 3 m g of the purified enzyme. Prei n c u b a t i o n with allantoin was performed in a m i x t u r e containing, per ml, 45/~moles allantoin, 220/,moles diethanolamine-HC1 buffer (pH 7.7) and 0. 3 mg of the purified enzyme. The allantoin conversion was m e a s u r e d in this m i x t u r e and at the indicated time intervals MnSO 4 was added (o.oi ml to I ml p r e i n c u b a t i o n mixture). The final concentration of Mn 2+ was IO -~ M. The velocity of t h e allantoinase reaction was m e a s u r e d again, and the values obtained are presented in the figure. P o i n t s A a n d B indicate the velocities m e a s u r e d w h e n Mn 2+ was added t o g e t h e r w i t h allantoin at the start, or w h e n these ions were o m i t t e d f r o m the medium, respectively.
Experiments with allantoinases from A. allantoicus and E. coli gave rise to the same conclusion. In contrast with these allantoinases, the enzymes from higher plants were inhibited strongly b y phosphate buffer even in the absence of Mn 2+ (Fig. 5); 75% inhibition was measured in o.18 M phosphate buffer (pH 6.9). Allantoinases from frog and goldfish liver were also strongly inhibited in the absence of Mn ~+. Since E D T A in absence of added Mn ~+ does not change the activities of higher plant and animal allantoinases (Table I), it is difficult to explain the phosphate inhibition of these allantoinases on the basis of removal of essential bivalent cations. DISCUSSION
In a preceding communication 5 some enzymic properties of nine allantoinases were studied: p H optimum curve, Kin, activation energy and stability on storage, Biochim. Biophys. Acta, 122 (1966) 497-5o9
50()
(;. I). VOI;I,]LS AND C. VAN I)t,;1,~ 1)RII:']
o•100 >,
[]
42
~d < 80
|
60
A
cl
40
20
o. o
01o
[ Phosphate ]
Fig. 4. Inhibition of allantoinases f r o m S. allantoicus (0), A. allantoicus (×), E. coli (A) and frog liver ([B) b y phosphate buffers (pH 7-7) with increasing molarities. Allantoin solutions in Tris-HC1 buffer (pH 7-7) and in K H ~ P O 4 - N a 2 H P O 4 buffer (pH 7.7) were mixed to obtain the indicated molarity of phosphate buffer. There were present, per ml incubation mixture, 42/*moles allantoin, o.I /,mole MnSO4, 5.5/,moles G S H and 25 ° / * m o l e s buffer (conlposed of Tris and phospilate buffer). MnSO 4 and GSH were absent in the e x p e r i m e n t s with frog liver allantoinase. The a m o u n t of purified allantoinase per ml w a s 21/*g (S. allantoicus), 26/*g (A. aUantoicus), 51/*g (E. coli) and 13. 5 / , g (frog liver). The velocities measured in the absence of phosphate buffers were 177 , 91.5, 153 and 12.2 m/*moles allantoin converted per rain per ml, respectively.
heating, urea treatment and acid pretreatment. Differences between the enzymes were found, but no satisfactory classification of the enzymes w-as possible on this basis. Nor did the specificity of the enzymes for allantoin derivatives and ( + ) - and (--)-allantoin allow a classification. A classification into four groups of allantoinases could be made on the basis of the influence of bivalent cations and reducing substances (Table V). The first group includes the enzymes from S. allantoicus, A. allantoicus and E. coll. These organisms can use allantoin as the main source of carbon, nitrogen and energy for growth, but only under anaerobic conditions. The allantoinases are not optically specific, in contrast with the other allantoinases, and are activated by Mn 2+ and reducing substances. The second group is formed by the allantoinases from two Pseudomonas species which grow in media containing a]lantoin as the sole organic substrate under aerobic conditions only. The enzymes are inhibited by Mn 2+ and several other bivalent cations, whereas reducing substances have no effect on the activity. In the third group two allantoinases from animal livers are classified. These allantoinases are inhibited slightBiochim. Biophys. Acta, 122 (~960) 497-509
ALLANTOINASES.
ii
507
TABLE V SOME PROPERTIES
OF ALLANTOINASES
Group Organism
optical specificity*
A cid pretreatment*
Effect of some additions M n ~+
Zn 2+
Reducing substances
Phosphate buffer (pH 7)
S. allantoicus Aspecific ,4. allantoicus E. coli
Activation
None
Activation
Slight or no inhibition
Inactivation
P. acido-
Specific
Strong inhibition
Strong inhibition
None
None
Inactivation
III
F r o g liver Specifc Goldfish liver
Inhibition
Strong inhibition
Inhibition
Strong inhibition
Inactivation
IV
Ph. hyste-
Activation
Slight activation
Strong inhibition
Inhibition
Activation
I
II
vorans
P. fluorescens
Specific
rinus
G. hi@ida * T a k e n f r o m ref. 5.
6O
] y,\
!
• 200
"~ %v
t
"r
I-
o
I I
"~60 40
"- " . \ 20
,,
5o
31 o
o.65
o/o
o.i~
I
0.20
0
[Ph o~p]~,:tte]
Fig. 5. I n h i b i t i n g effect of p h o s p h a t e buffer (pH 6.9) on t h e a l l a n t o i n a s e activities of S. allantoicus (i), Ph. hysterinus (2) a n d s o y b e a n s (3) in t h e presence (closed symbols) a n d a b s e n c e (open s y m bols) of M n 2+. A l l a n t o i n s o l u t i o n s in Tris-HC1 buffer (pH 6.9) a n d K H 2 P O 4 - N a 2 H P O 4 buffer (pH 6.9) were m i x e d to o b t a i n t h e i n d i c a t e d molarities of p h o s p h a t e buffer. T h e i n c u b a t i o n m i x t u r e s a t 3 o° c o n t a i n e d , per ml, 1 8 2 / , m o l e s buffer ( m i x t u r e s of Tris-HC1 a n d p h o s p h a t e buffer), 46/~moles a l l a n t o i n a n d i i 4 / ~ g (cell-free e x t r a c t of S. allantoicus), 3 o o p g (soybeans) or 34o/~g (Ph. hysterinus) protein. I n one series o f e x p e r i m e n t s (closed s y m b o l s ) , m o r e o v e r , o.i # m o l e M n 2+ w a s 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 .
Biochim. Biophys. Acta, 122 (1966) 497-599
50~
~i. l). V()(iEI_S AN1) ( . V.kN I)1:.1,: I)I,~I1;I
ly by Mn 2 ~, and strongly t)y other bivalent cations. Reducing substancos also) inhibit these enzymes. The fourth group is fi~rmcd by enzymes from higher plants. These allantoinases are activated bv Mn" ~ and several other bivalent cations, but are strongly inhibited by reducing substances. This classification, however, does not seeln to be complete. Preliminary results with allantoinase from Pe~zicillium notatum indicated that this enzyme was inhibited by IO 4 M MnSO a (24~)o inhibition) and actiwtted by IO " M GSH (24~}.;) activation). Baker's yeast allantoinase was strongly inhibited by cysteine but seems not to be activated by Mn 2+ (ref. 3}- Allantoinases from wheat and gherkin were not activated by acid pretreatment, while Mn"~ inhibited these enzymes. Manganous ions activate allanff)inases of groups I and IV. These ions seem not to be essential for the activity of these enzymes, since addition of chelating compounds did not lower the activity or lowered it only slightly. Mn 2÷ inhibited the activity of allantoinases from groups I1 and I[I. Similar results were obtained with reducing substances. Cysteine, GSH or thioglycolate activated the enzymes from group I. Allantoinases from groups I I I and IV were inhibited. In the latter group the inhibition could be counteracted by Mn"~. SII
S
F/
l~oducing •-
/ +
E
SIII)St~MIC(?S i S ] t - M n ''+
¢
SI1 I Mn 2~ '
+ G
Mn 2 ~ - - -
/1 Reducing E ' -->Mn~+--"\ ! substances S
SIt / IL \ SIt
S c h e m e i. F o u r possible c o n f i g u r a t i o n s of a l l a n t o i n a s e s .
The most likely explanation for these differences among the allantoinases is to accept four structural configurations with different activities for all these enzymes (Scheme I). In group i all four configurations are active, the Mn2+-bound reduced form being most active. In group II only the two Mn2+-free configurations are active and the activities of both forms are equal. In group I I I the Mn2+-free oxidized form 0 / active and is most active, the Mn2+-bound oxidized configuration a little less (IO/o) the reduced forms are much less active. In group IV the Mni+-bound oxidized con. figuration has the highest activity; the Mn2+-bound reduced and the Mn2+-free oxidized form have a lower activity (about 5o%), and the Mn2
5o 9
ALLANTOINASES. I I
counteracts the activating effect of Mn 2+ on allantoinases from soybeans and S. allantoicus. Furthermore, the inhibiting effect of Cd 2+ on the latter enzyme is abolished almost completely upon preincubation of the enzyme with allantoin. Some effects observed by KANAMORI AND WIXOM 7 a n d SATYANARAYANA AND RADHAKRISHNAN8 with dihydroxyacid dehydratase (2,3-dihydroxyacid hydro-lyase, EC 4.2.1.9) from Phaseolus radiatus and spinach leaves can be explained by assuming a similar interconversion of active configurations. Mg ~+-, Mn 2+- and Co2+-bound enzymes were 2- 3 times more active than the cation-free configuration. Cysteine, in contrast with GSH, inhibited the reaction. The inhibiting effect of cysteine was largely reversed b y the addition of Mg 2+. MYERS9 observed that the same enzyme from E. coli required cysteine for activity and was, furthermore, activated by Mg 2+ and Mn2+; GSH could replace cysteine with this enzyme. It would be interesting to determine whether any other enzyme has several active configurations of the active site, as postulated here for the allantoinases. REFERENCES i 2 3 4 5 6 7 8 9
G. D. VOGELS, Thesis, I n s t i t u t e of Technology, Delft, 1963. Y. NAGAI AND S. FUNAHASHI, Agr. Biol. Chem. Tokyo, 25 (I96I) 265. K. W. LEE AND A. H. ROUSH, Arch. Biochem. Biophys., lO8 (1964) 460. CHR. VAN hER DRIFT AND G. D. VOGELS, Acta Bo/an. Neerl., 15 (1966) 209. G. D. VOGELS, F. TRIJBELS AND A. UFFINK, Biochim. Biophys. Acta, 122 (I966) 482. U. ]~ACHRACH, J. Sen. Microbiol., 17 (1957) I. M. KANAMORI AND R. L. WIXOM, J. Biol. Chem., 238 (1963) 998. T. SATYANARAYANA AND A. ~N]-.RADHAKRISHNAN, Biochim. Biophys. Acta, 92 (1964) 367 . J. W. MYERS, J. Biol. Chem., 236 (1961) 1414.
Biochim. Biophys. Acta, 122 (1966) 497-5o9