Competitive inhibition of adenosine deaminase by purine and pyrimidine bases

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BBA 63313

Competitive inhibition of adenosine deaminase by purine and pyrimidine bases The factors underlying the specificity of the binding of bases, ribosldes, nbotzdes and base-containing coenzymes to enzyme proteins, as well as related chemical mechanisms are still largely unknown One approach to this problem is through the study of enzymes which use simple purine and pyrimidme derivatives as substrates 1 For this s t u d y we have used adenosine deammase (adenosine aminohydrolase, EC 3-5 4-4) from calf intestinal mucosa Adenosine, 2,6-dlamlnopurme and 6-chloropurine rlbosides are substrates for the enzyme2, a The natural ribosldes moslne, guanoslne, xanthosine and cytmdlne do not inhibit the enzyme activity, whde purine and 6-methylammopurme rlbosldes are mhabltors a. A study of the inhibitory action of the purlne and pyrlmldine bases on adenosine deaminase activity is reported m this paper The direct utilization of the bases rather than the rlbosides, allows one to compare the Ki values of the bases whose nbosldes are enzyme substrates, with those of the bases whose nbosldes are competitive lnhlbitors. Furthermore, it seems to be easier to correlate the degree of lnhabltlon of the various bases with thelr chemical structure. Adenosine deammase has been purified according to the method of BRADY AND O'CONNELL4 The enzyme activity was measured spectrophotometrmally5 in o_05 M phosphate buffer at pH 7 and 2o°. The K~ values have been calculated from LineweaverBurk and from Dixon plots. For the nonlnhibltory compounds, the lower b o u n d a r y of the K~ values is reported In Table I the K, values of a series of bases and the corresponding ribosldes are reported The bases of the rlbomdes which are enzyme substrates or competitive mhlbitors are competlhve lnhlbitors. The lack of the rlbose moiety greatly decreases TABLE

I

KINETIC CONSTANTS FOR SLIBSTRATES AND INHIBITORS OF ADENOSINE DEAMINASE

Rzbos~des

K~ or Km

Adenosme ,-Ammoadenosme 6-Chloropurlne rlbomde N6-Methyladenosme Purine nboside

Inosme Guanosme Xanthosme 6-Mercaptopurme

nbomde

Bases

K, I0 t

X ro 5

X

(M)

(M)

4 2 64 o o

> 3° > 3° > 3° >3 °

(Km) (Km)

(Km)* 5 7

Ademne 2-Ammoadenme 6-Chloropurme N6-Methylade nme Purlne 2-Ammopurme 2-Methyladenlne 2-Hydroxyademne Hypoxanthme Guamne Xanthme 6-Mercaptopurme 2-Ammo-6-mercaptopurme 2-Hydroxy-6-mercaptopurme

2 i 13 3 9 3 I z > 30 > 3° > 3° >3 ° > 3o >3 °

8 7 o 7 o 2 5 4

* From ref 6

Bzochzm Bzophys Acta, 159 ( 1 9 6 8 ) 2 0 3

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the affinity for the enzyme, however the bases show a m e a s u r a b l e i n h i b i t o r y effect H y p o x a n t h l n e , guanine, a n d xanthlne, whose corresponding ribosldes do n o t inhibit the enzyme activity, are n o t Inhlbltors (K, over 30- IO 4 M) I t a p p e a r s t h a t adenosine d e a m m a s e has different affinities for the bases t e s t e d , however it does not a p p e a r t h a t an amino group at the 6-position of the purlne nucleus is necessary for the b i n d i n g at the active site, since purxne, 2 - a m m o p u r l n e a n d 6-chloropurine are r e l a t i v e l y good lnhlbltors T h e s u b s t i t u t i o n of the h y d r o g e n a t o m in the 2-position of adenine with an amino, h y d r o x y l or m e t h y l group leads to a decrease of the K , values with respect to t h a t of adenine. The mechanlsrn w h e r e b y the enzyme discriminates between t h e classes of i n h i b i t o r y c o m p o u n d s a n d n o n m h l b l t o r y c o m p o u n d s IS not I m m e d i a t e l y a p p a r e n t H o w e v e r a striking s t r u c t u r a l d i v e r s i t y exists between the bases which are m h i b l t o r s a n d t h e c o m p o u n d s which have K , values over 3o" lO -4 M_ All i n h i b i t o r c o m p o u n d s b e a r a double b o n d between N - I a n d C-6 a t o m s of the purine nucleus, while in aqueous solution h y p o x a n t h i n e and 6 - m e r c a p t o p u r m e are most p r e v a l e n t in the t a u t o m e r l c keto a n d thlone form, respectivelyT, s ; furthermore, the most basic nitrogen is N - I for the inhlbitors a n d N-7 for the other compoundsT, 9 As Table I I shows, there IS a correTABLE II BASICITY OF ADENOSINE DEAMINASE INHIBITORS

Bases

K~ x lO 4 (AI)

plXa*

Pos~tzon of the most baszc nztrogen °

2-Methyladenme 2-Ammoademne 2-Hydroxyademne Adenine 2-Ammopurme Purme 6-Chloropurme Hypoxanthme Guanine Xanthme 6-Mercaptopurme 2-Arnmo-6-mercaptopurme

i I 2 2 3 9 13 >3 ° >3 ° > 3° > 3° >3 °

5 5 4 4 3 2 <2 2 3 o < 2

N-I N-I N-I N-I N-I N-I N-I N- 7 N-7 N- 7 N-7 N- 7

" From

refs

7

5 7 4 8 2 o o

I** I 5 2 8 4 o o 3 8 5

IO

"" p K a v a l u e o f 2 m e t h y l - 6 - m e t h y l a m l n o p u r m e

latxon b e t w e e n the b a s i c l t y of the N - I and t h e K , values, i n d e p e n d e n t of the n a t u r e of the s u b s t l t u e n t . The p u r m e c o m p o u n d s in which the most basic nitrogen is N- 7 show Kz values higher t h a n 3o IO 4 M The presence of a m e t h y l group or a m e t h y l a t e d s u b s t l t u e n t in the 6-position leads to noninhibatory c o m p o u n d s : 6-methyl-, 6 - m e t h o x y - , 6 - m e t h y l t h l o - 6-dlm e t h y l a m i n o p u r l n e , which have a pK= of 2 6, 2.2, o and 3-9, respectively 1°, e x h i b i t inhibition constants higher t h a n 3 ° . I o -4 M W e t h i n k t h a t the lack of inhibition can be ascribed to t h e low p o l a r i t y of the m e t h y l - , m e t h o x y - , m e t h y l t h i o - a n d d l m e t h y l amino radicals r a t h e r t h a n to a sterac hindrance, since 6-1nethylammopurlne (pKa of 4-2) IS a good inhibitor Bzochzm Bzophys Acta, 159 (1968) 2 o 3 - 2 o 5

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T h e basicity of N - I does n o t seem to be the only factor responsible of the b i n d i n g of t h e purine. Other results show the i m p o r t a n c e of the imidazole m o i e t y of the p u r i n e nucleus. A l t h o u g h imldazole (20 mM) does not inhibit t h e enzyme, the modifications of t h e lmldazole ring of th e p u r m e nucleus result in a decrease of the affinity for adenosine d e a m m a s e , 8 - a z a a d e n m e (pKa of 2 6), in which t h e m o s t basic nitrogen is N - I (ref 8) does not inhibit the e n z y m e a c ti v i ty . Cytosine in which the most basic nitrogen is N-3 of t h e p y r i m i d i n e ring, w i t h a p K a close to t h a t of adenine s, shows a K , v a l u e higher t h a n 3 o - l o -4 M. H o w e v e r when the baslcity of t h e p y n m l d l n e c o m p o u n d s increases, we again find good c o m p e t i t i v e inhlbltors This is t h e case with 2,4,6-tria m m o p y r l m l d m e which has a p K a of 6 8 and a K , v al u e of 4" lO-4 M W e h a v e also studied th e effect of the p r o t o n a t l o n of 2 , 4 , 6 - t r i a m m o p y n m i d l n e , 2 - a m m o a d e n m e and 2 - m e t h y l a d e m n e on t h e K , values of these bases. Th e K , values of ttle three c o m p o u n d s increase w i t h decrease of p H and the increase of the K , values parallels the p r o t o n a t i o n of the inhibitor H o w e v e r , c o n s t a n t K~ values are found when t h e y are calculated from the c o n c e n t r a t i o n of the d e p r o t o n a t e d form of the m h l b i t o r s at the various p H ' s tested T h e p r o t o n a t l o n of N - I of the purine ring and of N-3 of t h e p y r i m l d m e ring results m n o n m h l b l t o r y c o m p o un d s A l t h o u g h t h e d a t a r e p o r t e d in this p a p e r suggest t h a t t h e N - I of the purIne nucleus plays a role m t h e b m d m g of the lnhlbltors and substrates to the a c t i v e site of adenosine d eam ma s e , at the present t i m e a direct d e m o n s t r a t i o n has not been made. C o m p o u n d s in which the various nitrogen at o m s of the p u r m e nucleus are s u b s t i t u t e d b y a carbon a t o m are now u n d e r i n v e s t i g a t i o n T h e au t h o r s wish to t h a n k Dr L B TOWNSEND for the gift of N e - m e t h y l adenosine and Mr R CHITI for technical assistance. This work was s u p p o r t e d b y t h e I t a l i a n C.N_R

Institute of B,ochem,stry, Umvers,ty of Pzsa, P,sa (Italy)

G. RONCA G ZUCCHELLI

R XVOLFENDEN,T K SHARPLESSAND R ALLAN,J Bzol Chem, 242 (1967) 977 A KORNBERGAND W E PRICER, JR, dr Btol Chem, 193 (I95 I) 481 J G CORY AND R J SUHADOLNIK,B~ochem~stry, 4 (1965) 1729 T G BRADYAND W O'CONNELL, Bzoch~m B*ophvs Acta, 62 (1962)216 H M KALCKAR,J Bzol Chem , 167 (1947) 445 J G CORY AND ]~ J SUHADOLNIK.B*ochem~str7, 4 (I965) 1733 J H LISTER, m A R KATRITZKYAND A J BOULTON, Advances *n Heterocvchc Chemtstry, Vol 6, Academic Press, New York, 1966, p I 8 B PULLMANAND A PULLMAN,Quantum B~ochem~strv, Intersclence, New York. 1963, p 230 9 A R KATRITZKYAND J M LAGOXVSKI,in A R KATRITZKY, Advances *n Heterocychc Chemzstry, Vol 2, Acadelmc Press, New York, 1963. p_ 27 IO A ALBERT, In A R KATRITZKY, Phvstcal Methods tn Heterocychc Chemzstry, Vol I, Acadermc Press, New York, 1963, p i i 2 3 4 5 (, 7

R e c e i v e d D e c e m b e r 27th, 1967

B*ochzm Bzophys Acta, 159 (1968) 203-205