8/8/2019 Phosphorus Cleavage Reaction

http://slidepdf.com/reader/full/phosphorus-cleavage-reaction 1/3

V o l . 1 7 1 , N o . 8

A M i c r o b i a l C a r b o n - P h o s p h o r u s Bond C l e a v a g e Enzyme R e q u i r e s

Two P r o t e i n C o m p o n e n t s f o r A c t i v i t y

KOUSAKU MURATA,* NORIKO HIGAKI, AND AKIRA KIMURA

R e s e a r c h I n s t i t u t e f o r Fo o d S c i e n c e , K y o t o U n i v e r s i t y , U j i , K y o t o 6 1 1 , Japan

R e c e i v e d 1 4 F e b r u a r y 1 9 8 9 / A c c e p t e d 2 4 A p r i l 1 9 8 9

E n t e r o b a c t e r aer ogenes IF O 1 2 0 1 0 c o n t a i n s a c a r b o n - p h o s p h o r u s ( C - P ) b o n d c l e a v a g e e n z y m e c a t a l y z i n g t h e

l i b e r a t i o n o f i n o r g a n i c p h o s p h a t e f r o m v a r i o u s a l k y l - a n d p h e n y l p h o s p h o n i c a c i d s . Th e e n z y m e i n t h e

b a c t e r i u m was f o u n d t o b e c o m p o s e d o f t w o p h y s i c a l l y d i f f e r e n t p r o t e i n c o m p o n e n t s , E2 a n d E 3 . Th e m o l e c u l a r

w e i g h t s o f E2 a n d E3 were 5 6 0 , 0 0 0 a n d 1 1 0 , 0 0 0 , r e s p e c t i v e l y , a n d E3 wa s r e s o l v e d i n t o t w o a p p a r e n t l y

h o m o g e n e o u s s u b u n i t s . N e i t h e r c o m p o n e n t a l o ne c o u l d c a t a l y z e t h e C-P b o n d c l e a v a g e r e a c t i o n , b u t t h e

r e a c t i o n was e f f i c i e n t l y c a t a l y z e d when t h e c o m p o n e n t s were m i x e d .

S i n c e t h e d i s c o v e r y o f a n a t u r a l p h o s p h o n i c a c i d , 2 -a m i n o e t h y l p h o s p h o n i c a c i d , i n 1 9 5 9 ( 8 ) , v a r i o u s k i nd s o f

p h o s p h o n i c a c i d s c o n t a i n i n g a d i r e c t c a r b o n - p h o s p h o r u s

( C - P ) b o n d h a v e b e e n f o u n d i n m a n y l i v i n g s y s t e m s . I n s e c -

t i c i d e s , h e r b i c i de s , f un g ic i d e s , n erve gases, f l a m e r e t a r -

d a n t s , a n d s e v e r a l o t h e r i m p o r t a n t s y n t h e t i c c h e m i c a l s a l s oc o n t a i n t h e C - P b o n d a n d h a v e b e e n u s e d i n l a r g e q u a n t i t i e s .S i n c e t h e C - P b o n d i s h i g h l y r e s i s t a n t t o c h e m i c a l h y d r o l y s i s

a n d t h e r m a l d e c o m p o s i t i o n , t h e b i o l o g i c a l b r e a k d o w n o f

p h o s p h o n i c a c i d s i s i m p o r t a n t n o t o n l y f o r b i o c h e m i c a l

s t u d i e s b u t a l so f or p r e v e n t i o n o f o v e r a c c u m u l a t i o n o f t h e s e

a c i d s i n n a t u r e . To d a t e , h o w e v e r , t h e m e t a b o l i c r o u t e o f

t h e s e unique b i o l o g i c a l a n d s y n t h e t i c m o l e c u l e s h a s r e -

m a i n e d o b s c u r e b e c a u s e o f a f a i l u r e t o d e t e c t t h e enzyme

a c t i v i t y r e s p o n s i b l e f o r t h e C - P b o n d c l e a v a g e r e a c t i o n i nc e l l - f r e e s y s t e m s ( 1 - 7 , 1 4 , 1 7 , 1 8 ) .

R e c e n t l y , we f o u n d a C - P b o n d c l e a v a g e enzyme i n c e l le x t r a c t s o f E n t e r o b a c t e r a e r o g e n e s IF O 1 2 0 1 0 ( 1 5 ) . Th e

enzyme i n t h e b a c t e r i u m c o u l d c l e a v e t h e C - P b o n d i n

various a l k y l - a n d p h e n y l p h o s p h o n i c a c i d s ( s u c h a s m e t h -y l p h o s p h o n i c a c i d , p h o s p h o n o a c e t i c a c i d , a n d p h e n y l p h o s -

p h o n i c a c i d ) a n d was shown t o b e d i s t i n c t f r o m t h e enzyme

p h o s p h o n a t a s e , w h i c h h y d r o l y z e s t h e C - P b o n d i np h o s p h o n o a c e t a l d e h y d e ( 9 - 1 1 ) .

I n o r d e r t o c h a r a c t e r i z e t h e p r o p e r t i e s o f t h e enzyme a n de l u c i d a t e t h e C - P b o n d c l e a v a g e m e c h a n i s m , we a t t e m p t e dt o p u r i f y t h e C - P b o n d c l e a v a g e enzyme f r o m E . a e r o g e n e s

IFO 1 2 0 1 0 . Th e c e l l s were a e r o b i c a l l y grown on a medium

[ 0 . 5 % g l u c o s e , 0.1% ( N H 4 ) 2 S O 4 , 0 . 0 1 % MgSO4 7 H 2 0 ,0 . 0 2 % y e a s t e x t r a c t ( P i - f r e e ) , a n d 0 . 4 % p h o s p h o n o a c e t i ca c i d ( p H 7 . 2 ) ] a t 3 0 ° C f o r 2 0 h . Th e c e l l s ( 3 8 g [ w e t w e i g h t ] )

f r o m a 1 5 - l i t e r c u l t u r e we re s u s p e n d e d i n 8 0 m l o f 1 0 mMT r i s h y d r o c h l o r i d e b u f f e r ( p H 7 . 5 ) c o n t a i n i n g 1 0 mM M g C l 2a n d 0 . 2 mM d i t h i o t h r e i t o l ( b u f f e r A ) ; c e l l e x t r a c t was pre-

p a r e d as d e s c r i b e d p r e v i o u s l y ( 1 5 ) . P i - f r e e y e a s t e x t r a c t was

p r e p a r e d a s d e s c r i b e d p r e v i o u s l y ( 1 5 ) . P h o s p h o n i c a c i d swere u s e d a f t e r f i l t r a t i o n w i t h a membrane f i l t e r ( M i l l i p o r e

C o r p . , B e d f o r d , M a s s . ) , a n d g l a s s w e a r was r i n s e d w i t h n i t r i ca c i d b e f o r e use.

Th e d i a l y z e d c e l l e x t r a c t ( p r o t e i n , 5 , 6 0 0 mg; 1 3 0 m l ) was

a p p l i e d t o a D E A E - c e l l u l o s e c o l u m n ( 4 b y 6 0 c m ) e q u i l i -b r a t e d w i t h b u f f e r A , a n d p r o t e i n s were e l u t e d w i t h a l i n e a rg r a d i e n t o f KC l ( 0 t o 0 . 6 M, 4 , 0 0 0 m l ) i n b u f f e r A . A 2 0 - m l

p o r t i o n was c o l l e c t e d every 9 m i n. T h e enzyme e l u t e d a s a

* C o r r e s p o n d i n g a u t h o r .

s i n g l e a c t i v e p e a k a t a b o u t 0 . 0 5 M K C I . Th e a c t i v e f r a c t i o n swere c o m b i n e d a n d c o n c e n t r a t e d b y u l t r a f i l t r a t i o n w i t h a

P M- 1 0 m em bra n e ( A m i c o n C o r p . , L e x i n g t o n , M a s s . ) , a n d

t h e c o n c e n t r a t e ( p r o t e i n , 1 , 4 0 0 m g; 2 0 m l ) was l o a d e d o n t o a

S e p h a d e x G - 1 5 0 c o l u m n ( 5 b y 7 0 c m ) e q u i l i b r a t e d w i t h b u f f e r

A . P r o t e i n s were e l u t e d w i t h b u f f e r A , a n d a 7 - m l p o r t i o n

was c o l l e c t e d every 1 0 m i n . A l m o s t a l l t h e a c t i v i t y was

r e c o v e r e d i n t h e v o i d f r a c t i o n o f t h e c o l u m n , a n d t h e f r a c t i o n

was c o n c e n t r a t e d as d e s c r i b e d a b o v e . Th e enzyme s o l u t i o n

( p r o t e i n , 4 8 5 m g; 5 m l ) was t h e n a p p l i e d t o t h e f i r s t TSK

HW65 c o l u m n ( 2 b y 7 0 c m ) e q u i l i b r a t e d w i t h b u f f e r A . A

m o l e c u l a r s i e v e g e l o f T S K H W6 5 was p u r c h a s e d f r o m Toyo

S o d a MFG, T o k y o , J a p a n . P r o t e i n s we re e l u t e d w i t h b u f f e r

A , a n d a 4 - m l p o r t i o n was c o l l e c t e d every 1 0 m i n . When t h e

enzyme i n e a c h f r a c t i o n was a s s a y e d, o nl y o n e a c t i v e p e a k( P 1 ) was o b s e r v e d . H o w e v e r , t h e a c t i v i t y i n P 1 was l o w

( a b o u t 0 . 1 % o f t h a t a p p l i e d t o t h e c o l u m n ) , a n d v i r t u a l l y a l l

a c t i v i t y h a d d i s a p p e a r e d . Th e c o l u m n f r a c t i o n s were t h e n

c o m b i n e d i n t o t h r e e p o o l s ( F i g . 1 ) , a n d t h e a c t i v i t i e s i n e a c h

p o o l a n d i n a l l p o s s i b l e c o m b i n a t i o n s we re d e t e r m i n e d . I twas f o u n d t h a t p o o l 2 c o n t a i n e d t h e C - P b o n d c l e a v a g e

enzyme a c t i v i t y , a n d v i r t u a l l y a l l o f t h e a c t i v i t y f r o m t h e

S e p h a d e x G - 1 5 0 c o l u m n was r e s t o r e d .

Our i n t e r e s t was t h e n f o c u s e d o n t h e p r o t e i n components

i n p o o l 2 . P o o l 2 ( f r a c t i o n s 5 5 t o 6 0 ) was c o n c e n t r a t e d t o 3 m l

( p r o t e i n , 1 1 0 m g ) a s d e s c r i b e d a b o v e a n d f r a c t i o n a t e d a g a i n

on t h e T SK H W65 c o l u m n ( F i g . 1 ) u n d e r t h e same c o n d i -

t i o n s a s t h o s e e m p l o y e d f o r t h e f i r s t TSK HW65 c o l u m n . A2 . 5 - m l p o r t i o n o f e l u a t e was c o l l e c t e d every 5 m i n . Th e C - Pb o n d c l e a v a g e enzyme a c t i v i t y was n o t f o u n d when e a c h

f r a c t i o n was a s s a y e d . To l o c a t e t h e f r a c t i o n s r e q u i r e d f o r t h e

enzyme a c t i v i t y , a p o r t i o n f r o m e a c h f r a c t i o n ( f r a c t i o n s 5 0 t o

7 0 ) was t a k e n a n d m i x e d w i t h s a m p l e s f r o m o t h e r f r a c t i o n s .As a r e s u l t , we c o u l d s e p a r a t e t wo p r o t e i n p e a k s ( P 2 a n d P 3 )

t h a t e x h i b i t e d t h e C - P b o n d c l e a v a g e a c t i v i t y when t h e ywere m i x e d ( F i g . 1 ) . Th e p r o t e i n c omp onents i n P 2 a n d P 3were named E2 a n d E 3 , r e s p e c t i v e l y , a n d f u r t h e r f r a c t i o n -a t e d b y d e t e r m i n i n g t h e a c t i v i t y w i t h P 2 o r P 3 .

F r a c t i o n s i n P 3 ( f r a c t i o n s 6 2 t o 6 6 ) were c o m b i n e d ( p r o -t e i n , 3 2 m g; 1 2 m l ) , s a t u r a t e d w i t h ammonium s u l f a t e ( 3 0 % ) ,

a n d a p p l i e d t o a b u t y l T o y o p e a r l 650M c o l u m n ( 1 b y 5 c m )e q u i l i b r a t e d w i t h b u f f e r A s a t u r a t e d w i t h ammonium s u l f a t e( 3 0 % ) . Th e enzyme was e l u t e d w i t h a l i n e a r g r a d i e n t o f

amm o nium s u l f a t e ( 3 0 t o 0 % , 5 0 m l ) i n b u f f e r A , a n d a 1 - m l

p o r t i o n was c o l l e c t e d every 4 m i n . Th e enzyme was e l u t e d a t

a b o u t 1 5% a mmonium s u l f a t e , a n d t h e n t h e a c t i v i t y i n e a c h

4 5 0 4

JOURNAL O F B A C T E R I O L O G Y , A u g . 1 9 8 9 , p . 4 5 0 4 - 4 5 0 6

0 0 2 1 - 9 1 9 3 / 8 9 / 0 8 4 5 0 4 - 0 3 $ 0 2 . 0 0 / 0C o p y r i g h t C ) 1 9 8 9 , A m e r i c a n S o c i e t y f o r M i c r o b i o l o g y

8/8/2019 Phosphorus Cleavage Reaction

http://slidepdf.com/reader/full/phosphorus-cleavage-reaction 2/3

N O T E S 4 5 0 5

1 st TSK HW65

-- - - ~ 3 0 . 4

15o A2sonm

* 0 . 3

*Activity10

0.2

5-

0.1

0 3

I - ; )os . . .

c m 2 n d TSK HW65 .a< E 2 a 0 .4

3P2 P 3 6Sk -

~ " r\3, *CjO2Q0.3

20

~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~

0k

l4 k

3 0 40 50 60 70 80

Fraction N o .

0.2

0 . 1

.. - 0

F I G . 1 . S e pa r a t i o n o f t wo p r o t e i n c o m p o n e n t s r e q u i r e d f ee x p r e s s i o n o f C - P b o n d c l e a v a g e r e a c t i o n . T h e enzyme was a s s

a t 3 7 ° C b y d e t e r m i n i n g P i l i b e r a t e d i n a r e a c t i o n m i x t u r e c o n s i

o f 5 0 mM p h o s p h o n o a c e t i c a c i d , 2 0 mM M g C l 2 , 5 0 mMh y d r o c h l o r i d e b u f f e r ( p H 7 . 5 ) , a n d t h e enzyme s a m p l e , a s d e s c

p r e v i o u s l y ( 1 5 ) . T h e f i r s t ( t o p ) a n d s e c o n d ( b o t t o m ) TSK F Ic o l u m n c h r o m a t o g r a p h i e s w er e c o n d u c t e d as d e s c r i b e d i n t h e

P o o l 2 ( s h a d e d a r e a ) was s u b j e c t e d t o t h e s e c o n d TSK I fc o l u m n . I n s e t s s h o w s o d i u m d o d e c y l s u l f a t e - p o l y a c r y l - a m i d

e l e c t r o p h o r e s i s o f t h e p u r i f i e d c o m p o n e n t s E2 ( l e f t ) a n d E 3 ( i( l a n e a , m ol e c u l a r w ei g h t m a r k e r s i n k i l o d a l t o n s [ k ] ) .

f r a c t i o n was d e t e r m i n e d b y u s i n g a s a m p l e o f P 2 . T h e a l

f r a c t i o n s were c o n c e n t r a t e d t o a b o u t 1 . 5 m l ( p r o t e i n , 4 . 8as d e s c r i b e d a b o v e a n d a p p l i e d t o a S e p h a d e x G - 2 0 0 c o l

( 1 b y 5 0 c m ) e q u i l i b r a t e d w i t h b u f f e r A . P r o t e i n s were e lw i t h b u f f e r A , a n d a 1 - ml p or t i on was c o l l e c t e d e v e r y 5T h r o u g h t h e s e c o l u m n c h r o m a t o g r a p h i e s , E3 was p u i

a p p r o x i m a t e l y 1 3 0 - f o l d w i t h 3 . 5 % y i e l d f r o m a s t e p o

s e c o n d TSK HW65 c o l u m n . E3 s h o w e d a m o l e c u l a r ma

1 1 0 k i l o d a l t o n s on S e p h a d e x G - 2 0 0 g e l f i l t r a t i o n ( F i g . 2s h o w e d a s u b u n i t m o l e c u l a r mass o f 5 5 k i l o d a l t o n s whwas s u b j e c t e d t o s o d i u m d o d e c y l s u l f a t e - p o l y a c r y l a m i d

e l e c t r o p h o r e s i s b y t h e m e t h o d o f L a e m m l i ( 1 2 ) ( F i g . 1 )F r a c t i o n s i n P 2 ( f r a c t i o n s 5 3 t o 5 8 ) were a l s o c o m b

( p r o t e i n , 2 8 m g; 1 2 m l ) a n d a p p l i e d t o a b u t y l T o y o ]

650M c o l u m n u n d e r t h e same c o n d i t i o n s a s t h os e e mp l i

f o r t h e E3 f r a c t i o n a t i o n . U n l i k e E 3 ; E2 was e l u t e d a t a

5% a mmonium s u l f a t e , a n d t h e n a c t i v i t y i n e a c h f r a c t i o na s s a y e d b y u s i n g a p o r t i o n o f P 3 . A c t i v e f r a c t i o n s

p o o l e d , d i a l y z e d o v e r ni g h t a g a i n s t b u f f e r A a t 4 ° C ,c o n c e n t r a t e d t o 1 . 3 m l ( p r o t e i n , 4 . 5 m g ) a s d e s c r i b e d a b

Th e enzyme s o l u t i o n was a p p l i e d t o a DEAE-Sephe

CL-6B c o l u m n ( 1 b y 9 0 c m ) e q u i l i b r a t e d w i t h b u f f e

P r o t e i n s were e l u t e d w i t h a l i n e a r g r a d i e n t o f KC l ( 0 t

M, 1 0 0 m l ) , a n d a 1 . 5- ml p o r t i o n was c o l l e c t e d every 5Th e a c t i v e f r a c t i o n s e l u t e d a t a b o u t 0 . 3 M KC l were

c e n t r a t e d t o 1 . 2 m l ( p r o t e i n , 1 . 8 m g ) a n d were t h e n a p p l ia S e p h a d e x G - 2 0 0 c o l u m n ( 1 b y 5 0 c m ) e q u i l i b r a t e db u f f e r A . P r o t e i n s were e l u t e d w i t h b u f f e r A , a n d a

p o r t i o n was c o l l e c t e d every 6 m i n . When t h e a c t i v i t y i n

f r a c t i o n was d e t e r m i n e d b y u s i n g a p o r t i o n o f P 3 on S e

d e x G - 2 0 0 c o l u m n c h r o m a t o g r a p h y , E2 s h o w e d o n l y o n e

p r o t e i n p e a k t h a t was c l o s e l y s u p e r i m p o s e d on t h e C - P b o n d

c l e a v a g e a c t i v i t y . T h e r e s u l t i n d i c a t e s t h a t E2 c o u l d b e

s u b s t a n t i a l l y p u r i f i e d . Th e f i n a l E2 p r e p a r a t i o n was p u r i f i e da b o u t 9 0 - f o l d w i t h 4 . 5 % y i e l d f r o m a s t e p o f t h e s e c o n d TSKHW65column and showed amolecularmassof 560 kilodal-

t o n s o n S e p h a d e x G - 2 0 0 c o l u m n c h r o m a t o g r a p h y ( F i g . 2 ) .E

-1 A l t h o u g h t h e s o d i u m d o d e c y l s u l f a t e - p o l y a c r y l a m i d e g e l

O e l e c t r o p h o r e t i c p a t t e r n o f E2 was s t i l l q u i t e c o m p l e x a n dE gave s e ve r a l p r o t e i n b a n d s w i t h m o l e c u l a r masses o f 2 4 , 2 3 ,- 1 7 , 1 6 , 1 5 , a n d 1 4 k i lo da l t o ns ( F i g . 1 ) , most o f t h e p r o t e i n s

O l a p p e a r e d t o b e a s s o c i a t e d w i t h t h e E2 c o m p o n e n t . F u r t h e r

, p u r i f i c a t i o n may b e r e q u i r e d f o r t h e d e t e r mi n a t i on o f t h e

* , s u b u n i t s t r u c t u r e o f E 2 .T h e f i n a l p r e p a r a t i o n s o f E2 a n d E 3 a f t e r S e p h a d e x G - 2 0 0

- c o l u m n c h r o m a t o g r a p h y were u s e d f o r t h e c h a r a c t e r i z a t i o n

X o f t h e C -P b on d c l e a v a g e r e a c t i o n . N e i t h e r p u r i f i e d c o m p o -

n e n t a l o n e c o u l d c a t a l y z e t h e C - P b o n d c l e a v a g e r e a c t i o n ,b u t t h e r e a c t i o n was e f f i c i e n t l y c a t a l y z e d when t h e compo-

n e n t s were m i x e d . Th e g r e a t e s t a c t i v i t y was f o u n d when s i xt i m e s a s much E2 a s E3 was u s e d ( d a t a n o t s h o w n ) , t h u s

i n d i c a t i n g t h a t E2 a n d E 3 c o u p l e d w e l l a t e q u i m o l a r c o n c e n -

t r a t i o n ; t h e r e f o r e , s u b s e q u e n t C - P b o n d c l e a v a g e r e a c t i o n s

were

p e r f o r m e d b y u s i n g 9 0 p L g o f E2 a n d 1 5 p , g o f E 3 . Th ei r t h e

r e a c t i o n was most a c t i v e a t a l k a l i n e p H ( p H 8 t o 9 ) . ATP a n d

a y e d o t h e r c o f a c t o r s (NAD, NADH, N A D P , NADPH, a n d p y r i -

s t i n g d o x a l p h o s p h a t e ) were n o t r e q u i r e d f o r t h e C - P b o n d c l e a v -

T r i s a g e r e a c t i o n . Th e r e a c t i o n was a c t i v a t e d b y d i v a l e n t c a t i o ns

: r i b e d such as M g 2 + , M n 2 + , and C o 2 ' a t a l m o s t t h e s a m e e f f i -1 W 6 5 c i e n c i e s . U n d e r t h e c o n d i t i o n s s p e c i f i e d i n t h e l e g e n d t o F i g .t e x t . 1 , t h e s p e c i f i c a c t i v i t y o f t h e enzyme was 3 . 5 , u m o l / h per mg

I W 6 5 o f p r o t e i n , a n d K , ? f o r p h o s p h o n o a c e t i c a c i d was c a l c u l a t e dl e g e l t o b e 3 . 5 mM f r o m a L i n e w e a v e r - B u r k p l o t ( 1 3 ) . Th e enzymer i g h t ) c l e a v e d C-P b o n d s i n a l k y l - a n d p h e n y l p h o s p h o n i c a c i d s

( r e l a t i v e a c t i v i t i e s : m e t h y l p h o s p h o n i c a c i d , 1 0 0 ; p h o s p h o n o -

a c e t i c a c i d , 8 5 ; a n d p h e n y l p h o s p h o n i c a c i d , 3 2 ) ( A l d r i c h

c t i v e C h e m i c a l C o . , I n c . , M i l w a u k e e , W i s . ) , i n c l u d i n g a h e r b i c i d e

3 m g ) ( g l y p h o s a t e [ r e l a t i v e a c t i v i t y , 1 5 ] ; Monsanto J a p a n C o . ,

l u m n R y u g a s a k i , I b a r a g i , J a p a n ) a n d a n a n t i b i o t i c ( f o s f o m y c i n e

l u t e d [ r e l a t i v e a c t i v i t y , 4 5 ] ; S i g m a C h e m i c a l C o . , S t . L o u i s , M o . ) .m i n . T h u s , E . a e r o g e n e s IF O 1 2 0 1 0 c o n t a i n s a C-P b o n d

r i f i e d c l e a v a g e e n z y m e , a n d t h e enzyme seems t o r e q u i r e t wo

I f t h e p r o t e i n c omp onents f o r i t s a c t i v i t y e x p r e s s i o n , a l t h o u g h t h e

ss o f f u n c t i o n o f e a c h c o m p o n e n t i s n o t y e t c l e a r . R e c e n t l y , a n

) . E 3 enzyme c a t a l y z i n g t h e f o r m a t i o n o f t h e C - P b o n d was

i e n i t p u r i f i e d b y S e i d e l e t a l . ( 1 6 ) . Our f i n d i n g o f t h e C-P bond

l e g e l c l e a v a g e e n z y m e , i n c o m b i n a t i o n w i t h t h e C-P b o n d - f o r m i n g

) i n e dp e a r lo y e dbout

was

were

a n d)ove.

a r o s e

r A .

o 0 . 8

m i n .

c o n -

i e d t o

w i t h

1 - m l

e a c h

e p h a -

1 0 0 0

a

3:

c j

500400

300

200

0 0

70

0 25 50 75

Elution volume ( m l )

F I G . 2 . M o l e c u l a r w e i g h t d e t e r m i n a t i o n o f t h e p u r i f i e d E 2 a n d

E 3 c o m p o n e n t s . Th e p u r i f i e d E2 and E3 c o m p o n e n t s were p a s s e d

t h r o u g h t h e S e p h a d e x G - 2 0 0 column ( 1 b y 5 0 c m ) e q u i l i b r a t e d with

b u f f e r A . P r o t e i n s were e l u t e d w i t h b u f f e r A , an d molecular w e i g h t s

were p l o t t e d a s f u nc t i o n s o f e l u t i o n v o l u m e .

T h y r o g l o b u l i n (669 k D o )E2

F e r r i t i n (430kDa)

C a t o l a s e ( 2 3 2 k D a )

A l d o l a s e ( 1 5 8 k D a )

_E

B o v i n e serum a l b u m i n ( 7 0 k D o )F - 1 -7 k

V O L . 1 7 1 , 1 9 8 9

8/8/2019 Phosphorus Cleavage Reaction

http://slidepdf.com/reader/full/phosphorus-cleavage-reaction 3/3

4 5 0 6 N O T E S

e n z y m e , ma y f a c i l i t a t e t h e r e s o l u t i o n o f p h y s i o l o g i c a l f u n c -t i o n s o f p h o s p h o n i c a c i d s a n d t h e i r m e t a b o l i c r o u t e i n l i v i n gs y s t e m s . T h e u n c o u p l i n g o f t h e C - P b o n d c l e a v a g e e n z y m er e a c t i o n a t t a i n e d h e r e b y t h e p h y s i c a l s e p a r a t i o n o f t w op r o t e i n c o m p o n e n t s a p p e a r s t o e l u c i d a t e a n a s p e c t o f t h er e a c t i o n m e c h a n i s m .

We t h a n k M o n s a n t o J a p a n C o . , R y u g a s a k i , I b a r a g i , J a p a n , f o r

s u p p l y i n g t h e h e r b i c i d e g l y p h o s a t e .

LITERATURE CITED

1 . B a l t h a z o r , T . M ., a n d L . E . H a l l i s . 1 9 8 6 . G l y p h o s a t e - d e g r a d i n gm i c r o o r g a n i s m s f r o m i n d u s t r i a l a c t i v a t e d s l u d g e . A p p l . E n v i -

r o n . M i c r o b i o l . 5 1 : 4 3 2 - 4 3 4 .

2 . C o o k , A . M . , C . G . D a ug h t o n , a n d M. A l e x a n d e r . 1 9 7 8 . P h o s -p h o n a t e u t i l i z a t i o n b y b a c t e r i a . J . B a c t e r i o l . 1 3 3 : 8 5 - 9 0 .

3 . C o o k , A . M . , C . G . D a u g h t o n , a n d M. A l e x a n d e r . 1 9 7 9 . B e n z e n ef r o m b a c t e r i a l c l e a v a g e o f t h e c a r b o n - p h o s p h o r u s b o n d o f

p h e n y l p h o s p h o n a t e . B i o c h e m . J . 1 8 4 : 4 5 3 - 4 5 5 .

4 . C o r d e i r o , M. L . , D . L . P o m p l i a n o , a n d J . W. F r o s t . 1 9 8 5 .D e g r a d a t io n a n d d e t o x i f i c a t i o n o f o r g a n o p h o s p h o n a t e s : c l e a v -a g e o f t h e c a r b o n t o p h o s p h o r u s b o n d . J . Am . C h e m . S o c .1 0 8 : 3 3 2 - 3 3 4 .

5 . D a u g h t o n , C . G . , A . M. C o o k , a n d M. A l e x a n d e r . 1 9 7 9 . B a c t e -r i a l c o n v e r si o n o f a l k y l p h o s p h o n a t e s t o n a t u r a l p r o d u c t s v i a

c a r b o n - p h o s p h o r u s b o n d c l e a v a g e . J . A g r i c . F o o d C h e m . 2 7 :1 3 7 5 - 1 3 8 2 .

6 . D a u g h t o n , C . G . , A . M. C o o k , a n d M. A l e x a n d e r . 1 9 7 9 . B i o d e g -r a d a t i o n o f p h o s p h o n a t e t o x i c a n t s y i e l d s m e t h a n e o r e t h a n e o nc l e a v a g e o f t h e C - P b o n d . FEMS M i c r o b i o l . L e t t . 5 : 9 1 - 9 3 .

7 . D a u g h t o n , C . G . , A . M. C o o k , a n d M. A l e x a n d e r . 1 9 7 9 . P h o s -

p h a t e a n d s o i l b i n d i n g : f a c t o r s l i m i t i n g b a c t e r i a l d e g r a d a t i o n o fi o n i c p h o s p h o r u s - c o n t a i n i n g p e s t i c i d e m e t a b o l i t e s . A p p l . E n v i -

r o n . M i c r o b i o l . 3 7 : 6 0 5 - 6 0 9 .8 . H o r i g u c h i , M . , a n d M. K a n d a t s u . 1 9 5 9 . I s o l a t i o n o f 2 - a m i n o e t h -

a n e p h o s p h o n i c a c i d f r o m r u m e n p r o t o z o a . N a t u r e ( L o n d o n )1 8 4 : 9 0 1 - 9 0 2 .

9 . La N a u z e , J . M . , J . R . C o g g i n s , a n d H . B . F . D i x o n . 1 9 7 7 .A l d o l a s e - l i k e i m i n e f o r m a t io n i n t h e m e c h a n i s m o f a c t i o n o f

p h o s p h o n o a c e t a l d e h y d e h y d r o l a s e . B i o c h e m . J . 1 6 5 : 4 0 9 - 4 1 1 .

1 0 . L a N a u z e , J . M . , a n d H . R o s e n b e r g . 1 9 6 8 . T h e i d e n t i f i c a t i o n o f

2 - p h o s p h o n o a c e t a l d e h y d e a s a n i n t e r m e d i a t e i n t h e d e g r a d a t i o n

o f 2 - a m i n o e t h y l p h o s p h o n a t e b y B a c i l l u s c e r e u s . B i o c h i m .

B i o p h y s . A c t a 1 6 5 : 4 3 8 - 4 4 7 .

1 1 . La N a u z e , J . M . , H . R o s e n b e r g , a n d D . C . S h a w . 1 9 7 0 . T h ee n z y m a t i c c l e a v a g e o f t h e c a r b o n - p h o s p h o r u s b o n d : p u r i f i c a t i o na n d p r o p e r t i e s o f p h o s p h o n a t e s . B i o c h i m . B i o p h y s . A c t a 2 1 2 :3 3 2 - 3 5 0 .

1 2 . L a e m m l i , U . K . 1 9 7 0 . C l e a v a g e o f s t r u c t u r a l p r o t e i n s d u r i n g t h ea s s e m b l y o f t h e h e a d o f b a c t e r i o p h a g e T 4 . N a t u r e ( L o n d o n )2 2 7 : 6 8 0 - 6 8 5 .

1 3 . L i n e w e a v e r , H . L . , a n d D . B u r k . 1 9 3 4 . T h e d e t er m i n a t i o n o f

e n z y m e d i s s o c i a t i o n c o n s t a n t s . J . Am . C h e m . S o c . 5 6 : 6 5 8 - 6 6 6 .

1 4 . M a s t a l e r z , P . , Z . W i e c z o r e k , a n d M. K o c h m a n . 1 9 6 5 . U t i l i z a t i o no f c a r b o n - b o u n d p h o sp h o r u s b y m i c r o o r g a n i s m s . A c t a B i o -

c h i m . P o l . 1 2 : 1 5 1 - 1 5 6 .

1 5 . M u r a t a , K . , N . H i g a k i , a n d A . K i m u r a . 1 9 8 8 . D e t e c t i o n o f

c a r b o n - p h o s p h o r u s l y a s e a c t i v i t y i n c e l l e x t r a c t s o f E n t e r o b a c -

t e r a e r o g e n e s . B i o c h e m . B i o p h y s . R e s . Commun. 1 5 7 : 1 9 0 - 1 9 5 .

1 6 . S e i d e l , H . M . , S . F r e e m a n , H . S e t o , a n d J . R . K no wl e s . 1 9 8 8 .

P h o s p h o n a t e b i o s y n t h e s i s : i s o l a t i o n o f t h e e n z y m e r e s p o n s i b l ef o r t h e f or ma t i on o f a c a r b o n - p h o s p h o r u s b on d . N a t u r e( L o n d o n ) 3 3 5 : 4 5 7 - 4 5 8 .

1 7 . W a c k e t t , L . P . , S . L . S h a m e s , C . P . V e n d i t t i , a n d C . T . W a l s h .

1 9 8 7 . B a c t e r i a l c a r b o n - p h o s p h o r u s l y a s e : p r o d u c t s , r a t e s , a n dr e g u l a t i o n o f p h o s p h o n i c a n d p h o s p h i n i c a c i d m e t a b o l i s m . J .B a c t e r i o l . 1 6 9 : 7 1 0 - 7 1 7 .

1 8 . Z e l e z n i c k , L . D . , T . C . M e y e r s , a n d E . B . T i t c h e n e r . 1 9 6 3 .G r o w t h o f E s c h e r i c h i a c o / i o n m e t h y l - a n d e t h y l p h o s p h o n i ca c i d s . B i o c h i m . B i o p h y s . A c t a 7 8 : 5 4 6 - 5 4 7 .

J . B A C T E R I O L .