On the Mechanism of Ribonuclease A

Transcription

1 Eur. J. Biochem. 46, (1974) On the Mechanism of Ribonuclease A Crystal and Molecular Structure of Uridine 3-0-Thiophosphate Methyl Ester Triethylammonium Salt W. SAENGER, D. SUCK, and F. ECKSTEIN Max-Planck-Institut fur Experimentelle Medizin, Abteilung Chemie, Gottingen (Received March 11, 1974) The triethylammonium salt of uridine 3 -O-thiophosphate methyl ester crystallized in space group P21 with cell dimensions a = pm, b = pm, c = pm, /3 = 92.8 and two molecules per asymmetric unit. Solution of the structure was accomplished by direct methods; the nucleotide molecules have been located unambiguously but the cations are disordered and appear only as highly noise background. The least-squares refinement converged at a conventional R-factor of 19.1 %. The base in both molecules of the asymmetric unit is anti with respect to the sugar, the conformation about the C(4 )-C(5 ) bond is gauche, gauche, the sugar pucker is C(2 )-endo. The conformation about the two P-0 ester bonds is not as observed in double helical RNA s, suggesting that the sulphur atom directs the orientation of the ester bonds. Uridine 3 -O-thiophosphate methyl ester was obtained by pancreatic ribonuclease A catalyzed methanolysis of the endo-isomer of uridine 2 : 3 -cyclothiophosphate. As the thiophosphate diester moiety is in the R configuration with respect to the methyl group, the enzyme must have attacked the cyclothiophosphate via an in-line mechanism. Pancreatic ribonuclease A (RNase A) is a well characterized enzyme which hydrolyses polyribonucleotides in a two-step mechanism : first a transesterification to yield a polynucleotide fragment with a terminal pyrimidine nucleoside 2 : 3 -cyclophosphate and second a hydrolysis of this cyclic ester to a terminal pyrimidine nucleoside 3 -phosphate. Although the threedimensional structure of RNase A itself and of several RNase. inhibitor complexes are known from X-ray crystallographic studies, the details of the mechanism by which the enzyme operates are still a matter of discussion. Particularly, it is of importance whether the 2 : 3 -cyclophosphate is opened by an inline mechanism or whether a pentacovalent phosphorous intermediate is formed which, after pseudorotation of its ligands, yields the pyrimidine nucleoside 3 -phosphate (adjacent mechanism) [ 11 (and references cited therein). In-line and adjacent mechanisms follow different stereochemical pathways. It has been shown recently that uridine 2 : 3 -cyclothiophosphate is a substrate for RNase A and that its two diastereomers can be separated by crystallization [2]. In an effort to differentiate between the in-line and the adjacent mechanism, the endo-isomer of uridine 2 : 3 -cyclothiophosphate [3] was incubated with RNase A in the presence of methanol to obtain uridine 3 -O-thiophosphate methyl ester (Fig.1). Since the absolute configuration of the substrate in this reaction is known, determination of the absolute configuration of the reaction product should allow one to determine the pathway of this reaction. In this contribution we report on the structural details of the triethylammonium salt of uridine 3 -thiophosphate methylester and its consequences for the mechanism of RNase A. A brief account of this work has already been published [4]. The structure of this molecule is also of interest for information on the structures of polyribonucleotides which contain thiophosphate instead of phosphate backbones. EXPERIMENTAL PROCEDURES Uridine 3 -O-thiophosphate methyl ester was obtained by incubation of the endo isomer of uridine 2 : 3 -cyclothiophosphate with RNase A in aqueous methanol [4]. Crystals of the triethylammonium salt of the ester were grown at 7 C from isopropanol sohtions. The crystals were of poor diffracting quality and yielded X-ray patterns of monoclinic, sometimes of

2 ~.~ 560 Structure of Uridine 3 -O-Thiophosphate Methyl Ester the sharpened Patterson map was not straightforward and the different possible P,S-vector sets could not be differentiated with enough confidence to yield the correct solution. Therefore direct methods in the form programmed by Germain eta!. [5] were applied, which involves a multi-solution tangent formula refinement. The normalized structure factor amplitudes, le;l, were calculated according to / T+ \1/2 \ H, H/ C(3 1 - c o I I oi31 S=P-OO I I oi2r ti HN(CH,-CH,)3 Fig. 1. Chemical formula and numbering scheme for uridine 3-0-thiophosphate methyl ester triethylammonium salt Table 1. Crystallographic data for uvidine 3 -O-thiophosphate methyl ester ProDertv Value Chemical formula CI~H~OOSN~PS Space group monoclinic, P21 Cell constants a = 685.4(2) pm b = (4) pm c = (7) pm fl = 92.8(3) Number of molecules/asymmetric unit 2 Formula weight 2x513 Calculated density g/cm data collected, resolution 10 pm orthorhombic symmetry. The cell constants of these two crystal forms were almost identical but the orthorhombic crystals, with space group P212121, contained one molecule per asymmetric unit while two molecules were arranged within an asymmetric unit of the crystals of monoclinic space group P21. A relatively well diffracting crystal of the monoclinic form was chosen for data collection (Table 1). Intensity data were measured using a four-circle diffractometer equipped with a Mo X-ray tube (Zrfiltered, MoK, radiation) and operated in the 0,20 scan mode. The data were corrected for geometrical factors and assigned weights according to counting statistics with 3 % allowance for machine error. Initially an attempt was made to solve the structure by Patterson methods. However, the interpretation of where Z; is the measured intensity at the reciprocal -+ lattice vector point h, I is the averaged measured intensity in the corresponding sin 0jA region and E is a multiplicity factor. With a starting set consisting of eight of the strongest 1E;l values, 64 different phase sets including the 323 IETl values >1.4 were obtained. A Fourier synthesis based on the most consistent phase set revealed the positions of 15 atoms. Several consecutive electron density calculations allowed us to locate all the atoms of the nucleotides but from the triethylammonium cations, only the nitrogen atoms were clearly visible while the ethyl groups were indicated as high noise background. Since only 46 out of the 58 non-hydrogen atoms were located, the least-squares methods could not refine the atomic parameters below a discrepancy index R = Zl IFo( - IFc/ \/Z\Fol = 19.1 %, with Fo and Fc observed and calculated structure factors. At this refinement stage geometric details of the structure cannot be stated but the conformational features and the configuration of the molecules are clear. RESULTS A list of the atomic parameters is presented in Table 2. Tables 3, 4, 5 and 6 give interatomic distances, angles, dihedral angles and a comparison of conformational angles in diester phosphate and thiophosphate groups. In Fig.2, 3 and 4 the conformation of diester phosphate and thiophosphate groups, a view of the crystal structure along a* and a projection of the asymmetric unit down the c-axis is shown. Models of the endo-isomer of uridine 2 : 3 -cyclothiophosphate and uridine 3 -thiophosphate methyl ester with R and S configuration are shown in Fig.5. Fig.6 illustrates the stereochemistry of the transesterification of uridine 2 : 3 -cyclothiophosphate for in-line and adjacent mechanisms. Configuration of Uridine 3 -O-Thiophosphate Methyl Ester As the configuration of the uridine moiety is not affected by the attack of RNase and is well known from other investigations,it can be used as a convenient

3 W. Saenger, D. Suck, and F. Eckstein 561 Table 2. Fractional atomic coordinates and isotropic temperature factors for nucleoside atoms, and anisotropic temperature factors fov the atoms of the thiophosphate group Results are expressed in the form T = exp - (PllhZ + Bz2kZ + P B12hk + 2&hZ + 2Bz3kZ). Average standard deviations obtained from the correlation matrix are 0.002, 0.004, and 0.006, 0.005, for x, y, z of third row and second row atoms, respectively Atomic parameters for molecule 1 Atom X Y Z 811 Pz B12 pi * Atomic parameters for molecule Atom X Y Z 811 B BIZ O(3') P O(5") C(5") S NU) C(2) O(2) N(3) C(4) O(4) C(5) C(6) CU') C(2') O(2') C(3') C(4') , 3.8 C(5') O(5') O(1' N handle to determine the absolute configuration of the chiral phosphorous atom. In the notation of Cahn et al. [6], both uridine 3'-U-thiophosphate methyl ester molecules in the crystal have an R configuration. As 90% of the uridine 3'-U-thiophosphate methyl ester formed in the RNase-catalyzed methanolysis of uridine 2': 3'-cyclothiophosphate could be obtained in crystalline form, the reaction proceeds stereospecifically.

4 ~~ 562 Structure of Uridine 3 -O-Thiophosphate Methyl Ester Bond Angles and Distances The estimated standard deviations of the geometrical data derived for the two molecules within the asymmetric unit (Table 3) are fairly large (5 pm for bonds and 3 for angles) due to the minor quality of the reflection data and difficulties to locate the cation atoms but do not show obvious violations with repects to standard nucieotide geometry. The atomic distances for the thiophosphate diester groups in both molecules, 196 pm and 200 pm for the Table 3. Interatomic bond distances in uridine 3 -O-thiophosphate methyl ester Average standard deviations are 5 pm Bond between atoms Distance Molecule 1 Molecule 2 N(l)-C(2) N(l)-C(6) I N( 1)-C( 1 ) 143 I51 C(2)-N(3) C(2)-O(2) 117 N(3)-C(4) C(4)-C(5) C(4)-0(4) C(5)-C(6) C(l )-C(2 ) C( 1 )-O( 1 ) C(2 )-C(3 ) C(2 )-0(2 ) C(3 )-C(4 ) C(3 )-O( 3 ) C(4 )-O(1 ) C(4 )-C(5 ) C(5 )-O(5 ) O(3 )-P P P-O( 5 ) P-s (5 )-C(5 ) Table 4. Bond angles of uridine 3 -O-thiophosphate methyl ester Average standard deviations are 3 Bond angles between atoms Value for C(6)-N( 1)-C( 1 ) C(6)-N( 1)-C(2) C(2)-N(l)-C(l ) N(l)-C(2)-N(3) N( 1)-C(2)-0(2) 0(2)-C(2)-N(3) C(2)-N(3)-C(4) N(3)-C(4)-0(4) N(3)-C(4)-C(5) 0(4)-C(4)-C(5) C(4)-C(5)-C(6) C(5)-C(6)-N( 1) N(1)-C(1 )-C(2 ) N( 1)-C( 1 )-O(1 ) O(l )-C(l )-C(Y) C( 1 )-C(T)-C(3 ) C(1 )-C(Z)-O(T) 0(2 )-C(2 )-C(3 ) C(Z)-C(3 )-C(4 ) C(2 )-C(3 )-0(3 ) O( 3 )-C(3 )-C(4) C(3 )-C(4 )-C(5 ) C(3 )-C(4)-0(1 ) C( 5 )-C(4)-0( 1 ) C(4 )-C(5 )-0(5 ) C(4)-0(1 )-C(1 ) C(3 )-0(3 )-P O(3 )-P-S O(3 )-P-0 0(3 )-P-0(5 ) 0-P-O(5 ) 0-P-s O(5 )-P-S P-O(5 )-C(5 ) molecule 1 molecule 2 degrees I I II. IE m v Fig. 2. Comparison of the conformations about the phosphodiester bonds in uvidine 3-0-thiophosphate methyl ester (I V) diethylthiophosphate isomer I (I) and isomer II (11) and in DNA in the A (Ill) and C (Vj conformations. Lone electron pair orbitals at the oxygen atoms are indicated

5 W. Saenger, D. Suck, and F. Eckstein 563 Fig. 3. Projection of the crystal structure along a *. Symmetry elements corresponding to monochic P21 space group are drawn in heavy lines, those of the pseudo-orthorhombic P space group are drawn in light lines. Hydrogen bonds to the ammonium groups of the cations are indicated by dashed lines, other hydrogen bonds are mentioned in the text P-S bonds but 142 pm and 148 pm for the unesterified P-0 bonds indicate the structure ur id I ne-o( 3 ) \,Oe,P. // \ S O-CCHs with double-bonded, uncharged sulphur atoms and negatively charged, unesterified oxygen atom in analogy to the results obtained for the analysis of the diethylthiophosphate anion [7]. Conformation of the Nucleoside Part The dihedral angles for both ester molecules within the asymmetric unit are similar within 10 but deviate as much as 24 when the methyl ester groups are involved (Table 5). The nucleotides assume the common anti conformation with dihedral angles C(2)-N(l)-C(l )- O(1 ) [8,9] at 129 and 137. The ribose moieties are in an almost ideal C(2 )-endo envelope form as expressed by the 0 found for the angles C(3 )-C(4 )-O{l )-C(l ). The C(5 )-O(5 ) bonds are directed towards the bases

6 ~ 564 Structure of Uridine 3 -O-Thiophosphate Methyl Ester \. Y 0 s OP 0 N oc Fig.4. Projection of one asymmetric unit along C. The pseudo-twofold screw axis which relates the two molecules is indicated in a gauche, gauche arrangement about the C(4 )- C(5 ) bond, i.e. the O(S )-C(S )- C(4 )-(O(1 ) and O(5 )- C(5 )-C(4 )-C(3 ) dihedral angles are -71 ; -79 and 41 ; 40, respectively. The orientation of the thiophosphate group about the C(3 )-O(3 ) bond is such that the dihedral angles C(4 )-C(3 )-0(3 )-P (269 and 274 ) are in the (-)gauche range. A similar conformation was observed in cytidine 3 -phosphate which exhibited a C(2 )-endo sugar puckering mode as well [lo, 111. In nucleoside 3 -phosphates and in polyribonucleotides with C(3 )-endo sugar puckering, however, the same angle is close to 200, i.e. in a trans conformation. This suggests that in C(2 )-endo nucleotides steric hindrance between the vicinal O(3 )-phosphate group and O(2 )-hydroxyl group is avoided when the C(4 )-C(3 )-0(3 )-P angle is twisted from trans position in C(3 )-endo nucleotides to a gauche position. Conformation around the P-0 Ester Bonds; Steric Influence of the Sulphur Atom In Table 6 and Fig.2 the conformation of the diester-thiophosphate groups in uridine 3 -O-thiophos- Table 5. Selected dihedral angles of widine 3 -O-thiophosphate methyl ester These angles are defined as zero, when, looking along the central bond, the four atoms are cis-planar and are counted positive when the far bond is rotated clockwise with respect to the near bond Dihedral angles C(1 )-C(Z )-C(3 )-C(4 ) C(2 )-C( 3 )-c(4 )-0( 1 ) C( 3 )-C(4)-0(1 )-C(1 ) C(4 )-0(1 )-C(l )-C(2 ) O( 1 )-C( l )-C(T)-C(3 ) C(2)-N(l)-C(l )-O(l ) O(l )-C(l )-C(2 )-0(2 ) N(l)-C(I )-C(2 )-0(2 ) C( 1 )-C(2 )-C(3 )-0(3 ) 0(2 )-C(2 )-C(3 )-0(3 ) C(2 )-C(3 )-0(3 )-P C(4 )-C( 3 )-0( 3 )-P C( 3 )-0( 3 )-P-s C(3 )-0( 3 )-P-0 C(3 )-0(3 )-P-0(5 ) 0(3 )-P-O(5 )-C(S ) O-P-O(5 )-C(5 ) s-p-o( 5 )-C( 5 ) C(2 )-C( 3 )-C(4 )-C(5 ) 0(3 )-C(3 )-C(4 )-C(5 ) C(3 )-C(<)-C(5 )-0( 5 ) 0(1 )-C(4 )-C(5 )-0(5 ) Value for molecule 1 molecule 2 degrees Table 6. Conformational angles about phosphodiester bonds Designations up (anti-periplanar), sc (syn-clinal) were defined by Klyne and Prelog [21]. Data for DNA in the C and A conformations were taken from [I71 and [20] Phosphate ester Bond angle C(3 )-0(3 )-P-0(5 ) C(5 )-0(5 )-P-0(3 ) degrees Urd-PS-OMea - 145, (-up) 46, 70 (+ sc) Diethylthiophosphate isomer I isomer I1-158 (-ap) (+ ap) 69 (+ sc) -69 (-sc) DNA (C conform.) (-up) -45 (-sc) DNA (A conform.) - 63 (-sc) - 68 (- SC) a Uridine 3 -O-thiophosphate methyl ester; results obtained in this study. phate methyl ester is compared with the conformation of the diester-phosphate groups in the C and A conformations of DNA, and in the two isomers I, I1 of the model compound diethylthiophosphate; the two isomers of diethylthiophosphate are related by an inversion center [7].

7 W. Saenger, D. Suck, and F. Eckstein 565 The dihedral angles C(3 )-0(3 )-P-0(5 ) and C(5 )- 0(5 )-P-0(3 ) are in about the same -ap (trans), +sc (gauche) range in uridine 3 -U-thiophosphate methyl ester and in diethylthiophosphate isomer I but differ from the corresponding angles observed in the C and A conformations of DNA. Similar angles as in the C conformation of DNA (-up, -sc) were observed, however, in isomer I1 of diethylthiophosphate (+up, -sc). The close similarity of the conformation angles in uridine 3 -O-thiophosphate methyl ester and in diethylthiophosphate isomer I suggests that repulsive interactions between the orbitals at the sulphur atom and the lone electron pair orbitals at both ester oxygen atoms O(3 ) and O(5 ) determine the orientation of the C-0 ester bonds [12]. In the diester thiophosphate groups, the C -0 bonds are oriented such that in no case both lone electron pair orbitals from the same oxygen atom are in agauche,gaucheposition with respect to the P=S bond but always one oxygen lone electron pair is oriented trans with respect to the P=S bond. If the diester thiophosphate groups were in conformations similar to the A conformation of DNA or if uridine 3 -O-thiophosphate methyl ester had a similar conformation to DNA in the C conformation, then the sulphur atoms would be in gauche, gauche position to both O(5 ) oxygen lone electron pair orbitals, an obviously unfavourable situation. The other alternative with the dihedral angles c(3 )-0(3!)-~-0(5//) and c(5 )-0(3 >-p-0(3 > both trans is as in this case the o(3 )- and O(5 ) oxygen lone electron Pair orbitals would interact strongly with each other, as discussed elsewhere [ 121. Fig. 5. Molecular models of the starting material, the endoisomer Of Uridine 2 : 3 -cyclothiophosphoro~hio~t~ (A) which has been converted by RNase-catalyzed methanolysis to uridine 3 -O-thiophosphate methyl ester of the R confguration (B). The diasteromeric methyl ester with the S configuration (C) is shown for comparison Crystal Structure A view of the - crystal structure - along a* is presented in Fig.3. The uracil residues are arranged close to the a,b-plane at c 1/4 and c 3/4 - whereas the ribose and thiophosphate parts of the molecules are arranged in the region near c = 0 and c 1/2. The empty space between the nucleotide molecules is occupied by the disordered, unlocated triethylammonium cations. Inspection of Fig. 3 and 4 shows that the crystal structure exhibits nearly orthorhombic symmetry. In addition to the P21 symmetry elements there are noncrystallographic two-fold screw axes parallel to a and c, introducing a pseudo P symmetry. The close relation between the two space group symmetries in this structure explains the fact that we obtained crystals of both symmetries under similar conditions. The hydrogen bonding scheme appears to be clear for the two recognizable nitrogen atoms of the triethylammonium cations : they are bonded to the thio- phosphate groups via N@-H..eO hydrogen bonds of 269 and 263 Pm respectively. Additional hydrogen bonds are indicated between atoms N(3)l and 0(4)2 (273 Pmh atoms N(3)2 and 0(4)1 (281 pm), atoms o(2 )1 and 0(5 )1 (279 pm) and between atoms o(2 )2 and 0(5 )2 (291 pm). DISCUSSION Mechanism of Pancreatic Ribonuclease A Uridine 3 -O-thiophosphate methyl ester, obtained by RNase-catalyzed methanolysis of the endo-isomer of uridine 2 : 3 -cyclothiophosphate [3,4] as described in this contribution, has the R configuration. Inspection of the models of the substrate and the product (Fig. 5) shows that the formation of this diastereomer can easily be explained by a nucleophilic attack of methanol on the phosphorous from the side opposite to the leaving group (2 -oxygen). Both incoming nucleo-

8 566 phile and leaving group can thus be accommodated in the two apical positions of a trigonal bipyramid which is in accord with the rules put forward by Westheimer [13] for the hydrolysis of 5-membered cyclic phosphates. This mechanism is classified as the in-line mechanism [14]. The methanolysis of the endo-isomer of uridine 2 : 3 -cyclothiophosphate has, thus, proceeded by an in-line mechanism (Fig. 6). No pseudorotation is required for this mechanism as opposed to the alternative adjacent mechanism where incoming and leaving groups are in apical and equatorial positions, respectively, and pseudorotation has to move the leaving group to an apical position for the reaction to proceed. Had the methanolysis followed this reaction path, the product would have to be of the S configuration. The establishment of the in-line mechanism for the methanolysis of endo-uridine 2 : 3 -cyclothiophosphate does not necessarily allow the extrapolation to the mechanism of the second step of RNase action, the hydrolysis as discussed by Richards and Wyckoff [l]. Employing HzlgO instead of methanol for the ring opening of the endo-isomer of uridine 2 : 3 -cyclothiophosphate Usher et al. [14a] could show, however, that also in this reaction the in-line mechanism applies. There still remains the question whether the hydrolysis or methanolysis of uridine 2 : 3 -cyclophosphate follow the same mechanism or whether the introduction of sulphur into the phosphate group induces a change in mechanism. Although this cannot be strictly excluded it appears unlikely as the endo-isomer of uridine 2 : 3 -cyclothiophosphate has the same K, value as uridine 2 : 3 -cyclophosphate and the rate of enzymatic hydrolysis is slower only by a factor of 8 for the cyclothiophosphate. This reduction in reaction rate is also observed in the non-enzymatic hydrolysis by alkali and reflects the decreased electrophilicity of the thiophosphate relative to the phosphate group [2]. These characteristics appear to indicate that the enzyme operates equally with both substrates. Are the mechanisms identical for the endo and exoisomers of uridine 2 : 3 -cyclothiophosphate? Reaction of the exo-isomer with methanol in the presence of RNase yields the uridine 3 -thiophosphate methyl ester with the S configuration, i.e. the diastereoisomer of the ester described in the present contribution [4]. Therefore, for both diastereoisomers of uridine 2 : 3 - cyclothiophosphate, the in-line mechanism applies. As discussed earlier [4], the topography of the RNase active site suggests that histidine-1 19 abstracts the proton from the nucleophile MeOH and histidine- 12 protonates the leaving O(2 ) group. Usher et al. [15] carried out the transesterification with cytidine instead of methanol. After isolation of the product and subsequent chemical ring closure to the endo-isomer of uridine 2 : 3 -cyclothiophosphate Structure of Uridine 3 -O-Thiophosphate Methyl Ester ROH- WU 8 Howu in-tine Howu RO Pseudorotation Howu Howu O \ R /I OR 0 Fig. 6. Reaction paths and reaction products for methanolysis of uridine 2 : 3 -cyclophosphorothioate via the in-line and adjacent mechanisms, respectively. Note that the two reaction products differ in configuration (R for itz-line and S for adjacent) at the phosphorous atom they could show independently that this reaction had also followed the in-line mechanism. The reaction scheme described here has also been used to establish an in-line mechanism for the transesterification step of RNase TI [16] using guanosine 2 : 3 -cyclothiophosphate as substrate.

9 W. Saenger, D. Suck, and F. Eckstein Uridine 3 -O-Thiophosphate Methyl Ester as a Model for Polynucleotides Containing Thiophosphate Groups One can look at the structure of uridine 3l-O-thiophosphate methyl ester as a model for a dinucleoside thiophosphate or as a fraction of a polynucleotide with thiophosphate groups. It is apparent that the ester in this particular conformation does not allow the formation of a regular helix without changes in the conformation about either the thiophosphate diester or the sugar bonds. With a conformational change about the P-O(S ) bond a structure like DNA in the C conformation would result [17]. This is in striking agreement with results obtained from a crystallographic study of the Mg2+ salt of diethylthiophosphate [7]. The conformations around the thiophosphate diester bonds in the isomers I and I1 of this conipound resemble those found for the diester bonds in uridine 3 -O-thiophosphate methyl ester and in DNA in the C conformation (Table 6). This observation is of interest since polyribonucleotides with thiophosphate backbones have been synthesized enzymatically [ls]. They are degraded more slowly by nucleases than the corresponding polymers with a phosphate backbone and they are more active in the induction of interferon at least in the case of poly[r(a-u)] and poly [r(i-c)] [ 191 (and references cited therein). The replacement of phosphodiester groups versus thiophosphate diester groups in polynucleotides cannot be described solely on the basis of a formal replacement of oxygen by sulphur but involves changes in structure and in charge distribution. All these alterations can contribute to the unusual biochemical properties of the polymers containing thiophosphate groups. A differentiation and more specific discussion, however, is not possible at present as more detailed studies on the polymers are not yet available. Theiauthors are indebted to Prof. F. Cramer for his interest in these investigations. D. S. acknowledges support by$ the Deutsche Forschungsgemeinschaft through grant Sa The computations were carried out at the Gesellschaft fur Wissenschaftliche Datenverarbeitung (Gottingen) 567 and were supported in part by the Deutsche Forschungsgemeinschaft. REFERENCES Richards, F. M. & Wyckoff, H. W. (1971) in The Enenzymes (Boyer, P. D., ed.) 3rd edit. Vol. 4, pp , Academic Press, New York. Eckstein, F. (1968) FEBS Lett. 2, Saenger, W. & Eckstein, F. (1970) J. Amer. Chem. SOC. 92, Eckstein, F., Saenger, W. & Suck, D. (1972) Biochem. Biophys. Res. Commun. 46, Germain, G., Main, P. & Woolfson, M. M. (1970) Acta Crystallogr. Sect. B, 26, Cahn, R. S., Ingold, C. K. & Prelog, V. (1965) Experientia (Basel) 12, Schwalbe, C. H., Goody, R. & Saenger, W. (1973) Acta Crystallogr. Sect. B, 29, Pullman, B., Saenger, W., Sasisekharan, V., Sundaralingam, M. & Wilson, H. R. (1972) Nomenclature Proposed on the V. Jerusalem Sympossium on Quantum Chemistry and Biochemistry (E. D. Bergman & B.Pullman, eds) The Israel Academy of Sciences and Humanities, Jerusalem (1973). Saenger, W. (1973) Angew. Chem. Int. Ed. 12, Sundaralingam, M. & Jensen, L. H. (1965) J. Mol. Biol. 13, Bugg, C. E. & Marsh, R. E. (1967) J. Mol. Biol. 25, Sundaralingam, M. (1969) Biopolymers, 7, Westheimer, F. H. (1968) Acc. Chem. Res. 1, Usher, D. A. (1969) Proc. Natl. Acad. Sci. U. S.A. 62, a. Usher, D. A., Richardson, D. I., Jr. & Eckstein, F. (1970) Nature (Lond.) 228, Usher, D. A., Erenrich, E. S. & Eckstein, F. (1972) Proc. Natl. Acad. Sci. U. S.A. 69, Eckstein, F., Schulz, H. H., Ruterjans, H., Haar, W. & Maurer, W. (1972) Biochemistry, 11, Marvin, D. A., Spencer, M., Wiikins, M. H. F. & Hamilton, L. D. (1961) J. Mol. Biol. 3, Eckstein, F. & Gindl, H. (1970) Eur. J. Biochem. 13, Black, D. R., Eckstein, F., DeClercq, E. & Merigan, T. C. (1973) Antimicrob. Agents Chemoth. 3, Arnott, S. & Hukins, D. W. L. (1972) Biochem. Biophys. Res. Commun. 47, Klyne, W. & Prelog, N. (1960) Experientia (Basel) 16, W. Saenger, D. Suck, and F. Eckstein, Max-Planck-Institut fur Experimentelle Medizin, Abteilung Chemie, D-3400 Gottingen, Hermann-Rein-Str& 3, Federal Republic of Germany