Eur. J . Biochem. 62, 365-372 (1976)

Deoxyribosyl Transfer Catalysis with trans-N-Deoxyribosylase Kinetic Study of Purine(Pyrimidine) to Pyrimidine(Purine) trans-N-Deoxyribosylase Charles DANZIN and Robert CARDINAUD Service de Biophysique, Dipartement de Biologie, Centre #Etudes NuclCaires de Saclay, Gif-sur-Yvette (Received October 15, 1975)

Kinetic studies were carried out in order to investigate the enzymic mechanism of a 215-foldpurified purine(pyrimidine) nucleoside :purine(pyrimidine) deoxyribosyl transferase fraction from Lactobacillushelveticus. A variety of natural deoxyribonucleosidesand bases were used as substrates. Initial velocity, product inhibition and isotopic exchange studies are consistent with a ping-pong bi-bi mechanism. The kinetic parameters are used to show that this fraction is free from any contamination by a specific purine nucleoside purine deoxyribosyl transferase also found in the same strain of L. helveticus.

It was shown recently that trans-N-deoxyribosylase extracted from Lactobacillus helveticus could be separated by affinity chromatography into two fractions one specifically catalyzing the transfer of the deoxyribosyl moiety between bases (trans-N-deoxyribosylase-I) and the other able to transfer the deoxyribosyl moiety between purine and pyrimidine bases [l, 21. With the second fraction trans-N-deoxyribosylaseI1 the following three possible transfers were observed : dRib-Pur dRib-Pur dRib-Pyr

+ Pur’ 2 dRib-Pur’ + Pur + Pyr dRib-Pyr + Pur + Pyr’ 2 dRib-Pyr’ + Pyr

(1)

All attempts to eliminate completely the purineto-purine transfer activity from the trans-N-deoxyribosylase-I1 fraction remained unsuccessful, nor was it possible to separate a distinct pyrimidine-to-pyrimidine transfer enzyme. The present paper reports a steady-state kinetic study suggesting that with this en~

_ _ Abbreviations. Pur, a purine base; Pyr, a pyrimidine base; (Pur 2 Pyr) is a transfer reaction where the deoxyribosyl moiety is transferred from a purine to a pyrimidine; deoxyribonucleosides are abbreviated as recommended by IUPAC-IUB; (dIno + Ade) denotes a transfer in which deoxyinosine and adenine are the substrates. Enzymes. rrans-N-Deoxyribosylase or purine@yrimidine) nucleoside : purine(pyrimidine) deoxyribosyl transferase (EC 2.4.2.6); xanthine oxidase or xanthine :oxygen oxidoreductase (EC 1.2.3.2); purine (deoxy)ribonucleoside phosphorylase or purine nucleoside : orthophosphate (deoxy)ribosyl transferase (EC 2.4.2.1);adenosine hydrolase or N-ribosyl-purine ribohydrolase (EC 3.2.2.1); inosine hydrolase or inosine ribohydrolase (EC 3.2.2.2); adenine deaminase or adenine aminohydrolase (EC 3.5.4.2); (deoxy)adenosine deaminase or (de0xy)adenosine aminohydrolase (EC 3.5.4.4).

zyme the transfer reaction is catalyzed by a pingpong bi-bi mechanism explaining why the three types of transfer could be observed.

MATERIALS AND METHODS Reagents Deoxyadenosine was purchased from Fluka, adenine from BGH, deoxyinosine, hypoxanthine, thymidine, cytosine from Calbiochem, deoxyguanosinefrom Serlabo, deoxycytidine . HCL from Sigma, inosine from NBC. Amino-pyrrolo-pyrimidine and hydroxypyrrolo-pyrimidine were a generous gift from Burroughs Welcome and Co. [8-14C]Hypoxanthine, [U-14C]adenine, [2-’4C]cytosine and [8-14C]deoxyinosine were obtained from the DCpartement des Radioelkments, Saclay. Protamine sulfate and adenosine were products from Mann Research. Enzymes Xanthine oxidase was a commercial preparation from Boehringer (10 mg x ml-’ suspension in ammonium sulfate). The specific activity was carefully checked before use. trans-N-deoxyribosylase was extracted from Lactobacillus helveticus (CNRZ 303 strain) grown under conditions described previously [l]. The bacterial material was ground in 0.1 M phosphate buffer, pH 6.0. After centrifugation the protein obtained by precipitation in 75 % saturated ammonium sulfate was

366

further purified by precipitating nucleic acids with 0.5% protamine sulfate. The precipitate was spun down and the supernatant dialyzed against a 0.1 M phosphate buffer, pH 6.0, then heated at 60 "C for 5 min. After centrifugation the enzyme in the supernatant was purified in a single step by affinity chromatography using a column of an adsorbent made of Sepharose-IV-B substituted with m-phenylenediamine onto which was coupled a diazonium salt of 5-(paminophenylpropyl-uracil) [l]. The purine-to-purine transfer enzyme (trans-N-deoxyribosylase-I)was not retained on this specific adsorbent. The trans-N-deoxyribosylase retained was eluted with a solution of 10 mM adenine. The eluate was exhaustively dialyzed against a 0.1 M phosphate buffer at pH 6.0 and after centrifugation the supernatant was stored at 4 "Cuntil use. No apparent loss of specific activity was observed after several months of storage under these conditions. Protein Determinations

Protein concentrations were measured by the Folin method as described [3] for concentrations ranging from 20-1000 pgxml-', and by the Schaffner method [4] for concentrations between 0.5 - 20 pg x ml-'. In both methods bovine serum albumin was used as a standard. Nucleic Acid Determination

The elimination of nucleic acids and nucleic acid constituents was followed by measuring absorbance at 260 nm. KINETIC MEASUREMENTS

Spec trophotome t ric Assays

All reactions were carried out at 40 OC in 0.1 M phosphate buffer, pH 6.0, in a quartz cuvette of 1-cm path length (final volume 3 ml). Deoxyribonucleoside and base solutions were mixed together with a sufficient volume of buffer. The mixture was equilibrated at 40 "C (15 min). The enzyme solution (100 pl) was added to start the reaction and the absorbance change was followed with time. A suitable wavelength was selected according to the nature of the substrate couples : deoxycytidine-adenine and deoxyadenosine= 3864 M-' x cm-'I); deoxycytosine: 280 nm guanosine: 290 nm = 2178 M-' x cm-'); deoxyinosine-cytosine: 280 nm ( A E~~~ = 2526 M-' x cm-'); thymidine-cytosine: 240 nm ( A E~~ = 2248 M-' x cm-'). All measurements were perfcirmed with a Beckman Kintrac VII spectrophotometer and a 10-in (25.4-cm) potentiometer recorder setting the span at 0-0.2 absorbance unit with a chart drive speed of 1in . min-' (2.54 cm x min-'). Under these conditions

Kinetic Studies of trans-N-Deoxyribosylase

in the extreme case of a kinetic measurement with 0.033 mM deoxycytidine and 0.0050 mM adenine, a linear absorbance decrease of 0.0066 (8.5 mm) was obtained over a period of 1 min. For the few similar kinetics reported here the determination of precise initial rates called for special care in operation but most other kinetics with higher substrate concentrations were obtained without difficulty. When deoxyinosine was used as donor, xanthine oxidase was used as an auxiliary enzyme and the absorbance measured at 290 nm (.zZg0for uric acid was taken as 12200 M-' x cm-'). trans-N-Deoxyribosylase Assays

A unit of enzyme activity (U) was defined as the quantity of enzyme necessary to produce 1 pmol of product in 1 min under standard conditions: 0.1 M phosphate buffer pH 6.0, 40 "C, 0.17 mM substrate concentration. The three possible types of transfer were followed, three specific activity units were defined corresponding respectively to the three transfer reactions: (dCyd -+ Ade), (dThd -+ Cyt) and (dIno -, Ade). Isotopic Method for Transfer Kinetics and Isotopic Exchange Studies

This method was used (a) to study the isotopic exchange in the two couples deoxy~ytosine-[2-'~C]cytosine and deoxyadenosine-[8-14C]adenine and (b) to measure the equilibrium constants for a certain number of reactions. The experimental conditions were: 0.2 pmol of each substrate in 0.4 ml of 0.01 M phosphate buffer, pH 6.0. The reaction was initiated by adding 0.1 ml of the enzyme solution (0.05 pg protein x ml-') in 0.01 M phosphate buffer pH 6.0 The reaction products were measured as described previously [2]. RESULTS Characteristics of Purified Enzyme Fraction

The different steps of the purification procedure (see Materials and Methods) are given in Table 1. Purification refers to the (dCyd -+ Ade) transfer. It was possible to obtain a 215-fold purification and a specific activity of 43 U x mg-'. These results are essentially identical with those of a parallel homogeneous preparation using the same bacterical culture [l]. The usual checks for deoxyadenosine deaminase, adenine deaminase, adenosine hydrolase and inosine hydrolase revealed no detectable amount of these activities. Traces of deoxyribonucleoside hydrolase were suspected but had a negligible effect in 5 min. It was ascertained that the transfer was independent of phos-

367

C. Danzin and R. Cardinaud Table 1 . Purification oftrans-N-deoxyribosylasefrom L. helveticus: comparison between different transfer activities Recovery and purification values refer to the (dCyd + Ade) transfer Fraction

Total orotein

Recovery Purification

Total activitv

Specific activity ratio

Specific activity ____

~-

(dCyd + Ade) (dThd + Cyt)

Initial extract Ammonium sulfate Protamine sulfate Heat denaturation Affinity column

(dlno

--f

Ade)

(dThd -+ Cyt) ____. (dIno + Ade) (dCyd ---t Ade) (dCyd +Ade)

mg

units

%

-fold

7750

1550

100

1

0.20

0.033

0.050

0.165

0.250

3915

1504

97

1.92

0.384

0.061

0.095

0.159

0.250

3396

1450

93.5

2.13

0.427

0.052

0.128

0.124

0.300

2360

1410

91

2.99

0.598

0.082

0.153

0.137

0.260

1210

78

7.75

0.80

0.180

0.019

28.1

215

units/mg __

~ _ _ _ _ _ _ - _ - ~

43

r

-.-; X x ,

I

I

I

1

F 5"

z

:

u

b

: 100

Kx)

0

0

50

Kx)

150

"

l/[Adenine] (rnt@)

I

I

10

x)

I

30

l/IDeorycytidine] (rnM-')

Fig. 1. Initial velocity pattern for the (dCyd+ Ade) reaction with ( A ) adenine and ( B ) deoxycytidine as variable substrate. Protein concentration. 0.13 pg/ml. (A) Deoxycytidine as fixed substrate. (0)0.17 mM; (A) 0.10 mM; (A) 0.067 mM, (0)0.050 mM, (0)0.033 mM. (B) Adenine as fixed substrate: (B) 0.067 mM; (0)0.033 mM; (A) 0.017 mM; (A) 0.010 m M ;(0)0.0067 mM; (0) 0.0050 mM

phate concentration and deoxyribose 1-phosphate was not a substrate. Table 1 indicates that the ratio of activities for the (dThd + Cyt) and (dCyd -+ Ade) transfer did not vary significantly at the different stages of purification, whereas the ( d h o -+ Ade)/(dCyd + Ade) transfer ratio was noticeably lower after the last purification step. However this non-negligible residual (dIno -+ Ade) transfer activity remained constant even after repeated chromatography on the specific adsorbent. This had been observed in earlier preparations also and indicates that this activity is not due to a contamination by trans-N-deoxyribosylase-I. KINETIC STUDIES

The initial velocity was shown to increase lineary with enzyme in the concentration range used in these studies.

Deoxycytidine-Adenine Transfer Kinetic Studies

Deoxycytidine + adenine 3 Cytosine deoxyadenosine (2) Initial velocity plots as a function of adenine concentration for various concentrations of deoxycytidine indicated an inhibitory effect of adenine. At adenine concentration below 0.07 mM a double-reciprocal plot ( l / u versus l/[S]) (Fig. 1) with varying concentrations of deoxycitidine or adenine yielded parallel lines suggesting a ping-pong bi-bi mechanism [ 5 ] . For inhibitory concentrations of adenine a very characteristic competitive inhibition of deoxycytidineby adenine was obtained, as seen in F2g. 2. On the other hand no competitive inhibition of adenine by deoxycytidine was observed, even at concentrations as high as 50 times those of adenine. A plot of l / u versus l/c, c being the concentration for both substrates, gave a straight line (Fig. 3). This

+

368

Kinetic Studies of trans-N-Deoxyribosylase I

< J

OO

I

I

10

m

33

I/[Deaycytidinel (mM?

Fig. 2. Competitive substrate inhibition by adenine. Protein concentration 0.13 pg/ml. Adenine as fixed substrate: (0) 0.10 mM; (A) 0.17 mM; (A) 0.33 mM; (0)0.50 m M ;(0)0.67 mM

Fig. 4. Product inhibition by deoxyadenosine with deoxycytidine as variable substrate. Protein concentration: 0.13 pg/ml. Adenine concentration: 0.017 mM. Deoxyadenosine concentration: (m) 0 ; (0) 0.033mM; (A) 0.066mM; (A) 0.10mM; (0) 0.13mM; (0)0.17 rnM

In Fig. 5 we have a linear non-competitive inhibition of adenine by cytosine, whereas cytosine gave a linear non-competitive inhibition of deoxycytidine (Fig. 7). Deoxyadenosine-Cytosine Transfer Kinetic Study

/

I

I

i

I

I

I

This is the reverse of the preceding transfer reaction. Similarly, a double-reciprocal plot with varying concentrations of deoxyadenosine or cytosine yielded parallel lines and the initial velocity pattern for identical concentrations of cytosine and deoxyadenosine was a straight line (Fig.8). The product inhibition behaved similarly but on the other hand neither substrate showed any sign of excess inhibition, in agreement with previous studies [7,8]. Kinetic Constants

Fig. 3. Initial velocity pattern for identical concentrations of both adenine and deoxycytidine substrates. Protein concentration: 0.13 pgg/rnl

test is taken as further evidence that the transfer proceeds via a ping-pong bi-bi mechanism, one of the substrates being a competitive inhibitor of the other [2,6] by formation of a dead-end complex with the free enzyme. A linear competitive inhibition of deoxycytidineby deoxyadenosine was observed, as reported in Fig. 4.

The, kinetic constants have been obtained for these two transfer reactions as described in [2] and are given in Table 2. The inhibition constants Ki were calculated from the observed non-competitive inhibition by the products. Isotopic Exchange Studies

It was checked that an isotopic exchange occurred between : (a) deoxycytidine and [2-14C]cytosinein the

369

C. Danzin and R. Cardinaud 400 350

300 -250 .c

-!

$ -m 150

1CU

50

0

so

0

-25

25

50

75

~/I~denine] (M')

Fig. 5. Product inhibition by deoxyadenosine with adenine as variable subsrrate. Protein concentration : 0.13 pg/ml. Deoxycytidine Concentration: 0.067 mM. Deoxyadenosine concentration: (a)0; (0)0.033 mM; (A)0.067 mM; (A) 0.10 mM; (0)0.13 mM; (0)0.17 mM

where U X - ~= isotopic exchange velocity between first substrate and first product (A = deoxycytidine, P = [2-'4C]cytosine). &$t isotopic exchange velocity between second substrate and second product (B = [8-14C]adenine; Q = deoxyadenosine). In the second relation a term is introduced to account for the excess inhibition by substrate B. For the exchange reaction (dCyd 2 Cyt*) the ratio U&,/U:& was 1.13. For the exchange reaction (dAdo 2 Ade*) this ratio was 1.05. Kinetic Studies of (dGuo i? Cyt) and (dIno 2 Cyt) Transfers

3

OO

25

50

75

loo

150

l/[Adenine] (mM-')

Fig.6. Product inhibition by cytosine with adenine as variable substrate. Protein concentration 0.13 pg/ml. Deoxycytidine concentration: 0.010 mM. Cytosine concentration: (B) 0; (0) 0.033 mM; (A)0.067 mM; (A) 0.13 mM; (0)0.20 mM; (0)0.30 mM

absence of adenine and deoxyadenosine and (b) deoxyadenosine and [8-14C]adenine in the absence of deoxycytidine and cytosine. Good agreement was obtained with the corresponding measured initial velocities calculated from the relations given by Cleland [9] for the exchange reaction in a ping-pong bi-bi mechanism.

With two other purine deoxyribonucleosides as donors the same kinetic patterns were obtained and the Michaelis constants, as well as maximum velocities corresponding to these two transfer reactions, are given in Table 3. DISCUSSION The initial velocity studies reported here supply evidence that the deoxyribosyl transfer catalyzed by trans-N-deoxyribosylase-11proceeds via a ping-pong bi-bi mechanism. In Cleland's representation the mechanism is summarized for the reaction given in Equation (2) by the short hand notation: *

E

Ade

dCyd

1

E-dCyd EdRib-Cyt /+Ade E-Ade

1

E-dRib

'

dAdo

E-dRib-Ade E-dAdo

*E (5)

370

Kinetic Studies of trans-N-Deoxyribosylase 400

350

xo ._ m = -E

'200

-5 -- 150 >

100

50 0 -15

5

-10

0

5 10 15 l/[[)eoxycytidine] (mM-')

20

25

30

35

Fig. I . Product inhibition by cytosine with deoxycytidine as variable substrate. Protein concentration : 0.13 pg/ml. Adenine concentration 0.017 mM. Cytosine concentration (m) 0; (0) 0.033 m M ;(A) 0.067 m M ; (A) 0.13 mM; (0)0.20 mM; (0) 0.30 mM

The validity of this model is again justified by the good agreement of Haldane's relations for the pingpong bi-bi mechanism [5,6] [Equation (6)]

l/[Cytosine]~1/[!3mxyadenosine] (rnM')

Fig. 8. Initial velocity pattern for identical concentrations of both cytosine and deoxyadenosine substrates. Protein concentration: 0.13 pg/ml

where A and B are respectively the first and second substrates and P and Q the first and second products. Using the constants obtained experimentally from the present study of dCyd + Ade and dAdo -+ Cyt transfers, the following values were calculated for the different members of these relations: 8.15,7.85,8.1,8.6,8.8 and 8.7. The ping-pong bi-bi mechanism for a (Pur st Pyr) and (Pyr 2 Pyr) transfers are both catalyzed by the same enzyme accounting for the 'residual' (Pur 2 Pyr) transfer activity found in homogeneous preparation of trans-N-deoxyribosylase-I1and (b) the enzyme

Table 2. Kinetic constantsfor the transfers (dCyd+ Ade) and (dAdo + Cytj ~~~~~

~

~~

Michaelis constant

Inhibition constant

mM

Maximum velocity

Equilibrium constant K

pmol min-' pg-'

Forward reaction Deoxycytidine Adenine

KdCyd= 0.090 KAde = 0.019

(Ki)dCyd = 0.095 (KJAdC = 0.021

(dCyd-tAde)

= 0.085

8.15

(K,)Ade = 0.41"

__ Reverse reaction Cytosine Deoxyadenosine a

Dead-end inhibition.

____________ Kc,, = 0.22 KdAdo= 0.12

(K)cyt = 0.17 (Ki)dAdo = 0.092

__

v(dAdo+Cyt)

= 0.113

-~

0.115

371

C . Danzin and K. Cardinaud

Table 3. Kinetic constuntsfor various tran.$ers to cytosine and adenine Michaelis constant of acceptor

Transfer reaction

Michaelis constant

mM

kmol min-' pg-'

_____

---

~

GY,= 0.22

(Deoxyadenosine +cytosine) (Deoxyguanosine + cytosine) (Deoxyinosine +cytosine) (Deoxycytidine +cytosine) (Deoxyadenosine +adenine) (Deoxycytidine + adenine) (Deoxyinosine +adenine) (Deoxyguanosine +adenine)

Equilibrium constant K

Maximum velocity

of donor (deoxyriboside)

KdAdo= 0.12 Kdciyo= 0.37 Kdlno = 3.5 KdCyd= 0.095

Kcy, = 0.077 K,,, = 0.073 K,,, = 0.17 KAde= 0.021 KAde= 0.019 K A d e = 0.0073" KAde= 0.008"

KdAdo

v[dAdo-Cyt)

= o.113

v(dGuo-Cyl)

= 0.038 = 0.034 = O.Og0

v(dIno-Cyt) v(dCyd+Cyt)

= 0.092

KdCyd= 0.090 Kdlno= 3.4" KdGuo= 0.346'

0.095

V(dAdo-Ade)

=

v[dCyd-Ade)

= 0.0855 = 0.033" = 0.036a

'(dlno-Ade) V(dGuo-Ade)

0.115 0.094 0.29 -

' Calculated values (see text).

catalyzes the isotopic exchanges between A and P in the absence of B and Q, and B and Q in the absence of A and P respectively:

+

Deoxycytidine ['4C]cytosine 2 Cytosine [14C]deoxycytidine

+

(7)

Deoxyadenosine [I4C]adenine 2 Adenine ['4C]deoxyadenosine.

(8)

+

+

A comparison of experimental initial isotopic exchange rates with the corresponding values calculated from the kinetic parameters of the transfer reaction (dCyd + Ade) showed a reasonably good agreement (Results). This suggests in particular that no trans-N-deoxyribosylase-Itakes any part in the (dAdo + Ade) exchange ( i e . the enzymic fraction studied here is free from trans-N-deoxyribosylase-I). The following reaction: Deoxyinosine + adenine 2 Hypoxanthine

+ deoxyadenosine

(9)

was used as an example of (dRib Pur 2 Pur) transfer. The experimental study of this transfer reaction proved to be difficult owing to the low activity of the purified fraction (Table 1). The theoretical values of the kinetic constants of this transfer were worked out by the following method, assuming that a single given protein catalyses the three possible kinds of transfer:

According to Alberty [lo] the maximum velocity is :

where et is the total enzyme concentration. Hence :

In Equation (11) k3 depends only on the nature of the deoxyriboside and k7 only on the nature of the base. Consequently : 1 V(dIno

1

-

-,Ade)

V(dIno

+

-t

Cyt)

1

-

1

v(dAdo

+

Ade)

i

1

v(dAdo

-

Cyt)

(13)

In this relation all the maximum velocities were determined (see Table 3) except v(/(dl,,o-,A&), which can therefore be calculated.

Calculation of V(dlno -t

The transfer reaction can be divided into two parts :

E

+ A+(EA-FP)+F

F+B?

hs

(FB-EQ)+E

+P

+Q

(10)

F is an intermediate form of the enzyme, (EA - FP) and (FB - EQ) are enzyme-substrate or enzyme-product complexes.

Calculation of

&fnO

and KAde

For all transfer reactions catalyzed by the same enzyme according to a ping-pong bi-bi mechanism it can easily be established that the Michaelis constants of the substrates are related to the maximum velocities by the following relations: = Cte (dRib-Bn+B)

372

C. Danzin and R. Cardinaud : Kinetic Studies of trans-N-Deoxyribosylase

where dRib - Bn is any purine or pyrimidine donor

( \

KdR;

'

-B

Purine

)

= Cte

(Kdlno) (dlno

+

Ade)

- V(dlno

Cyt)

V(dIno

-.

+ Ade) +

4 for trans-N-deoxyribosylase I

1 (dRib-B+Bn)

where Bn is any purine or pyrimidine base. In the present case we can write: (Kdlno)(dlno

Table 4. Influence of substituents on the 6 position of purines

11"

mM dAdo dIno Ade HPX

Cyt) a

0.092 0.38 0.021 0.21

0.43 0.34 0.039 0.079

From[2].

and

-.

Thus (KdIno)(dIno Ade) and (KAde)(dIno Ade) may easily be calculated since all the other constants are known (Table 3). A value of the initial velocity for the substrate concentrations under normal conditions (see Materials and Methods) was obtained by means of the following equation : +

these groups in the formation of the enzyme-substrate complex can be ruled out, since it has been shown in a specificity study [ l l ] that purine bases with various bulky substituents on position 6 (6-iodopurine, 6-nhexylaminopurine, 6-benzylaminopurine) are substrates. Conversely purine is also a good substrate. Incidentally this phenomenon is not found with trunsN-deoxyribosylase-I [2] for which the corresponding constants of hypoxanthine and adenine are much closer. Plots not given in this paper are available upon request to one of the authors (R. C.). C.D. wishes to thank the Commissariat ri I'Energie Atomique for a predoctoral fellowship.

In the (dIno+Ade) transfer [A] and [B] are the initial deoxyinosine and adenine concentrations, K A and KB the Michaelis constants and V = V(&,wAde); K I is the constant of the E-adenine abortive complex [5]. The calculated value is consistent with the v value measured under the same concentration conditions ( b p . / L l c . ) = 0.89. The method described in this paragraph was also used to calculate the kinetic constants (Michaelis constants and maximum velocity) of the (deoxyguanosine + adenine) transfer (Table 3). The apparent dissociation constants for the substrates assembled in Table 4 show the effect of the substituent in position 6 of the purine bases. It may be observed that the purine derivatives with an amino group in position 6 have a better a f h i t y than those with a hydroxyl group both for the bases and for the deoxyribosides. Nevertheless a direct participation of

REFERENCES 1. Holguin, J. & Cardinaud, R. (1975) Eur. J. Biochem. 54, 505- 514. 2. Danzin, C. & Cardinaud, R. (1974) Eur. J. Biochem. 48, 387395. 3 . Leggett Bailey, J. (1967) in Techniques in Protein Chemistry, 2nd edn, pp. 340-341, Elsevier, Amsterdam. 4. Schaffner, W. & Weissmann, C. (1973) Anal. Biochem. 56, 502-514. 5. Cleland, W. W. (1963) Biochim. Biophys. Acta, 67, 104- 137, 173- 187and 188 - 196. 6. Garces, E. & Cleland, W. W. (1969) Biochemistry,8,633-640. I. Beck, W. S. & Levin, M. (1963) J. Biol. Chem. 238, 702-709. 8. Uerkwitz, W. (1971) Eur. J. Biochem. 23, 387-395. 9. Cleland, W . W. (1970) The Enzymes, 3rd edn, vol. 11, pp. 1- 65, (Boyer, P. D., ed.) Academic Press, New York. 10. Alberty, R. A. (1956) Adv. Enzymol. 17, 1-64. 11. Holguin, J. & Cardinaud, R. (1975) Eur. J. Biochem. 54, 515- 520.

C. Danzin and R. Cardinaud, Service de Biophysique, Dtpartement de Biologie, BLtiment 142, Centre d'Etudes NuclCaires de Saclay, Boite postale 2, F-91190 Gif-sur-Yvette, France