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The relationship of mono- and polynucleotide conformation to catalysis by polynucleotide phosphorylase
I8
BIOCHIMICA ET BIOPHYSICA ACTA
BBA 96575
T H E R E L A T I O N S H I P OF MONO- AND POLYNUCLEOTIDE CONFORMATION TO CATALYSIS BY POLYNUCLEOTIDE P H O S P H O R Y L A S E A M K A P U L E R ", C. MONNY AND A M. M I C H E L S O N
Inst*tut de B~olog~e Phys~co-chzmzque, Par~s (France) (Received F e b r u a r y 26th, 197 o)
SUMMARY
8-Bromoguanosine 5'-dlphosphate, 8-oxoguanosine 5'-dlphosphate, 6-methylcytidine 5'-diphosphate and 2,6-quinazoline dione I'-ribosyl 5'-diphosphate are mactive as substrates for homopolymer synthesis with polynucleotlde phosphorylase They inhibit polymerization and exchange but not phosphorolysis. Poly 8-bromoguanyhc acid (poly BrG), prepared chemically, is not phosphorolysed by polynucleotlde phosphorylase. The polymer inhibits polymerization reversibly and competitively whereas phosphorolysls is inhibited irreversibly and non-competitively. Thus the tenacity of polymer binding is a function directly or mdirectly of Pi concentration. The behaviour of these molecules with polynucleotide phosphorylase suggests that the catalytic activities of this enzyme require substrates which are capable of assuming the ant~ conformation. The ant~ conformation normally present in nucleoslde monomers and in polynucleotides, requires close apposition of the 2'-H of the ribofuranose and either the C-8 hydrogen of purines or the C-6 hydrogen of pyrimidlnes Replacement of these ring protons by larger substituents such as bromine or methyl inhibits the assumption of a normal antz conformation. Hence the sterically restricted nucleoside 5'diphosphates are predominantly In the syn conformation, as are residues in poly BrG. Since molecules containing residues in the syn (non-ant~) conformation interact strongly with polynucleotide phosphorylase, a syn - ~ ant~ conformatlonal isomerism IS possibly a natural part of the various catalytic processes Consistent with this formulation, ApBrG and BrGpA do not act as primers for the Micrococcus enzyme.
INTRODUCTION
Many nucleotide analogs have been examined as substrates for polynucleotide phosphorylase These derivatives fall into three general classes The first comprises * Rockefeller University, New York Present address Mmroblology Section, Life Scmnces, University of Connecticut, Storrs. Conn U S A Abbreviations NDP, nucleoslde 51-dlphosphate, BrGDP, 8-bromoguanosme 5'-d~phosphate, 8-0 GDP. 8-oxoguanosme5'-dlphosphate, QDP, 2,6-qumazohne dlone i ' - n b o s y l 5'-dlphosphate, poly BrG, poly 8-bromoguanyhc acld, copoly G, BrG (23 77) a r a n d o m copolymer of g u a n y h c acid and 8-bromoguanyhc acid with a e o m p o s m o n of 23 % GMP and 77 % 13rGMP.
Bzoch*m B~ophys Acta, 217 (197 ° ) 18-29
POLYNUCLEOTIDE PHOSPHORYLASE
19
those molecules which serve as substrates and which behave in a manner similar to that of natural nucleotides. Included in this class are the 5'-dlphosphate derivatives of the nucleosides pseudouridme 1, 5-bromourldine 2, 6-hydroxyethyladenosine3, 8azaguanoslne 4, I-methylguanosine 5, 2,6-dlaminopurine riboside e and 2-aminopurine rlboside 7. The second class comprises derivatives that are neither good substrates nor strong mhibltors, such as dlhydro UDP s and dADP 1°,12. The third class encompasses strongly inhibitory nucleoside diphosphates and is represented by 6-aza UDP 9 and 6-mercaptopurlne riboslde 5'-diphosphate 11. A fundamental difference between the substrate requirements of nucleic acid polymerases and polynueleotide phosphorylase concerns the Watson-Crick hydrogenbonding positions of the bases: for template-directed reactions the hydrogen-bonding position must not be blocked. In contrast, polynucleotlde phosphorylase appears to be indifferent to the status of these functional groups, since the 5'-dlphosphates of xanthosine, N~-methyluridine and NS-dlmethyladenosme are readily polymerized. Nevertheless, those nucleoside derivatives not polymerized by polynucleotide phosphorylase either fail to serve or are poorly utlhzed as substrates (as 5'-triphosphates) by DNA-dependent polymerases 13. In this paper, we describe the interaction of polynucleotlde phosphorylase with 8-bromoguanoslne 5'-diphosphate (BrGDP) and poly BrG. The salient features of this class of mhlbltors (which includes also the 5'-diphosphates of 6-methylcytldine, 8-oxoguanosine and quinazohne-2,6-dione I'-riboside) are (I) the normal hydrogenbonding positions and the ribofuranose ring are preserved and (2) the chemically unrelated substltuents introduced into the bases share an important property, namely, they influence the stereochemical relationship between the heterocyclic ring and the sugar. The effect of all these groups is to restrict the rotation around the glycosyl bond of the corresponding monomers and polymers, in a manner which makes the s y n conformation energetically more favorable" t h a n t h e normal anti conformation 14-16. These monomers and polymers are lnhibitors of polynucleotide phosphorylase. An interpretation of the results based on polynueleotide stereochemlstry is discussed. The interaction of these and related molecules with nucleic acid polymerases is described elsewhere lv,~l Since this work was completed IKEHARA et al. 2e have described the incorporation of 8-bromoguanosine and 8-hydroxyguanosine dlphosphates into polymer materials by polynucleotide phosphorylase. The present results provide conformation of their studies on polymerisation and extend them to consideration of the behaviour of the monomers with respect to the phosphate exchange and phosphorolysis activities of polynucleotide phosphorylase. In addition, the action of poly BrG on all three activities catalysed by polynucleotide phosphorylase is described.
* Single crystal X - r a y diffraction studies of 8 - b r o m o g u a n o s l n e and 8 - o x o g u a n o s m e h a v e confirmed this assertion which w a s based on model b m l d l n g 18 a n d calculation. These t w o nucleosides h a v e t h e syn c o n f o r m a t i o n in crystals le. Unlike p u r m e nucleosides where the energy b a r r i e r b e t w e e n syn a n d ant* is r a t h e r small, pyrlmldlne nucleosldes exist m a i n l y in the ant* conforrnatlonl,,15,2, A b u l k y s u b s t l t u e n t at C-6 p r o b a b l y reduces the e x t e n t of antz c o n f o r m a t i o n w i t h o u t necessarily I m p o s i n g a p u r e syn f o r m Bzoch,m. B,ophys. Acta, 217 (197o) I 8 - 2 9
20
A.M. KAPULER et al.
MATERIALS AND METHODS
Poly BrG, a random copolymer o/GMP and 8-bromoguanylic acid with a compositwn O~ 23 % GMP and 77 % 8-bromoguanylic acid (copoly G,BrG (23:77), ApBrG and BrGpA The synthesis and characterisation of these dlnucleotides and polymers is described m the preceding publication is
Polynucleot~de phosphorylase The isolation and purification of this enzyme from Azotobacter vzneland~L Escherwhm coli and Mzcrococcus lysodezktzcus has been described previously 19,2°. Unless otherwise noted, reactions were performed in final volumes of IOO/~1 for 15 min at 37 °, incubation mixtures were as follows (/zmoles/ml). phosphorolysis, Tlis-HC1 (pit 8 2), 50; poly A, 3.o; K22PO4 (pH 8.2), IO, EDTA, 0.5; MgC12, 0.5; polymerization, Trls-HC1 (pH 8 2), IOO; MgC12, IO, ADP, 25-50; EDTA, o 5, exchange, TrisHC1 (pH 8.2), IOO; MgC12, 5; EDTA, o 5; K2~PO4 (pH 8 2), 5; ADP, 5.0 (limiting). The procedures employed for these assays have already been described in detail 3°.
8-Bromoguanosfne 5'-dzphosphate (BrGDP) A suspension of finely powdered GDP (o.5 mmole) In formamide (4 ml) at o ° was treated with Br2 (I mmole) in chloroform (I ml) and the mixture kept at o ° with frequent agitation for 80 rain. The solution was then extracted twice with ether and the extracts discarded. Crude BrGDP was then precipitated by the addition of ethanol-ether ( I : I , by vol.), the precipitate was collected, washed with ether and dried. The material was purified on Dowex I × 2 (C1- form) using a gradient of o.oi M HC1 to o.oi M HC1, I.O M LIC1 Appropriate fractions were combined, neutrahsed with trl-n-butylamine and concentrated under reduced pressure. The lithium salt of BrGDP was precipitated with ethanol-acetone, collected, washed and dried. Yield 80 9'0 Paper chromatography showed that the material was homogeneous and free from GDP.
8-Bromo-E8-14CJguanos~ne 5'-d~phosphate (BrI14CJGDP) 5 °/~C of I8-14CJGTP (specific radioactivity 28/~C//zmole, C.E.A., Saclay, France) were dissolved in IOO/~1 formamide and bromlnated by addition of a Io-fold excess of Br 2. The product was isolated and purified by chromatography on DEAE-cellulose and analyzed for purine composition by depurlnation and chromatography of the bases in n-butanol-98 °/o formic acid-water (76.1o:13, by vol )31. It consisted of 99.2 °/o BrGTP and o 8 ~o GTP. The trlphosphate was converted to the diphosphate with hexokmase and purified by DEAE-eellulose chromatography.
8-Oxoguanoszne 5'-d@hosphate (8-0 GDP), 6-methylcytzd~ne 5'-diphosphate(6MeCDP), 2,6-qmnazolzne d, one z'-r~bosyl 5'-d@hosphate (QDP) The synthesis of these molecules from crystalline nucleosldes (8-oxoguanosme, 6-methyleytidine and 2,6-quinazohne dlone I'-riboside were generously supplied by Dr. R Robins) followed standard procedures 22,23. Details of the synthesis of the corresponding 5'-triphosphates will be published elsewhere 24. B~ochzm Bzophys dcta, 217 (197o) I 8 - 2 9
POLYNUCLEOTIDE PHOSPHORYLASE
21
RESULTS
Interaction o/polynucleotidephosphorflase with con/ormationally restricted nucleoside 5'-diphosphates (NDPs) (a) BrGDP: Using polynucleotlde phosphory]ase isolated from both A. wneland,~ and E. colh the activity of BrGDP was studied with respect to polymerization, phosphorolysls and a2Pt-NDP exchange. A variety of experimental conditions were employed including the normal conditions at 37 ° for both enzymes, as well as the higher temperatures and the use of Mn ~+ which are known to promote poly G synthesis by the E. colt enzyme 25. Under all conditions examined, BrGDP did not serve as a substrate for homopolymer synthesis by either enzyme. For example, at 6o °, with the E. col~ enzyme, under standard Mn 2+ conditions 2s, less than o.15 re#mole of Br[t4CIGDP was converted into acid-insoluble form while in a separate, control reaction, 20 m/~moles of [14CJGDP were polymerlzed. When a mixture of BrGDP/[14CJGDP -= 3.0 was studied, polymerization of GDP proceded at 1/8 the control rate. Furthermore, BrGDP strongly inhibited the utlhzation of any of the normal substrates when these were present in equimolar amounts (Figs. Ia, Ib). The effect of different concentrations of 30 F 50
UDP o ~& 2c
?
4C
f
® cgP
~3c ?
g
g 2c
8
12
L6
20
24
::L
Tirne (hours)
"•3E C
GDP
x~--x ~--x
GDP gr GOIP
/
:k
4
..._...... ~ ×
2
4 6 8 Tlrne (hours)
I0
UDP
BrGDP ~co
o-2 C
I0
IC
33
16
~
,'2
,'~
2'o
2'4
Time (hours)
F i g I a I n h l b l t l o n of p o l y m e r i z a t i o n of C D P , U D P a n d A D P b y B r G D P I n c u b a t i o n s i o t i m e s n o r m a l scale c o n t a i n e d 5 o u n i t s of t h e A. v*neland** p o l y n u c l e o t l d e p h o s p h o r y l a s e a t 3 °o A l l q u o t s of 2 0 / z l w e r e r e m o v e d as i n d i c a t e d , b I n h i b i t ] o n of p o l y m e r i z a t i o n of G D P b y B r G D P R e a c t i o n s i o t i m e s n o r m a l scale w e r e I n c u b a t e d a t 60 ° w i t h 2 o u n i t s of t h e E col, p o ] y n u c ] e o t l d e phosp h o r y l a s e u n d e r s t a n d a r d M n ~÷ c o n d i t i o n s (o o02 M MnC12, o OlO M G D P ) . c E f f e c t of v a r y i n g a m o u n t s of B r G D P o n t h e p o l y m e r i z a t i o n of U D P C o n d i t i o n s as in a. B*och,m. B,ophys
Acta, 217 (197 o) 1 8 - 2 9
22
A.M. KAPULER et al.
B r G D P on the synthesis of poly U by the A. vznelandz, polynucleotide phosphorylase IS shown in Fig. IC A more detailed analysis was performed with the E. coh polynucleotide phosphorylase. Whereas the dependence on substrate concentration of the polymerization of ADP (corrected for free ADP) follows classical enzymological relationships, the action of BrGDP results in non-linear Lineweaver-Burks plots (Fig. 2a). Inhibition is most pronounced at low ratios of A D P / B r G D P and a disproportionate decrease in mhibition is observed when this ratio as increased. The observation of a xT=1 5 x IO-3M
b
I t0,1o"u ~i=l
O x 10-3M i-6s~lo 4M I=6 5 x 10-4M
-I > 2
-I>
4o zo io
tO
N-M xI03
20
le
2o ~'o L~ M-Ix $0 3
I o 40
Fig 2 a. I n h i b i t : o n of polymerization of A D P b y ]3rGDP. I n c u b a t i o n s were s t a n d a r d and similar to t h a t described in Fig 9 except t h a t copoly G, BrG (23 77) was replaced b y ]3rGDP E a c h incub a t i o n c o n t a m e d o 13 u n i t of E coh polynucleotlde p h o s p h o r y l a s e and the polymerlzatlon of A D P (specific radioactivity i 14- lO 6 counts/rain per/xmole) was measured, b. I n h i b i t i o n of polymerization of A D P b y 8 - O G D P Conditions as in a except t h a t 8 - O G D P replaced B r G D P The specific r a d t o a c t i v l t y of A D P was I 45' lO5 counts/rain per # m o l e v = nmoles [3H]AMP incorporated
IKEHARA et al. 26 that low rates of polymerization of C-8-substituted purlne nucleoside diphosphates occur only in the presence of natural nucleoside diphosphates provides a basis for interpreting these kmetlcs. Polymerization of BrGDP occurs only at those ratios of B r G D P / A D P that do not reduce the rate of synthesis too drastically (BrGDP >> ADP) and do not completely eliminate the ability of BrGDP to compete with A D P (ADP >> BrGDP). The BrGMP residues, once incorporated into oligonucleotide become part of a family of inhibitory ohgonucleotides, some of which are more potent inhibltors than BrGDP. We have characterized the limit polymer of this family, namely poly BrG, as described below. The z2Pi-NDP exchange reaction, like the polymerization reaction, was strongly inhibited b y BrGDP (Fig 3). In contrast, only a feeble inhibitory effect on phosphorolysis of poly A was seen (Fig. 4) Exchange of 32Pi into BrGDP was not observed. (b) Other con/ormationally restrzcted N D P s : 8-OGDP, 6-MeCDP, QDP It appeared possible that the effect of BrGDP on polynucleotide phosphorylase was due to the chemical properties of the bromine atom, or to specific characteristics unique to BrGDP. To evaluate this possibility, other conformationally restricted NDPs were B~och~m. B*ophys
Acla, 217 (197 ° ) I 8 - 2 9
23
POLYNUCLEOTIDE PHOSPHORYLASE 2O
4"
o
E
\ ~030
Poqy
v
Poy ,~
,o
\
,\ /
E
5
\~ADP \\
•
)
/I /
u o p X ~
poly
i// //* p y A =mOO
0
05
l ~
[I0
BrG~)P ADP(uoP)
,
, Poly A - 20
Time (hours)
Fig. 3. E f f e c t of v a r i o u s r a t i o s of B r G D P t o A D P or U D P on Pt e x c h a n g e . R e a c t i o n s i o t i m e s n o r m a l scale c o n t a i n e d 5 o u n i t s of t h e A. wnelandzz p o l y n u c l e o t l d e p h o s p h o r y l a s e a n d w e r e s t o p p e d a f t e r IO rain a t 3 o°. A h q u o t s of 2o btl were t a k e n a n d a n a l y z e d . Fig. 4- E f f e c t of B r G D P a n d p o l y B r G on t h e p h o s p h o r o l y s I s of p o l y A R e a c t i o n s i o t i m e s n o r m a l scale c o n t a i n e d 5.0 u n i t s of t h e A. vznelandz, p o l y n u c l e o t l d e p h o s p h o r y l a s e A h q u o t s of 2o/zl were t a k e n as i n d i c a t e d .
synthesized and their effects on polynucleotide phosphorylase studied. 8-OGDP is not polymerized by polynucleotide phosphorylase and, as seen m Fig. 2b, this analog acts as an inhibitor of polynucleotide phosphorylase in a manner quahtatlvely identical to BrGDP. Likewise, neither 6-MeCDP nor QDP were polymerized by the enzyme, and both inhibit the synthesis of poly A; furthermore, 6-MeCDP and QDP resemble BrGDP also in their failure to inhibit phosphorolysis of poly C (Table I). TABLE I INHIBITORY REACTIONS
PROPERTIES OF
OF
6-MeCDP AND Q D P IN
POLYNUCLEOTIDE
THE
POLYMERIZATION
AND
PHOSPHOROLYSIS
PHOSPHORYLASE
C o n d i t i o n s for t h e s e r e a c t i o n s are specified in MATERIALS AND METHODS. The specific r a d i o a c t i v i t y of [ 3 H ] A D P w a s 18oo c o u n t s / r a i n p e r n m o l e a n d t h a t of p o l y [3H]C 34 c o u n t s / m l n p e r n m o l e , i o o m/~moles of t h e l a t t e r were a d d e d t o each p h o s p h o r o l y s l s r e a c t i o n All r e a c t i o n s c o n t a i n e d o o4o u n i t of E. col* p o l y n u c l e o t l d e p h o s p h o r y l a s e (specific a c t i v i t y i o u m t s / m g ) . U n i t s as d e f i n e d b y GRUNBERG-MANAGO3°
-
-
Q D P , I I mM Q D P , 8.6 mM -
-
6-MeCDP, 5 7 mM 6-MeCDP, I I rnM 6-MeCDP, 27 mM
Polymer*zat~on (nmoles [3H~AM P zncorporated)
Phosphorolyszs (nmolespoly E3HICphosphorolyzed)
95 44 i.o 7 9 6.6 4 8 3-9
III 12o 16.9 25 .2 27 7 26 5 24 7
Interaction o~ polynucleotide phosphorylase with poly BrG and copoly G,BrG (23:77) The effect of poly BrG on polynucleotide phosphorylase was examined with respect to polymerization, phosphorolysis and exchange. In contrast to BrGDP, B,och~m. B,ophys Acta, 217 (197 o) 18-29
24
A . M . KAPULER dt
al.
which does not affect phosphorolysis, all three catalytic activities are inhibited by poly BrG and copoly G,BrG (23'77) Poly BrG, like poly G, is itself resistant to phosphorolysls; however, unlike poly G, it is a potent irreversible, non-competitive inhibitor of the phosphorolysls of poly A (Fig 5)- The similar dose-response curve obtained for the case of poly U (Fig 6), demonstrates that the inhibition of phosphorolysis is independent of the nature of the polymer being degraded. The efficacy of 3O
Po]y U PoLy B,G
./ o
o0
o.tO *
~-
080
o
I=5,1o-%
2o
~,~o,
_'~
S=Mxl0-5
•
CO
E
/
,oo
,~u 20 400
I=5×10-6M ~ r
ooo 04D
~
i o
0
/5 d
o/
200
/-
__--~
I00
0o I
@ =M-IxFo 5
2
3
T~me ( h o u r s )
Fig 5 N o n - c o m p e t l t w e inhibition of phosphorolyms of p o l y A b y copoty G,BrG (23 77) ReactmnS contained o Ol 5 unit of E coh polynucleotlde phosphorylase and 5oo m/~moles Ka2PO4 (pH 8 2) (specific radioactivity 286o e o u n t s / m m per nmole) Increasing a m o u n t s of p o l y A were added to a series of reactions prepared in duplicate and o 5 nmole of copoly G,BrG (23 77) was added to one of each pair. Fig 6. Effect-of p o l y BrG~on-phosphorolyms of p o l y U Conditions as in Fig. 4
poly BrG as an inhibitor of phosphorolysis IS illustrated by the fact that immediate and total suppression of the degradation of poly A could be produced by addition of poly BrG at any time during the reaction (Fig 7) N" 3O .
'_o
x
No Poly 8r G
l /-/
x . . . . . . . . . .
//
•
Polyr a/Poly 8r G,20
ix/z / /
1'/ / //
~_ . . . . 5
IO
z
Poky tt/Poly BrG=20
. . . . . . . . . . . . . 20
~
}
* Poly l/Poly GOt=40 Poly A 1henPo~y BrG Poly ~ 20 60Poly BrGthenPoly A Po[y Br =
Time (ram)
Fig 7 Addltmn of poly BrG at different times during the phosphorolysls of p o l y .k Conditions as m Fig 4
The polymerization of UDP is also strongly inhibited by poly BrG (Fig. 8). With respect to the kinetics of Inhibition, however, poly BrG behaves as a classical competitive inhibitor of the NDP being polymerized (Fig. 9) The nature of the binding of poly BrG to polynucleotlde phosphorylase during polymerization was tested by pre-incubating a fixed quantity of the enzyme with increasing amounts of polymer followed by the addition of ADP and subsequent measurement of poly A synthesis B~och~m Bzophys .4cta, 217 (i97o) 18 29
POLYNUCLEOTIDE
25
PHOSPHORYLASE
/ 1 : 3 0
X LO'SM
/
060-
I = [ 6 X 10-5M
050-
/ /
O4O 030-
&
Kb poly G, B r = 5 0 ~ I O - 6 H K m ADP=I 3 x IO-~'M
z
4 Tbrne Chours)
~
8
I I0
210
410
---S=m"1 x iO 3
Fig 8 Effect of poly ]3rG on the polymerization of U D P Conditions as in Fig I a Fig. 9 Competitive l n h i b m o n of p o l y m e r l z a t m n of A D P b y copoly G,BrG (23 77) Three parallel series of reactions were p r e p a r e d containing increasing q u a n t m e s of A D P (specific radioactivity = i 08. i o 5 c o u n t s / m m per pmole). To one series was added n o t h i n g (I concn = o) to each reacUon of the second 1.6 nmoles of copoly G, BrG (23 '77) (I concn = 16 pM) and to each reaction of the third 3 o nmoles of copoly G,BrC (23 77) (I conch. = 3°/~M) All incubations contained o.13 u m t of the E coh polynucleotlde p h o s p h o r y l a s e v = nmoles [3H]AMP incorporated
The results (Fig. IO) which are consistent with c o m p e t m v e inhibition kinetics, d e m onstrate that polynucleotide phosphorylase is reversibly bound to poly BrG. It is of interest that poly BrG interacts w~th polynucleotide phosphorylase in at least t w o d]fferent ways: under c o n d m o n s which favor polymerization poly BrG binds ~OP
Poly BrG + polynucleotlde ~
I
phosphor ylose 5 rain 3 0 r a i n
o r~
6o
ao
o ' ; ' ,%' 2'. ' ~,' 2o'," nmoles
po~y BrG pre,ncuboted polynucleohde phosphorylose
wtlh
Fig IO Reversible binding of poly BrG to polynucleotlde p h o s p h o r y l a s e during p o l y m e n z a t l o n o 3 ° u m t of E coh polynueleotlde p h o s p h o r y l a s e was added to a series of reactions containing increasing q u a n t m e s of poly BrG I n addition, each i n c u b a t i o n contained i o F m o l e s T n s - H C 1 (pH 8 2), 5 ° nmoles E D T A , I / , m o l e MgC12 and ioo/~g bovine s e r u m a l b u m i n in a final v o l u m e of 90 #1 These reactions were p r e m c u b a t e d at 37 ° for 5 m m and synthesis was begun b y the addition of I oo m/2moles of A D P (specific radioactivity 18o0 c o u n t s / r a m per nmole). The final v o l u m e of all reactions was IOO~1, incubations were continued at 37 ° for 3 ° m m after addition of A D P The s y n t h e m s of acld-preclpltable p o l y m e r was m e a s u r e d b y the mflhpore technique I n the reaction lacking poly BrG, 9 6 nmoles of poly A were synthesized
Bzochzm. B~ophys. Acta, 217 (197 o) 18-29
26
A.M. KAPULER et al.
to the enzyme reversibly, whereas under conditions which promote phosphorolysls, an irreversible complex between enzyme and polymer is formed. In the former case, poly BrG behaves as a competitive inhibitor, and m the latter case as a non-competitive inhibitor. Thus, the concentration of P i appears to determine the mode of attachment of the polymer to the enzyme. These observations do not provide a basis for assessing the possible identity of the binding sites under the two sets of conditions Since polynucleotide phosphorylase has been shown to degrade polynucleotides sequentially from the 3'-OH and without release of the polynucleotide substrate 2v,~s and since polynucleotide phosphorylase can synthesize mixed polymers containing residues that exist largely in the syn conformation z6, it is hkely that poly BrG brads to the enzyme in a manner comparable to that of natural polymers. The immediate arrest of phosphorolysls by addition of poly BrG does not necessarily require displacement of the polymer being degraded and could depend on some other mechanism. The striking affimty of poly BrG for polynucleotide phosphorylase suggests that assumption of the syn conformation b y polymer residues m a y play a role in the enzymatic process. When tested in the P I - A D P exchange reaction, increasing concentrations of poly BrG produce corresponding reductions in exchange; however, a m a x i m u m of 80 % inhibition is achieved (Fig. I I ) . The observation that a residual level of exchange occurs at ratio of poly BrG/enzyme sufficient to inhibit phosphorolysls com-
x~
\ 0 Or05
I 0010
I 0015
0~02
Poly BrG ADP
Fig. I I E f f e c t of dafferent r a t i o s of p o l y ]3rG to A D P on t h e e x c h a n g e of P l Fig 3
C o n d i t i o n s as m
pletely implies that a certain fraction of the exchange actlvlty m a y be catalyzed by a process (or at a site) independent of synthesis and breakdown: otherwase, total inhibition of phosphorolysis should also completely abohsh the exchange reaction. The residual exchange could reflect the activity of a separate site which normally catalyzes the de novo initiation of new chains, if such a site exists*. However recent stud* S e v e r a l r e p o r t s in t h e h t e r a t u r e are consastent w i t h s uc h a f o r m u l a t i o n T h u s d m u c l e o t t d e s , w h i c h m u s t be p r o d u c e d as t h e first s t e p in t h e i n i t i a t i o n of n e w c h a i n s de novo, are r e s i s t a n t t o p h o s p h o r o l y s l s 3°. Moreover, t h e s y n t h e s i s of t h e first d m u c l e o t l d e , w h i c h r e v o l v e s t h e m m u l t a neous c o n d e n s a t i o n of t w o nucleos~de d l p h o s p h a t e s , is a c a t a l y t i c process q m t e d i s t i n c t from c h a i n propagation Bzochzm
Bzophys. Acta, 217 (197 ° ) 18-29
POLYNUCLEOTIDE PHOSPHORYLASE
27
ies 82 have shown that inhibition of phosphorolysis of oligonucleotides by, for example, polymers terminating with a 3'-phosphate, is much less marked than that of polynucleotides. This is also true for inhibition by poly BrG (T. GODEFROY, private communication), which satisfactorily explains the above results
Interact,on o/ polynucleot,de phosphorylase w,th BrGpA and ApBrG Both ApBrG and BrGpA were unable to serve as chain initiators for the Micrococcus lysodeikticus polynucleotide phosphorylase under conditions where their natural counterparts function well (Fig. 12).
12000
8000
/
..'"
4000 /...//s"'*" ApBrG 3O
6O
90 minutes
120
150
Fig. 12. Primer activity of ]3rGpA and ApBrO for the polymerization of [14CIADP by M~crococcus lysode,htwus polynucleotlde phosphorylase. The incubation mixture, io times normal scale, contamed 0.025 1V[ADP (o.I/zC//~mole), o.15 M Tns (pH 8 2); 0.0025 M MgClo and 2 5 units of enzyme. Apart from the control (absence of primer) the incubation mixture also contained o 125 #mole/ml of dmnucleomde phosphate (ratio of ADP/prlmer = 200 I) Incubatmn was at 3°° ahquots of o 02o ml were placed on W h a t m a n No. i paper at the indicated times and ADP removed by chromatography In I M a m m o n m m acetate-ethanol (3° 7o, by vol ) as solvent. After drying, the chromatogram was passed through a scanner coupled to an integrator to give the total number of counts in each spot. Similar results were obtained with a ratio of A D P / p n m e r = 5 ° i. Counts obtained in the absence of primer were subtracted at each point At 2 5-h incubation, this count was approx. IOOO.
DISCUSSION
In the preceding commumcatlon, we have discussed in detail the concept that poly BrG, ApBrG, BrGpA and the NDPs reported above share in common the steric constraints imposed by substituents which act to impede the assumption of the ant, conformation. The bromine atom at C-8 of purine nucleosides increases the probability of assuming the syn rather than the anti conformation, particularly w h e n s u c h r e s i d u e s a r e in p o l y m e r s . T h e r e f o r e it is n o t e w o r t h y t h a t p o l y B r G is n o t a s u b s t r a t e for a n y of t h e c a t a l y t i c a c t i v i t i e s of p o l y n u c l e o t l d e p h o s p h o r y l a s e a n d t h a t t h e c o n f o r m a t i o n a l l y r e s t r i c t e d N D P s a r e s u b s t r a t e s o n l y in t h e p r e s e n c e of n a t u ral N D P s 26 a n d t h e n p o o r l y . N e v e r t h e l e s s , b o t h t h e c o n f o r m a t i o n a l l y r e s t r i c t e d N D P s a n d p o l y m e r s i n t e r a c t w i t h t h e e n z y m e u n d e r a v a r i e t y of c o n d i t i o n s . T h u s , although NDPs and polynucleotides can bind to polynucleotide phosphorylase when
Bzoch,m. B,ophys. Acta, 217 (i97 o) 18-29
28
A. M KAPULER et al.
they are in the syn conformation, the successful completion of a n y polynucleotide phosphorylase reaction requires that the product be capable of assuming the ant~ conformation. Possibly a change in conformation of the substrate (NDP or polymer) from syn to anti m a y be associated with the course of enzyme action and could indeed represent an obligatory intermediate step in the catalytic process. Recent results with 4-thiouridine dlphosphate and polymers containing 4-thlouridine can also be interpreted in this sense (K. SCHEIT, private communication, in the press). Another conclusion which m a y be drawn from the present studies derives from the effect of poly BrG on the polymerization reaction. The kinetic analysis reveals that poly BrG behaves formally as a competitive inhibitor of the N D P substrate and is therefore bound reversibly at the polymerizing site (we have no way of assessing the relationship between poly BrG and the growing polynucleotide chain). It is thus of interest that poly BrG, BrGpA and ApBrG fail to function as primers under these conditions although these molecules possess free 3'-OH groups. It appears reasonable to suppose that the confornlation of the 3'-terininal nucleotide is an important determinant of primer function. In addition, conformation of the penultimate nucleotide is also of Importance in view of the characteristics of BrGpA. The present experiments do not provide a basis for the formulation of a specific model for the enzymatic action of polynucleotide phosphorylase. However, they contribute several new phenomena which must be accounted for in any model, and they make available a new reagent, poly BrG Poly BrG is unique in its potent Inhibition of all enzymatic processes catalyzed by polynucleotide phosphorylase; this polymer, and analogous oligomers and polymers containing lower proportions of 8-bromoguanme should prove to be effective tools for elucidating the mechanism of polynucleotide phosphorylase. In particular, it would be instructive to examine the effect of poly BrG simultaneously on polymerization, phosphorolysls and exchange throughout a range of reaction conditions calculated to alter the ratio of these catalytic activities.
ACKNOWLEDGEMENT
This work was supported by grant No. 66 oo 2o6 from the Ddl6gation Gdn6ral la Recherche Sclentlfique et Technique. REFERENCES I L SASSE, M RAmNOWlTZ AND I. H GOLDBERG, Bwch*m B w p h y s Acta, 72 (1963) 353. 2 A M MICHELSON, J DONDON AND M. GRUNBERG-MANAGO, Bwoch*m. B*ophys Acta, 55 (1962) 529 3 A. M MICHELSON AND M GRUNBERG-MANAGO, Bwch*m B w p h y s Acta, 91 (1964) 92 4 D H LEVlN, B*och*m B*ophys Acta, 61 (1962) 75 5 F POCHON AND A M MICHELSON, B~och*m B w p h y s Acta, 145 (1967) 321. 6 F 13 HOWARD, J. FRAZlER AND H. T MILES, J. B w l Chem, 241 (1966) 4293 . 7 A WACKER, E LODEMANN, A. GAURI AND P. CHANDRA, J 1Viol B , o l , 18 (1966) 382. 8 \V SZER AND D. SHUGAR, Acta Bwch*m. Polon, 8 (1961) 235 9 J SKODA, J KARA,z SORMOVAAND F SORM, B*och*m B*ophys Acta, 33 (1959) 579IO S S COHEN, Progr Nucleic Acid Res, 5 (1966) I I I J A CARBON, B~ochem B w p h y s Res Commun, 7 ( 1 9 6 2 ) 3 6 6 . 12 G KAUFMANN AND g Z LITTAUER, F E B S Letters, 4 (1969) 79 13 A M KAPULER, P h D Thesis, R o c k e f e l l e r U n l v e r m t y , N e w York, 1968 14 A E V HAESCHEMEYER AND A. RICH, J Mol B , o l , 27 ( 1 9 6 7 ) 3 6 9
Bwchzm
Bzophys Acta, 217 (197 ° ) I 8 - 2 9
POLYNUCLEOTIDE PHOSPHORYLASE
29
15 I. TINOCO Jr., R. C. DAVIS AND S R. JASKUNAS, in B. PULLMAN, Molecular Assoczatzons *n B,ology, Academic Press, London, 1968, pp. 77-91. 16 S. S. TAVALE AND H. M. SOBELL, in preparation. 17 A. M. KAPULER, D C WARD, N. •ENDELSOHN, H. KLETT AND G. ACS, V~rology, 37 (I969) 7Ol. 18 A. M. MICHELSON, C. MONN¥ AND A. i . KAPULER, B$och, m. B*ophys Acta, 217 (197 o) 719 M F SINGER, in G. L. CANTONI AND D R. DAVIES, Procedures *n Nucle,c Aczd Research, H a r p e r and Row, N e w York, 1966, pp. 245-262. 20 F. R WILLIAMS AND M. C-RUNBERG-MANAGO, B,och~m Bzophys Acta, 89 (1964) 66 21 J DUVAL AND J. P ]~BEL, Bull. Soc. Chum B$ol., 46 (1964) lO59. 22 G TENER, J A m Chem. Soc, 83 (1961) 159 23 IV[ SMITH AND H G KHORANA, J. A m Chem Soc, 8o (1958) 1141 24 A M. KAPULER AND E. REICH, in preparation. 25 M. N. THANG, M. GRAFFE AND M GRUNBERG-MANAGO,B*och*m. B*ophys Acta, lO8 (1965)125. 26 ]V[ IKEHARA, I. TAZAWA AND T. FOKUI, B*ochem,stry, 8 (1969) 736. 27 C. B. KLEE AND M SINGER, J. B,ol Chem, 243 (1968) 923. 28 ]~ N THANG, W GUSCHLBAUER, H G ZACHAU AND M GRUNBERG-MANAGO, J Mol. B , o l , 26 (1967) 403 . 29 M. SUNDARALINGAM, B*opolymers, 7 (1969) 821. 3 ° •. GRUNBERG-MANAGO, Progr. Nucle*c Ac*d Res, I (1963) 93 31 A M KAPULER AND S. SPIEGELMAN, Proc Natl Acad Sc*. U S , m the press. 32 T GODEFRO¥, European J B,ochem, in the press.
B~och, m. B~ophys
Acta, 217 (197 o) 18-29
BIOCHIMICA ET BIOPHYSICA ACTA
BBA 96575
T H E R E L A T I O N S H I P OF MONO- AND POLYNUCLEOTIDE CONFORMATION TO CATALYSIS BY POLYNUCLEOTIDE P H O S P H O R Y L A S E A M K A P U L E R ", C. MONNY AND A M. M I C H E L S O N
Inst*tut de B~olog~e Phys~co-chzmzque, Par~s (France) (Received F e b r u a r y 26th, 197 o)
SUMMARY
8-Bromoguanosine 5'-dlphosphate, 8-oxoguanosine 5'-dlphosphate, 6-methylcytidine 5'-diphosphate and 2,6-quinazoline dione I'-ribosyl 5'-diphosphate are mactive as substrates for homopolymer synthesis with polynucleotlde phosphorylase They inhibit polymerization and exchange but not phosphorolysis. Poly 8-bromoguanyhc acid (poly BrG), prepared chemically, is not phosphorolysed by polynucleotlde phosphorylase. The polymer inhibits polymerization reversibly and competitively whereas phosphorolysls is inhibited irreversibly and non-competitively. Thus the tenacity of polymer binding is a function directly or mdirectly of Pi concentration. The behaviour of these molecules with polynucleotide phosphorylase suggests that the catalytic activities of this enzyme require substrates which are capable of assuming the ant~ conformation. The ant~ conformation normally present in nucleoslde monomers and in polynucleotides, requires close apposition of the 2'-H of the ribofuranose and either the C-8 hydrogen of purines or the C-6 hydrogen of pyrimidlnes Replacement of these ring protons by larger substituents such as bromine or methyl inhibits the assumption of a normal antz conformation. Hence the sterically restricted nucleoside 5'diphosphates are predominantly In the syn conformation, as are residues in poly BrG. Since molecules containing residues in the syn (non-ant~) conformation interact strongly with polynucleotide phosphorylase, a syn - ~ ant~ conformatlonal isomerism IS possibly a natural part of the various catalytic processes Consistent with this formulation, ApBrG and BrGpA do not act as primers for the Micrococcus enzyme.
INTRODUCTION
Many nucleotide analogs have been examined as substrates for polynucleotide phosphorylase These derivatives fall into three general classes The first comprises * Rockefeller University, New York Present address Mmroblology Section, Life Scmnces, University of Connecticut, Storrs. Conn U S A Abbreviations NDP, nucleoslde 51-dlphosphate, BrGDP, 8-bromoguanosme 5'-d~phosphate, 8-0 GDP. 8-oxoguanosme5'-dlphosphate, QDP, 2,6-qumazohne dlone i ' - n b o s y l 5'-dlphosphate, poly BrG, poly 8-bromoguanyhc acld, copoly G, BrG (23 77) a r a n d o m copolymer of g u a n y h c acid and 8-bromoguanyhc acid with a e o m p o s m o n of 23 % GMP and 77 % 13rGMP.
Bzoch*m B~ophys Acta, 217 (197 ° ) 18-29
POLYNUCLEOTIDE PHOSPHORYLASE
19
those molecules which serve as substrates and which behave in a manner similar to that of natural nucleotides. Included in this class are the 5'-dlphosphate derivatives of the nucleosides pseudouridme 1, 5-bromourldine 2, 6-hydroxyethyladenosine3, 8azaguanoslne 4, I-methylguanosine 5, 2,6-dlaminopurine riboside e and 2-aminopurine rlboside 7. The second class comprises derivatives that are neither good substrates nor strong mhibltors, such as dlhydro UDP s and dADP 1°,12. The third class encompasses strongly inhibitory nucleoside diphosphates and is represented by 6-aza UDP 9 and 6-mercaptopurlne riboslde 5'-diphosphate 11. A fundamental difference between the substrate requirements of nucleic acid polymerases and polynueleotide phosphorylase concerns the Watson-Crick hydrogenbonding positions of the bases: for template-directed reactions the hydrogen-bonding position must not be blocked. In contrast, polynucleotlde phosphorylase appears to be indifferent to the status of these functional groups, since the 5'-dlphosphates of xanthosine, N~-methyluridine and NS-dlmethyladenosme are readily polymerized. Nevertheless, those nucleoside derivatives not polymerized by polynucleotide phosphorylase either fail to serve or are poorly utlhzed as substrates (as 5'-triphosphates) by DNA-dependent polymerases 13. In this paper, we describe the interaction of polynucleotlde phosphorylase with 8-bromoguanoslne 5'-diphosphate (BrGDP) and poly BrG. The salient features of this class of mhlbltors (which includes also the 5'-diphosphates of 6-methylcytldine, 8-oxoguanosine and quinazohne-2,6-dione I'-riboside) are (I) the normal hydrogenbonding positions and the ribofuranose ring are preserved and (2) the chemically unrelated substltuents introduced into the bases share an important property, namely, they influence the stereochemical relationship between the heterocyclic ring and the sugar. The effect of all these groups is to restrict the rotation around the glycosyl bond of the corresponding monomers and polymers, in a manner which makes the s y n conformation energetically more favorable" t h a n t h e normal anti conformation 14-16. These monomers and polymers are lnhibitors of polynucleotide phosphorylase. An interpretation of the results based on polynueleotide stereochemlstry is discussed. The interaction of these and related molecules with nucleic acid polymerases is described elsewhere lv,~l Since this work was completed IKEHARA et al. 2e have described the incorporation of 8-bromoguanosine and 8-hydroxyguanosine dlphosphates into polymer materials by polynucleotide phosphorylase. The present results provide conformation of their studies on polymerisation and extend them to consideration of the behaviour of the monomers with respect to the phosphate exchange and phosphorolysis activities of polynucleotide phosphorylase. In addition, the action of poly BrG on all three activities catalysed by polynucleotide phosphorylase is described.
* Single crystal X - r a y diffraction studies of 8 - b r o m o g u a n o s l n e and 8 - o x o g u a n o s m e h a v e confirmed this assertion which w a s based on model b m l d l n g 18 a n d calculation. These t w o nucleosides h a v e t h e syn c o n f o r m a t i o n in crystals le. Unlike p u r m e nucleosides where the energy b a r r i e r b e t w e e n syn a n d ant* is r a t h e r small, pyrlmldlne nucleosldes exist m a i n l y in the ant* conforrnatlonl,,15,2, A b u l k y s u b s t l t u e n t at C-6 p r o b a b l y reduces the e x t e n t of antz c o n f o r m a t i o n w i t h o u t necessarily I m p o s i n g a p u r e syn f o r m Bzoch,m. B,ophys. Acta, 217 (197o) I 8 - 2 9
20
A.M. KAPULER et al.
MATERIALS AND METHODS
Poly BrG, a random copolymer o/GMP and 8-bromoguanylic acid with a compositwn O~ 23 % GMP and 77 % 8-bromoguanylic acid (copoly G,BrG (23:77), ApBrG and BrGpA The synthesis and characterisation of these dlnucleotides and polymers is described m the preceding publication is
Polynucleot~de phosphorylase The isolation and purification of this enzyme from Azotobacter vzneland~L Escherwhm coli and Mzcrococcus lysodezktzcus has been described previously 19,2°. Unless otherwise noted, reactions were performed in final volumes of IOO/~1 for 15 min at 37 °, incubation mixtures were as follows (/zmoles/ml). phosphorolysis, Tlis-HC1 (pit 8 2), 50; poly A, 3.o; K22PO4 (pH 8.2), IO, EDTA, 0.5; MgC12, 0.5; polymerization, Trls-HC1 (pH 8 2), IOO; MgC12, IO, ADP, 25-50; EDTA, o 5, exchange, TrisHC1 (pH 8.2), IOO; MgC12, 5; EDTA, o 5; K2~PO4 (pH 8 2), 5; ADP, 5.0 (limiting). The procedures employed for these assays have already been described in detail 3°.
8-Bromoguanosfne 5'-dzphosphate (BrGDP) A suspension of finely powdered GDP (o.5 mmole) In formamide (4 ml) at o ° was treated with Br2 (I mmole) in chloroform (I ml) and the mixture kept at o ° with frequent agitation for 80 rain. The solution was then extracted twice with ether and the extracts discarded. Crude BrGDP was then precipitated by the addition of ethanol-ether ( I : I , by vol.), the precipitate was collected, washed with ether and dried. The material was purified on Dowex I × 2 (C1- form) using a gradient of o.oi M HC1 to o.oi M HC1, I.O M LIC1 Appropriate fractions were combined, neutrahsed with trl-n-butylamine and concentrated under reduced pressure. The lithium salt of BrGDP was precipitated with ethanol-acetone, collected, washed and dried. Yield 80 9'0 Paper chromatography showed that the material was homogeneous and free from GDP.
8-Bromo-E8-14CJguanos~ne 5'-d~phosphate (BrI14CJGDP) 5 °/~C of I8-14CJGTP (specific radioactivity 28/~C//zmole, C.E.A., Saclay, France) were dissolved in IOO/~1 formamide and bromlnated by addition of a Io-fold excess of Br 2. The product was isolated and purified by chromatography on DEAE-cellulose and analyzed for purine composition by depurlnation and chromatography of the bases in n-butanol-98 °/o formic acid-water (76.1o:13, by vol )31. It consisted of 99.2 °/o BrGTP and o 8 ~o GTP. The trlphosphate was converted to the diphosphate with hexokmase and purified by DEAE-eellulose chromatography.
8-Oxoguanoszne 5'-d@hosphate (8-0 GDP), 6-methylcytzd~ne 5'-diphosphate(6MeCDP), 2,6-qmnazolzne d, one z'-r~bosyl 5'-d@hosphate (QDP) The synthesis of these molecules from crystalline nucleosldes (8-oxoguanosme, 6-methyleytidine and 2,6-quinazohne dlone I'-riboside were generously supplied by Dr. R Robins) followed standard procedures 22,23. Details of the synthesis of the corresponding 5'-triphosphates will be published elsewhere 24. B~ochzm Bzophys dcta, 217 (197o) I 8 - 2 9
POLYNUCLEOTIDE PHOSPHORYLASE
21
RESULTS
Interaction o/polynucleotidephosphorflase with con/ormationally restricted nucleoside 5'-diphosphates (NDPs) (a) BrGDP: Using polynucleotlde phosphory]ase isolated from both A. wneland,~ and E. colh the activity of BrGDP was studied with respect to polymerization, phosphorolysls and a2Pt-NDP exchange. A variety of experimental conditions were employed including the normal conditions at 37 ° for both enzymes, as well as the higher temperatures and the use of Mn ~+ which are known to promote poly G synthesis by the E. colt enzyme 25. Under all conditions examined, BrGDP did not serve as a substrate for homopolymer synthesis by either enzyme. For example, at 6o °, with the E. col~ enzyme, under standard Mn 2+ conditions 2s, less than o.15 re#mole of Br[t4CIGDP was converted into acid-insoluble form while in a separate, control reaction, 20 m/~moles of [14CJGDP were polymerlzed. When a mixture of BrGDP/[14CJGDP -= 3.0 was studied, polymerization of GDP proceded at 1/8 the control rate. Furthermore, BrGDP strongly inhibited the utlhzation of any of the normal substrates when these were present in equimolar amounts (Figs. Ia, Ib). The effect of different concentrations of 30 F 50
UDP o ~& 2c
?
4C
f
® cgP
~3c ?
g
g 2c
8
12
L6
20
24
::L
Tirne (hours)
"•3E C
GDP
x~--x ~--x
GDP gr GOIP
/
:k
4
..._...... ~ ×
2
4 6 8 Tlrne (hours)
I0
UDP
BrGDP ~co
o-2 C
I0
IC
33
16
~
,'2
,'~
2'o
2'4
Time (hours)
F i g I a I n h l b l t l o n of p o l y m e r i z a t i o n of C D P , U D P a n d A D P b y B r G D P I n c u b a t i o n s i o t i m e s n o r m a l scale c o n t a i n e d 5 o u n i t s of t h e A. v*neland** p o l y n u c l e o t l d e p h o s p h o r y l a s e a t 3 °o A l l q u o t s of 2 0 / z l w e r e r e m o v e d as i n d i c a t e d , b I n h i b i t ] o n of p o l y m e r i z a t i o n of G D P b y B r G D P R e a c t i o n s i o t i m e s n o r m a l scale w e r e I n c u b a t e d a t 60 ° w i t h 2 o u n i t s of t h e E col, p o ] y n u c ] e o t l d e phosp h o r y l a s e u n d e r s t a n d a r d M n ~÷ c o n d i t i o n s (o o02 M MnC12, o OlO M G D P ) . c E f f e c t of v a r y i n g a m o u n t s of B r G D P o n t h e p o l y m e r i z a t i o n of U D P C o n d i t i o n s as in a. B*och,m. B,ophys
Acta, 217 (197 o) 1 8 - 2 9
22
A.M. KAPULER et al.
B r G D P on the synthesis of poly U by the A. vznelandz, polynucleotide phosphorylase IS shown in Fig. IC A more detailed analysis was performed with the E. coh polynucleotide phosphorylase. Whereas the dependence on substrate concentration of the polymerization of ADP (corrected for free ADP) follows classical enzymological relationships, the action of BrGDP results in non-linear Lineweaver-Burks plots (Fig. 2a). Inhibition is most pronounced at low ratios of A D P / B r G D P and a disproportionate decrease in mhibition is observed when this ratio as increased. The observation of a xT=1 5 x IO-3M
b
I t0,1o"u ~i=l
O x 10-3M i-6s~lo 4M I=6 5 x 10-4M
-I > 2
-I>
4o zo io
tO
N-M xI03
20
le
2o ~'o L~ M-Ix $0 3
I o 40
Fig 2 a. I n h i b i t : o n of polymerization of A D P b y ]3rGDP. I n c u b a t i o n s were s t a n d a r d and similar to t h a t described in Fig 9 except t h a t copoly G, BrG (23 77) was replaced b y ]3rGDP E a c h incub a t i o n c o n t a m e d o 13 u n i t of E coh polynucleotlde p h o s p h o r y l a s e and the polymerlzatlon of A D P (specific radioactivity i 14- lO 6 counts/rain per/xmole) was measured, b. I n h i b i t i o n of polymerization of A D P b y 8 - O G D P Conditions as in a except t h a t 8 - O G D P replaced B r G D P The specific r a d t o a c t i v l t y of A D P was I 45' lO5 counts/rain per # m o l e v = nmoles [3H]AMP incorporated
IKEHARA et al. 26 that low rates of polymerization of C-8-substituted purlne nucleoside diphosphates occur only in the presence of natural nucleoside diphosphates provides a basis for interpreting these kmetlcs. Polymerization of BrGDP occurs only at those ratios of B r G D P / A D P that do not reduce the rate of synthesis too drastically (BrGDP >> ADP) and do not completely eliminate the ability of BrGDP to compete with A D P (ADP >> BrGDP). The BrGMP residues, once incorporated into oligonucleotide become part of a family of inhibitory ohgonucleotides, some of which are more potent inhibltors than BrGDP. We have characterized the limit polymer of this family, namely poly BrG, as described below. The z2Pi-NDP exchange reaction, like the polymerization reaction, was strongly inhibited b y BrGDP (Fig 3). In contrast, only a feeble inhibitory effect on phosphorolysis of poly A was seen (Fig. 4) Exchange of 32Pi into BrGDP was not observed. (b) Other con/ormationally restrzcted N D P s : 8-OGDP, 6-MeCDP, QDP It appeared possible that the effect of BrGDP on polynucleotide phosphorylase was due to the chemical properties of the bromine atom, or to specific characteristics unique to BrGDP. To evaluate this possibility, other conformationally restricted NDPs were B~och~m. B*ophys
Acla, 217 (197 ° ) I 8 - 2 9
23
POLYNUCLEOTIDE PHOSPHORYLASE 2O
4"
o
E
\ ~030
Poqy
v
Poy ,~
,o
\
,\ /
E
5
\~ADP \\
•
)
/I /
u o p X ~
poly
i// //* p y A =mOO
0
05
l ~
[I0
BrG~)P ADP(uoP)
,
, Poly A - 20
Time (hours)
Fig. 3. E f f e c t of v a r i o u s r a t i o s of B r G D P t o A D P or U D P on Pt e x c h a n g e . R e a c t i o n s i o t i m e s n o r m a l scale c o n t a i n e d 5 o u n i t s of t h e A. wnelandzz p o l y n u c l e o t l d e p h o s p h o r y l a s e a n d w e r e s t o p p e d a f t e r IO rain a t 3 o°. A h q u o t s of 2o btl were t a k e n a n d a n a l y z e d . Fig. 4- E f f e c t of B r G D P a n d p o l y B r G on t h e p h o s p h o r o l y s I s of p o l y A R e a c t i o n s i o t i m e s n o r m a l scale c o n t a i n e d 5.0 u n i t s of t h e A. vznelandz, p o l y n u c l e o t l d e p h o s p h o r y l a s e A h q u o t s of 2o/zl were t a k e n as i n d i c a t e d .
synthesized and their effects on polynucleotide phosphorylase studied. 8-OGDP is not polymerized by polynucleotide phosphorylase and, as seen m Fig. 2b, this analog acts as an inhibitor of polynucleotide phosphorylase in a manner quahtatlvely identical to BrGDP. Likewise, neither 6-MeCDP nor QDP were polymerized by the enzyme, and both inhibit the synthesis of poly A; furthermore, 6-MeCDP and QDP resemble BrGDP also in their failure to inhibit phosphorolysis of poly C (Table I). TABLE I INHIBITORY REACTIONS
PROPERTIES OF
OF
6-MeCDP AND Q D P IN
POLYNUCLEOTIDE
THE
POLYMERIZATION
AND
PHOSPHOROLYSIS
PHOSPHORYLASE
C o n d i t i o n s for t h e s e r e a c t i o n s are specified in MATERIALS AND METHODS. The specific r a d i o a c t i v i t y of [ 3 H ] A D P w a s 18oo c o u n t s / r a i n p e r n m o l e a n d t h a t of p o l y [3H]C 34 c o u n t s / m l n p e r n m o l e , i o o m/~moles of t h e l a t t e r were a d d e d t o each p h o s p h o r o l y s l s r e a c t i o n All r e a c t i o n s c o n t a i n e d o o4o u n i t of E. col* p o l y n u c l e o t l d e p h o s p h o r y l a s e (specific a c t i v i t y i o u m t s / m g ) . U n i t s as d e f i n e d b y GRUNBERG-MANAGO3°
-
-
Q D P , I I mM Q D P , 8.6 mM -
-
6-MeCDP, 5 7 mM 6-MeCDP, I I rnM 6-MeCDP, 27 mM
Polymer*zat~on (nmoles [3H~AM P zncorporated)
Phosphorolyszs (nmolespoly E3HICphosphorolyzed)
95 44 i.o 7 9 6.6 4 8 3-9
III 12o 16.9 25 .2 27 7 26 5 24 7
Interaction o~ polynucleotide phosphorylase with poly BrG and copoly G,BrG (23:77) The effect of poly BrG on polynucleotide phosphorylase was examined with respect to polymerization, phosphorolysis and exchange. In contrast to BrGDP, B,och~m. B,ophys Acta, 217 (197 o) 18-29
24
A . M . KAPULER dt
al.
which does not affect phosphorolysis, all three catalytic activities are inhibited by poly BrG and copoly G,BrG (23'77) Poly BrG, like poly G, is itself resistant to phosphorolysls; however, unlike poly G, it is a potent irreversible, non-competitive inhibitor of the phosphorolysls of poly A (Fig 5)- The similar dose-response curve obtained for the case of poly U (Fig 6), demonstrates that the inhibition of phosphorolysis is independent of the nature of the polymer being degraded. The efficacy of 3O
Po]y U PoLy B,G
./ o
o0
o.tO *
~-
080
o
I=5,1o-%
2o
~,~o,
_'~
S=Mxl0-5
•
CO
E
/
,oo
,~u 20 400
I=5×10-6M ~ r
ooo 04D
~
i o
0
/5 d
o/
200
/-
__--~
I00
0o I
@ =M-IxFo 5
2
3
T~me ( h o u r s )
Fig 5 N o n - c o m p e t l t w e inhibition of phosphorolyms of p o l y A b y copoty G,BrG (23 77) ReactmnS contained o Ol 5 unit of E coh polynucleotlde phosphorylase and 5oo m/~moles Ka2PO4 (pH 8 2) (specific radioactivity 286o e o u n t s / m m per nmole) Increasing a m o u n t s of p o l y A were added to a series of reactions prepared in duplicate and o 5 nmole of copoly G,BrG (23 77) was added to one of each pair. Fig 6. Effect-of p o l y BrG~on-phosphorolyms of p o l y U Conditions as in Fig. 4
poly BrG as an inhibitor of phosphorolysis IS illustrated by the fact that immediate and total suppression of the degradation of poly A could be produced by addition of poly BrG at any time during the reaction (Fig 7) N" 3O .
'_o
x
No Poly 8r G
l /-/
x . . . . . . . . . .
//
•
Polyr a/Poly 8r G,20
ix/z / /
1'/ / //
~_ . . . . 5
IO
z
Poky tt/Poly BrG=20
. . . . . . . . . . . . . 20
~
}
* Poly l/Poly GOt=40 Poly A 1henPo~y BrG Poly ~ 20 60Poly BrGthenPoly A Po[y Br =
Time (ram)
Fig 7 Addltmn of poly BrG at different times during the phosphorolysls of p o l y .k Conditions as m Fig 4
The polymerization of UDP is also strongly inhibited by poly BrG (Fig. 8). With respect to the kinetics of Inhibition, however, poly BrG behaves as a classical competitive inhibitor of the NDP being polymerized (Fig. 9) The nature of the binding of poly BrG to polynucleotlde phosphorylase during polymerization was tested by pre-incubating a fixed quantity of the enzyme with increasing amounts of polymer followed by the addition of ADP and subsequent measurement of poly A synthesis B~och~m Bzophys .4cta, 217 (i97o) 18 29
POLYNUCLEOTIDE
25
PHOSPHORYLASE
/ 1 : 3 0
X LO'SM
/
060-
I = [ 6 X 10-5M
050-
/ /
O4O 030-
&
Kb poly G, B r = 5 0 ~ I O - 6 H K m ADP=I 3 x IO-~'M
z
4 Tbrne Chours)
~
8
I I0
210
410
---S=m"1 x iO 3
Fig 8 Effect of poly ]3rG on the polymerization of U D P Conditions as in Fig I a Fig. 9 Competitive l n h i b m o n of p o l y m e r l z a t m n of A D P b y copoly G,BrG (23 77) Three parallel series of reactions were p r e p a r e d containing increasing q u a n t m e s of A D P (specific radioactivity = i 08. i o 5 c o u n t s / m m per pmole). To one series was added n o t h i n g (I concn = o) to each reacUon of the second 1.6 nmoles of copoly G, BrG (23 '77) (I concn = 16 pM) and to each reaction of the third 3 o nmoles of copoly G,BrC (23 77) (I conch. = 3°/~M) All incubations contained o.13 u m t of the E coh polynucleotlde p h o s p h o r y l a s e v = nmoles [3H]AMP incorporated
The results (Fig. IO) which are consistent with c o m p e t m v e inhibition kinetics, d e m onstrate that polynucleotide phosphorylase is reversibly bound to poly BrG. It is of interest that poly BrG interacts w~th polynucleotide phosphorylase in at least t w o d]fferent ways: under c o n d m o n s which favor polymerization poly BrG binds ~OP
Poly BrG + polynucleotlde ~
I
phosphor ylose 5 rain 3 0 r a i n
o r~
6o
ao
o ' ; ' ,%' 2'. ' ~,' 2o'," nmoles
po~y BrG pre,ncuboted polynucleohde phosphorylose
wtlh
Fig IO Reversible binding of poly BrG to polynucleotlde p h o s p h o r y l a s e during p o l y m e n z a t l o n o 3 ° u m t of E coh polynueleotlde p h o s p h o r y l a s e was added to a series of reactions containing increasing q u a n t m e s of poly BrG I n addition, each i n c u b a t i o n contained i o F m o l e s T n s - H C 1 (pH 8 2), 5 ° nmoles E D T A , I / , m o l e MgC12 and ioo/~g bovine s e r u m a l b u m i n in a final v o l u m e of 90 #1 These reactions were p r e m c u b a t e d at 37 ° for 5 m m and synthesis was begun b y the addition of I oo m/2moles of A D P (specific radioactivity 18o0 c o u n t s / r a m per nmole). The final v o l u m e of all reactions was IOO~1, incubations were continued at 37 ° for 3 ° m m after addition of A D P The s y n t h e m s of acld-preclpltable p o l y m e r was m e a s u r e d b y the mflhpore technique I n the reaction lacking poly BrG, 9 6 nmoles of poly A were synthesized
Bzochzm. B~ophys. Acta, 217 (197 o) 18-29
26
A.M. KAPULER et al.
to the enzyme reversibly, whereas under conditions which promote phosphorolysls, an irreversible complex between enzyme and polymer is formed. In the former case, poly BrG behaves as a competitive inhibitor, and m the latter case as a non-competitive inhibitor. Thus, the concentration of P i appears to determine the mode of attachment of the polymer to the enzyme. These observations do not provide a basis for assessing the possible identity of the binding sites under the two sets of conditions Since polynucleotide phosphorylase has been shown to degrade polynucleotides sequentially from the 3'-OH and without release of the polynucleotide substrate 2v,~s and since polynucleotide phosphorylase can synthesize mixed polymers containing residues that exist largely in the syn conformation z6, it is hkely that poly BrG brads to the enzyme in a manner comparable to that of natural polymers. The immediate arrest of phosphorolysls by addition of poly BrG does not necessarily require displacement of the polymer being degraded and could depend on some other mechanism. The striking affimty of poly BrG for polynucleotide phosphorylase suggests that assumption of the syn conformation b y polymer residues m a y play a role in the enzymatic process. When tested in the P I - A D P exchange reaction, increasing concentrations of poly BrG produce corresponding reductions in exchange; however, a m a x i m u m of 80 % inhibition is achieved (Fig. I I ) . The observation that a residual level of exchange occurs at ratio of poly BrG/enzyme sufficient to inhibit phosphorolysls com-
x~
\ 0 Or05
I 0010
I 0015
0~02
Poly BrG ADP
Fig. I I E f f e c t of dafferent r a t i o s of p o l y ]3rG to A D P on t h e e x c h a n g e of P l Fig 3
C o n d i t i o n s as m
pletely implies that a certain fraction of the exchange actlvlty m a y be catalyzed by a process (or at a site) independent of synthesis and breakdown: otherwase, total inhibition of phosphorolysis should also completely abohsh the exchange reaction. The residual exchange could reflect the activity of a separate site which normally catalyzes the de novo initiation of new chains, if such a site exists*. However recent stud* S e v e r a l r e p o r t s in t h e h t e r a t u r e are consastent w i t h s uc h a f o r m u l a t i o n T h u s d m u c l e o t t d e s , w h i c h m u s t be p r o d u c e d as t h e first s t e p in t h e i n i t i a t i o n of n e w c h a i n s de novo, are r e s i s t a n t t o p h o s p h o r o l y s l s 3°. Moreover, t h e s y n t h e s i s of t h e first d m u c l e o t l d e , w h i c h r e v o l v e s t h e m m u l t a neous c o n d e n s a t i o n of t w o nucleos~de d l p h o s p h a t e s , is a c a t a l y t i c process q m t e d i s t i n c t from c h a i n propagation Bzochzm
Bzophys. Acta, 217 (197 ° ) 18-29
POLYNUCLEOTIDE PHOSPHORYLASE
27
ies 82 have shown that inhibition of phosphorolysis of oligonucleotides by, for example, polymers terminating with a 3'-phosphate, is much less marked than that of polynucleotides. This is also true for inhibition by poly BrG (T. GODEFROY, private communication), which satisfactorily explains the above results
Interact,on o/ polynucleot,de phosphorylase w,th BrGpA and ApBrG Both ApBrG and BrGpA were unable to serve as chain initiators for the Micrococcus lysodeikticus polynucleotide phosphorylase under conditions where their natural counterparts function well (Fig. 12).
12000
8000
/
..'"
4000 /...//s"'*" ApBrG 3O
6O
90 minutes
120
150
Fig. 12. Primer activity of ]3rGpA and ApBrO for the polymerization of [14CIADP by M~crococcus lysode,htwus polynucleotlde phosphorylase. The incubation mixture, io times normal scale, contamed 0.025 1V[ADP (o.I/zC//~mole), o.15 M Tns (pH 8 2); 0.0025 M MgClo and 2 5 units of enzyme. Apart from the control (absence of primer) the incubation mixture also contained o 125 #mole/ml of dmnucleomde phosphate (ratio of ADP/prlmer = 200 I) Incubatmn was at 3°° ahquots of o 02o ml were placed on W h a t m a n No. i paper at the indicated times and ADP removed by chromatography In I M a m m o n m m acetate-ethanol (3° 7o, by vol ) as solvent. After drying, the chromatogram was passed through a scanner coupled to an integrator to give the total number of counts in each spot. Similar results were obtained with a ratio of A D P / p n m e r = 5 ° i. Counts obtained in the absence of primer were subtracted at each point At 2 5-h incubation, this count was approx. IOOO.
DISCUSSION
In the preceding commumcatlon, we have discussed in detail the concept that poly BrG, ApBrG, BrGpA and the NDPs reported above share in common the steric constraints imposed by substituents which act to impede the assumption of the ant, conformation. The bromine atom at C-8 of purine nucleosides increases the probability of assuming the syn rather than the anti conformation, particularly w h e n s u c h r e s i d u e s a r e in p o l y m e r s . T h e r e f o r e it is n o t e w o r t h y t h a t p o l y B r G is n o t a s u b s t r a t e for a n y of t h e c a t a l y t i c a c t i v i t i e s of p o l y n u c l e o t l d e p h o s p h o r y l a s e a n d t h a t t h e c o n f o r m a t i o n a l l y r e s t r i c t e d N D P s a r e s u b s t r a t e s o n l y in t h e p r e s e n c e of n a t u ral N D P s 26 a n d t h e n p o o r l y . N e v e r t h e l e s s , b o t h t h e c o n f o r m a t i o n a l l y r e s t r i c t e d N D P s a n d p o l y m e r s i n t e r a c t w i t h t h e e n z y m e u n d e r a v a r i e t y of c o n d i t i o n s . T h u s , although NDPs and polynucleotides can bind to polynucleotide phosphorylase when
Bzoch,m. B,ophys. Acta, 217 (i97 o) 18-29
28
A. M KAPULER et al.
they are in the syn conformation, the successful completion of a n y polynucleotide phosphorylase reaction requires that the product be capable of assuming the ant~ conformation. Possibly a change in conformation of the substrate (NDP or polymer) from syn to anti m a y be associated with the course of enzyme action and could indeed represent an obligatory intermediate step in the catalytic process. Recent results with 4-thiouridine dlphosphate and polymers containing 4-thlouridine can also be interpreted in this sense (K. SCHEIT, private communication, in the press). Another conclusion which m a y be drawn from the present studies derives from the effect of poly BrG on the polymerization reaction. The kinetic analysis reveals that poly BrG behaves formally as a competitive inhibitor of the N D P substrate and is therefore bound reversibly at the polymerizing site (we have no way of assessing the relationship between poly BrG and the growing polynucleotide chain). It is thus of interest that poly BrG, BrGpA and ApBrG fail to function as primers under these conditions although these molecules possess free 3'-OH groups. It appears reasonable to suppose that the confornlation of the 3'-terininal nucleotide is an important determinant of primer function. In addition, conformation of the penultimate nucleotide is also of Importance in view of the characteristics of BrGpA. The present experiments do not provide a basis for the formulation of a specific model for the enzymatic action of polynucleotide phosphorylase. However, they contribute several new phenomena which must be accounted for in any model, and they make available a new reagent, poly BrG Poly BrG is unique in its potent Inhibition of all enzymatic processes catalyzed by polynucleotide phosphorylase; this polymer, and analogous oligomers and polymers containing lower proportions of 8-bromoguanme should prove to be effective tools for elucidating the mechanism of polynucleotide phosphorylase. In particular, it would be instructive to examine the effect of poly BrG simultaneously on polymerization, phosphorolysls and exchange throughout a range of reaction conditions calculated to alter the ratio of these catalytic activities.
ACKNOWLEDGEMENT
This work was supported by grant No. 66 oo 2o6 from the Ddl6gation Gdn6ral la Recherche Sclentlfique et Technique. REFERENCES I L SASSE, M RAmNOWlTZ AND I. H GOLDBERG, Bwch*m B w p h y s Acta, 72 (1963) 353. 2 A M MICHELSON, J DONDON AND M. GRUNBERG-MANAGO, Bwoch*m. B*ophys Acta, 55 (1962) 529 3 A. M MICHELSON AND M GRUNBERG-MANAGO, Bwch*m B w p h y s Acta, 91 (1964) 92 4 D H LEVlN, B*och*m B*ophys Acta, 61 (1962) 75 5 F POCHON AND A M MICHELSON, B~och*m B w p h y s Acta, 145 (1967) 321. 6 F 13 HOWARD, J. FRAZlER AND H. T MILES, J. B w l Chem, 241 (1966) 4293 . 7 A WACKER, E LODEMANN, A. GAURI AND P. CHANDRA, J 1Viol B , o l , 18 (1966) 382. 8 \V SZER AND D. SHUGAR, Acta Bwch*m. Polon, 8 (1961) 235 9 J SKODA, J KARA,z SORMOVAAND F SORM, B*och*m B*ophys Acta, 33 (1959) 579IO S S COHEN, Progr Nucleic Acid Res, 5 (1966) I I I J A CARBON, B~ochem B w p h y s Res Commun, 7 ( 1 9 6 2 ) 3 6 6 . 12 G KAUFMANN AND g Z LITTAUER, F E B S Letters, 4 (1969) 79 13 A M KAPULER, P h D Thesis, R o c k e f e l l e r U n l v e r m t y , N e w York, 1968 14 A E V HAESCHEMEYER AND A. RICH, J Mol B , o l , 27 ( 1 9 6 7 ) 3 6 9
Bwchzm
Bzophys Acta, 217 (197 ° ) I 8 - 2 9
POLYNUCLEOTIDE PHOSPHORYLASE
29
15 I. TINOCO Jr., R. C. DAVIS AND S R. JASKUNAS, in B. PULLMAN, Molecular Assoczatzons *n B,ology, Academic Press, London, 1968, pp. 77-91. 16 S. S. TAVALE AND H. M. SOBELL, in preparation. 17 A. M. KAPULER, D C WARD, N. •ENDELSOHN, H. KLETT AND G. ACS, V~rology, 37 (I969) 7Ol. 18 A. M. MICHELSON, C. MONN¥ AND A. i . KAPULER, B$och, m. B*ophys Acta, 217 (197 o) 719 M F SINGER, in G. L. CANTONI AND D R. DAVIES, Procedures *n Nucle,c Aczd Research, H a r p e r and Row, N e w York, 1966, pp. 245-262. 20 F. R WILLIAMS AND M. C-RUNBERG-MANAGO, B,och~m Bzophys Acta, 89 (1964) 66 21 J DUVAL AND J. P ]~BEL, Bull. Soc. Chum B$ol., 46 (1964) lO59. 22 G TENER, J A m Chem. Soc, 83 (1961) 159 23 IV[ SMITH AND H G KHORANA, J. A m Chem Soc, 8o (1958) 1141 24 A M. KAPULER AND E. REICH, in preparation. 25 M. N. THANG, M. GRAFFE AND M GRUNBERG-MANAGO,B*och*m. B*ophys Acta, lO8 (1965)125. 26 ]V[ IKEHARA, I. TAZAWA AND T. FOKUI, B*ochem,stry, 8 (1969) 736. 27 C. B. KLEE AND M SINGER, J. B,ol Chem, 243 (1968) 923. 28 ]~ N THANG, W GUSCHLBAUER, H G ZACHAU AND M GRUNBERG-MANAGO, J Mol. B , o l , 26 (1967) 403 . 29 M. SUNDARALINGAM, B*opolymers, 7 (1969) 821. 3 ° •. GRUNBERG-MANAGO, Progr. Nucle*c Ac*d Res, I (1963) 93 31 A M KAPULER AND S. SPIEGELMAN, Proc Natl Acad Sc*. U S , m the press. 32 T GODEFRO¥, European J B,ochem, in the press.
B~och, m. B~ophys
Acta, 217 (197 o) 18-29