The enzymic phosphorylation of vitamin B6 derivatives and their effects on tyrosine decarboxylase

496

BIOCHIMICA ET BIOPHYSICA ACTA

VOL. 9

(1952)

T H E ENZYMIC PHOSPHORYLATION OF VITAMIN B 6 DERIVATIVES AND T H E I R EFFECTS ON TYROSINE DECARBOXYLASE* by JERARD HURWITZ Department o/Biochemistry, Western Reserve University School o/Medicine, Cleveland 6, Ohio (U.S.A.)

One type of inhibition found with vitamin analogues has been suggested to be via its ability to be further metabolized and thus successfully compete for the same site on an enzyme surface normally occupied by coenzymes. The antagonism found with desoxypyridoxine1 and pyrithiamine2 has been attributed to enzymic phosphorylation of these compounds to an inhibitory form. Recently, KEARNEY3 has demonstrated the direct enzymic phosphorylation of vitamin 132 antagonists with a purified yeast enzyme. The present investigation demonstrates the direct phosphorylation of vitamin B 6 analogues and their effect on tyrosine apodecarboxylase. The enzyme system used for these studies was an enzyme preparation capable of phosphorylating pyridoxal. The enzyme has been purified about IOOO-fold from brewer's yeast strain BSC** by (NH4)2SO 4 fractionations, alcohol fractionation, isoelectric precipitation of contaminating proteins, and heat inactivation. The enzyme was followed through the above purification by its ability to convert pyridoxal and ATP to pyridoxal phosphate and ADP. Pyridoxal phosphate was assayed by a modification of the tyrosine apodecarboxylase method4. The enzyme preparation does not exhibit strict specificity regarding pyridoxal. Pyridoxine, pyridoxamine, desGxypyridoxine, 4,5-dihydroxy-2-methyl pyridine, and 3- amino- 4, 5- dihydroxymethyl-2- methyl pyridine appear to be phosphorylated since labile phosphate disappears when ATP and the analogues are incubated together in the presence of the kinase. The unphosphorylated analogues have no effect on the union of pyridoxal phosphate with tyrosine apodecarboxylase, while the phosphorylated analogues are found to inhibit tyrosine apodecarboxylase, apparently by blocking the union of pyridGxal phosphate with the enzyme (Table I). The same inhibition is observed when the phosphorylated analogue and pyridoxal phosphate are incubated together as when the phosphorylated analogue is preincubated with tyrosine apodecarboxylase before the addition of pyridoxal phosphate. However, if pyridoxal phosphate is preincubated with tyrosine apodecarboxylase before the addition of the analogue phosphate, no inhibition is found1. Both chemically and enzymically synthesized pyridoxamine phosphate are found to stimulate the decarboxylase, and this may be due to its conversion to pyridoxal phosphate. The compound, 2-ethyl-3-amino* This w o r k w a s a i d e d in p a r t b y g r a n t s from Th e N a t i o n a l V i t a m i n F o u n d a t i o n , a n d t h e Elisabeth Severance Prentiss Foundation. ** A n h e u s e r - B u s c h , w a s h e d , l o w - t e m p e r a t u r e dried y e a s t .

Re/erences p. 498.

VOL. 9 (1952)

PHOSPHORYLATIONOF VITAMIN l{6 DERIVATIVES

497

TABLE I THE

EFFECT

8 6 ANALOGUES

ON

PHOSPHORYLATION

OF

OF VITAMIN AND

Compounds tested

TYROSINE

DECARBOXYLATION

PYRIDOXAL

Inhibition of tyrosine apodecarboxylase by product of kinase reaction

2 - methyl- 3 - hydroxy- 4- hydroxymethyl pyridine 2-ethyl-3-amino-4-ethoxymethyl-5-aminomethylpyridine desoxypyridoxine 3, 4- dihydroxymethyl- 2, 6- dimethyl pyridine 4, 5 - dihydroxymethyl- 2 - methyl pyridine 3 - amino- 2 - methyl - 4, 5, 6- trihydroxy methyl pyridine 5 - hydroxymethyl- 2, 4, 6 - trimethyl pyridine 3 - hydroxy- 5, 6- dihydroxymethyl- 2 - methyl pyridine 3 - amino- 4, 5 - dihydroxymethyl- 2 - methyl pyridine pyridoxine pyridoxamine

--

Inhibition oJ pyridoxal phosphate formation from pyridoxal plus A TP

÷

+

-}stimulation

(+) indicates inhibition while (--) indicates no inhibition. Those compounds which were found to inhibit the union of pyridoxal phosphate with tyrosine apodecarboxylase were not assayed for their ability to inhibit pyridoxal phosphorylation since separation of pyridoxal phosphate and the phosphorylated analogue would have been necessary for such tests.

4 - e t h o x y m e t h y l - 5 - a m i n o m e t h y l pyridine, does not i n h i b i t the u n i o n of pyridoxal phosphate with tyrosine apodecarboxylase, b u t it m a r k e d l y i n h i b i t s the p h o s p h o r y l a t i o n of p y r i d o x a l b y the kinase when i n c u b a t e d with ATP. The i n h i b i t i o n is competitive a n d the KI of this c o m p o u n d is 7.3" IO-5 M which indicates t h a t this c o m p o u n d has a greater affinity for the kinase t h a n p y r i d c x a l (K m of pyridoxal is 1. 4. Io .4 M). T h u s there are at least 2 possible m e t h o d s w h e r e b y these v i t a m i n B 6 analogues can i n h i b i t : (I) b y i n h i b i t i n g the u n i o n of p y r i d o x a l phosphate with the tyrosine apodecarboxylase b y v i r t u e of their being phosphorylated, a n d (2) b y i n h i b i t i n g the p h o s p h o r y l a t i o n of pyridoxal. Also listed in Table I are those c o m p o u n d s which had no effect on the kinase or on the tyrosine decarboxylase after i n c u b a t i e n with ATP. A c o m m o n feature of those c o m p o u n d s which appear to be p h o s p h o r y l a t e d a n d affect the kinase is the free 6 position of the p y r i d i n e ring a n d the presence of a h y d r o x y m e t h y l or m e t h y l a m i n e group in the 5 position. The e n z y m e is c o m p e t i t i v e l y i n h i b i t e d b y ADP, adenylic acid, adenGsine, and a d e n i n e while h y p o x a n t h i n e , inosine, a n d inosinic acid have no effect on the reaction. I T P c o m p e t i t i v e l y i n h i b i t s the reaction only when high c o n c e n t r a t i o n s are used a n d the i n h i b i t i o n can be completely reversed b y increasing the c o n c e n t r a t i o n of Mg ++, suggesting t h a t it is i n h i b i t o r y b y v i r t u e of its a b i l i t y to chelate Mg ++~. I t appears t h a t the 6-amino group is responsible for the u n i o n of the e n z y m e a n d p u r i n e ring. A D P a n d I T P are u n a b l e to p a r t i c i p a t e in the p h o s p h o r y l a t i o n of pyridoxal, a n d the system is i n a c t i v e in the absence of a d i v a k n t metal. A recent report 6 i n d i c a t e d t h a t an e n z y m e p r e p a r a t i o n o b t a i n e d b y repeated (NH4).~SO4 f r a c t i o n a t i o n of a n e x t r a c t ef yeast, p h o s p h o r y l a t e d p y r i d e x i n e a n d also p h o s p h o r y l a t e d t h i a m i n e leading to p y r i d o x i n e phosphate a n d d i p h o s p h o t h i a m i n e , respectively. The p r e p a r a t i o n described here does n o t appear to p h o s p h o r y l a t e t h i a m i n e as d e t e r m i n e d b y cocarboxylase a c t i v i t y . The a u t h o r is deeply i n d e b t e d to Dr W. W. UMBREIT for a gift of s y n t h e t i c pyridoxal Re/erences p. 498.

498

j . HURWITZ

VOL. 9 (1952)

phosphate; to D r R E U B E N J O N E S o f t h e L i l l y R e s e a r c h L a b o r a t o r i e s , SINGER f o r a g e n e r o u s g i f t o f s o m e o f t h e v i t a m i n B 6 a n a l o g u e s .

and

Dr T. P.

T h i s p r o b l e m w a s k i n d l y s u g g e s t e d t o t h e a u t h o r b y D r T . P . SINGER w h o s e g e n e r o u s guidance was indispensible during the course of this work. Protocol Inhibition o[ tyrosine apodecarboxylase: 5' lO-3 M a n a l o g u e , 1.87. io - t ~V/ A T P , 0.04 M m a l e a t e buffer p H 6.9, 4" io 3 M Mg++, a n d 1. 5 m g of e n z y m e in a t o t a l v o l u m e of 2 m l were i n c u b a t e d 12 h o u r s a t 33.5 ° C. A f t e r h y d r o l y z i n g t h e r e m a i n i n g ATt? w i t h a p y r a s e , t h e r e a c t i o n p r o d u c t s were a s s a y e d for t h e i r ability to i n h i b i t t y r o s i n e a p o d e c a r b o x y l a s e u n i o n w i t h 5.5' io s 3/I of p y r i d o x a l phosphate. Inhibition o[ pyridoxal phosphate /ormation: 2. lO -3 M analogue, i . lO -2 M A T P , 0.02 M phosp h a t e buffer p H 6.9, 3' lO-a M Mg ++, 0.64 m g of protein, i n c u b a t e d in presence of 3 different concent r a t i o n s of p y r i d o x a l (i. IO 3 ~/, 2. IO 3 M , a n d i • lO -3 M) in a t o t a l v o l u m e of 2 ml. T h e s e reaction m i x t u r e s were i n c u b a t e d 2 h o u r s at 33.5 ° C a n d t h e n a s s a y e d for p y r i d o x a l p h o s p h a t e f o r m a t i o n . SUMMARY A k i n a s e s y s t e m purified f r o m y e a s t is c a p a b l e of p t l o s p h o r y l a t i n g p y r i d o x a l in t h e presence of A T P . T h i s s y s t e m h a s been f o u n d capable of p h o s p h o r y l a t i n g o t h e r v i t a m i n B 6 d e r i v a t i v e s p r o v i d e d t h e 6 p o s i t i o n of t h e p y r i d i n e ring is free a n d t h e 5 position c o n t a i n s a h y d r o x y m e t h y l group. T h e c o m p o u n d s w h i c h are p h o s p h o r y l a t e d a n d r e p o r t e d in t h i s p a p e r are also f o u n d to i n h i b i t t h e u n i o n of p y r i d o x a l p h o s p h a t e w i t h t y r o s i n e a p o d e c a r b o x y l a s e . T h e r e q u i r e m e n t s for inhibition of this u n i o n a n d o t h e r m e t h o d s of i n h i b i t i n g t h e kinase are discussed. RI~SUMI~ U n s y s t ~ m e de k i n a s e purifi6e p e u t p h o s p h o r y l e r le p y r i d o x a l en presence d ' A T P . Ce s y s t ~ m e est 6 g a l e m e n t c a p a b l e de p h o s p h o r y l e r d ' a u t r e s d6riv6s de la v i t a m i n e B6, p o u r v u q u e la position 6 d u n o y a u p y r i d i n i q u e soit libre et q u e le position 5 c o n t i e n n e u n g r o u p e h y d r o x y m e t h y l . L e s compos6s qui s o n t p h o s p h o r y M s et q u i se t r o u v e n t cit6s d a n s ce m 6 m o i r e e m p 6 c h e n t aussi l ' u n i o n d u p h o s p h a t e de p y r i d o x a l ~ la t y r o s i n e - a p o d e c a r b o x y l o s e . L e s conditions d ' i n h i b i t i o n de cette u n i o n et d ' a u t r e s m 6 t h o d e s d ' i n h i b i t i o n de la k i n a s e s o n t discut6es. ZUSAMMENFASSUNG E i n y o n Here gereinigtes K i n a s e - S y s t e m k a n n P y r i d o x a l in G e g e n w a r t y o n A T P p h o s p h o r y lieren. Dieses S y s t e m k a n n a u c h a n d e r e V i t a m i n - B s - D e r i v a t e p h o s p h o r y l i e r e n , v o r a u s g e s e t z t , d a s s die Stellung 6 i m P y r i d i n r i n g frei ist u n d in Stellung 5 eine O x y m e t h y l - G r u p p e sitzt. Die Verbind u n g e n , welche p h o s p h o r y l i e r t w e r d e n u n d die in dieser A r b e i t aufgez/ihlt sind, v e r h i n d e r n a u c h die B i n d u n g v o n P y r i d o x a l - P h o s p h a t a n T y r o s i n - A p o d e c a r b o x y l a s e . Die I3edingungen, u n t e r welchen diese V e r b i n d u n g g e h e m m t wird u n d a n d e r e I - I e m m u n g s - M e t h o d e n der K i n a s e w e r d e n er6rtert. REFERENCES 1 w . }v. UMBREIT AND J. G. WADDEL, Proc. Soc. Exptl. Biol. Med., 7° (1949) 293. D. W. WOOLEY, J. Biol. Chem., 191 (1951) 43. 3 E. B. I~EARNEY, J. Biol. Chem., 194 (1952) 747. 4 W. W. UMBREIT, ~V. D. I3IgLLAMY, AND I. C. GUNSALUS, Arch. Biochem., 7 (1945) 185. li C. ~EUBERG AND I. MANDL, Arch. Biochem., 23 (1949) 499. 6 L. CHEVILLARD AND N. V. THOAI, Bull. soc. chim. biol., 33 (1951) 1147. Received

March 25th, 1952