Biochimica et Biaphysica Acta, 1076 (1991) 49-60 © 1991 Ekerier Science Publishers B.V. (Biomedical Division) 0167-a838/01/$03.5¢ ADONIS 016748389100064G

BBAPRO 33798

~H-, ~C-, 3~ P-NMR studies and conformational analysis of NADP ÷, NADPH eoenzymes and of dimers

from electrochemical reduction of NADP ÷

Enzio Ragg 1, Leonardo Seaglioni t, R. Mondel l i ~, I. Carell i 2, A Casini 3 and S. Tortorel la 3

t Dipartimentodi Scienze Malecotan'Agraalimentan, Universit A di Mdang Milano (Italy), ~ Dipartimento di Chimica~ LC.M., Uaiversitd de L'AclUila, L'Aquila fltaly) and J Dipartimento di Studi di Chimica e Teotologia dells Soslanza Biologicameme Auive,

Uni~tsit~ di Roma ' La Sapienz a ; Rama ( haly )

(Receiwd 9 August t990)

49

Key words: NMR; Conformation analysis; Electrochemical reductiom Nic~linamide adenine nuclemide

All H,H, H,P and several C,P coupling constants, including those between C-4' and the vicinal phosphorus atom, have been determined for NADP ÷, NADPH coensymes and for a 4,4-dimer obtained from one-deetron electrodamical reduction of NADP +. From these data the preferred conformation of the ribese, that of the 1,4-dihydtonieolinamide rings, and the conformation about bonds C(4')-C(5') and C(5')-O(5') were deduced. The preferred [arm of the 1,4- and l~-dihydrolffridiue rings and the conformation about the ring-ring junetlon were also obtained far all the other 4,,L and 4,6-directs fonued in the same reduction. All Ihe dimers show a puckered stng'hn~, i.e., a boat form for the 1,4fi and a twist-beat for the 1,6-dihydronlcotinamide ring; both protons at the rlng-ring junctions are equatorial and have preferred gauche orientation. On the contrary, the reduced coenzyme NADPH displays a planar or highly flexible conformation, rapidly flipping between two limiting boat structures. The conformation of the ribose rings, already suggested for the NADP ¢uenzymes to be an equilibrium mixture of C(2")-endo (S.~pe) anal C(3')-ende (N.~pe) puckering modes, has been reexamined by using the Mtona procedure and the relative proportien of the two modes has been obtained. The S and N fmnflles of conformers have almost equal population for the adeniue.rlbnse, whereas for the nkollnamide.rlbose rings the S-type reaches the 90%, The rotatlon about the ester bond C(5')-O(5') and about C(4')-C(5'), defmad by torsion angles fl and ¥ respectively, displays a constant high preference for the trans conformer ~ (7S-80%), whereas Ihe retainers ¥ are spread out in a range of different populations. The values are distributed between the gauche ¥ + (48-69%) and the truns yt forms (28-73%). The 7 + conformer reaches a 90% value in the ease o | NA1DP + and NMN +. The conformations of the mononuelcotides 5'.AMP, NMN + and NMNH were also calculated from the experimental coupling constant values of the literature.

lnlrnduelion

In the preceding paper [1] we have reported the 1H-NMR analysis and the structure determination of the 4,4- and 4,6-1inked dimers (Scheme i) obtained from orb, e-electron electrochemical reduefioa of NADP*

We report here on a 13C and 3,p study of the a,4-di- mar (XI in the accompanying paper [!]) isolated as a 90f[ pure compound. In the course of this study, we needed to perform parallel NMR analyses, includkng ~H, on coeazymes NADPH and NADP +, as chemical

Correspondence: It, Mondelli, Dipanimento di Scienze Molecolmi Agtoalimentari, Via C.eloria 2, 20133, Milano, Italy.

shifts and coupling constants for the nuclei of file phosphodbose chain were not available. Actually, al- though a few errors have been corrected [2,4], there is great confusion in the literature [5] about these NMR parameters, which were generally derived through in- complete and often approximate approaches [2,3,6-9]. Therefore all H,H, P,H and the most important of the P,C coupling constants were obtained and are here reported together with those of dimer XI.

The conformation of nicotinamid¢ coenzymes has been the subject of many papers [3,4,7-M], hat the preferred conformations in solution have not yet been unambiguously determined. Nevertheless. much of the discussion, for instance, on the stereoselectivity of en- zymatic transfer of hydrogen from NADH [15l, has

50

been based on the assumption that the 1,4-dihydro- pyrldine ring exists in a boat form. Furthermore, it is usually accepted [5], with a few exceptions [10,12,13,16], that NADH occurs in folded forms, with adenine and nicotinamide rings stacked in parallel. The existence of folded structures was based on chemical shifts argu- ments [5] and again on the puckering of the dihydro- nicotinamide ring ]8]. The conclusion is that this model, after 30 years, still lacks sound experimental founda- tion, as recently pointed out by Bonnet and Duni!z [17]. Actually the determination of the crystal structure for N-substituted dihydronicotinamides showed that the ring is essentially planar [17] and NAD +, as free-acid and as Li salt, appears to be in the "extended" confor- mation in solid phase [16,18]. In addition X-ray studies of NADPH bound to a dihydrnfolate reductase, and of a number of dehydrogenases hound to NAD + demon- strated that generally the coenzyme exists in an 'ex~ tended' form [19].

We report here on the results of a conformational analysis of NADP +, NADPH and of 4,4- and 4,6-di- mars [1] in aqueous solutions; specifically, on the pre- fel'red conformation of the ribose and dihydronico- tinamide rings; on the conformation around bonds C(4 ' ) -C(5 ' ) and C(5')-O(5"), for dimer XI and for the coenzymes; on the conformation around the ring-ring junction, and on the preferred forms of ff~e 1,4- and 1,6-dihydropyridine tings for the dimers.

Materials and Methods

#-NADP + monosodium salt, tetrahydrate, M, 837.5 from U.S.B. (U.S.A.) and NADPH tetrasodium salt, M~ 833.4, 98% pure from Boehringer (F.R.G.) were used for NMR studies. The 4,4.dimer Xl was prepared by one- electron electrochemical reduction of fl-NADP+, free acid. as described in the accompanying paper [l].

NMR spectra were ~ceordcd with Bruker CXP-300 and AM-500 spectrometers. Chemical shifts are in ppm (8) values from external 3-(trimethylsilyl)-propane-1- mlphonic acid sodium salt hydrate (DSS) for lH and ~*C, and from external 85~o H~PO4 for 3tp. Estimated accuracy for tH and tZc 4_.0.005 ppm and +0.01 for ~tP. Coupling constants are in Hz, accuracy __+.0.1 Hz, unless specified in the tables. The spectra were mea- sured in D~O (20 rag, ml -~) at pH 9.3 _+ 0.l (NADPH and Xl) and at pH 7.2 + 0.1 (NADP ÷); the solutions were adjusted to the desired pH with NH 3 and kept under nitrogen; NADPH and dimcr XI were also mea- sured in 0.15 M sodium tetraborate buffer (pH 9.3). The variation in ~H and ~3C chemical shift with concentra- tion in the range 20-50 mg-ml -I are within 0.1 ppm. All the compounds are stable for more than 24 h in the experimental conditions used. UC assignments were performed by heteronuclear shift-correlated two-dimen- sional experiments, with concentrated solutions;

quaternary carbons ~ere assigned on the basis of chem- ical shift, relaxation time and long-range coupling infor- mation. For COSY spectra and HOD signal suppres, stun, see the accompanying paper [1]. Spectra simula- tion was performed by using the PANIC program.

Calculations of the populations relative to the differ- ent conformers were performed by solving the ap- propriate linear equation:

where Job~ is the experimental coupling constant, x~

denotes the molar fraction of the i-th species and J~ is the coupling constant relative to the pure i, th confor- mation. The equations are solved as sets of linear equa- tions, with the additional constrain that V,x, = 1. The relative populations of S-type conformers are calculated as arithmetic means of two values, obtained indepen- dently from the experimental values of J (HI ' ,H2 ' ) and J(H3',H4"). The estimated error on the populations is within _+ 5%.

Molecular mechanics calculations were performed by using the CVFF force field [38], with the partial charges calculated with the MOPAC program [39].

Results and Discussion

~H-NMR spectra of N,4DPH and NADP + Both forms of the coenzyme were analysed following

the procedure used for dimer XI [1] and the same experimental conditions, except for NADP +, which is not stable at pH higher than 7.5. The results are given in Tables 1 and 11; and partial proton spectra arc reported in Figs. 1, 2 and 3.

The spectral pattern of some rihose protons, for the coenzyme and dimer X], are strongly dependent on th~ relative chemical shift difference between the two phos- phorus nuclei of the pyrophosphate chain. Whenever this difference is very small, as is the case with NADPH (Fi~, 1) and with dimer XI in D20 /NH ~ solution (Table I|l), the patterns of the adenine-rlbos¢ protons at C-4' and C-5' an: deceptively simple due to second order effects, while the corresponding protons of the nicotinamidc fragment lie together at 4.05 6, A first- order analysis of A4' * and A5',5" leads to incorrect results, whereas the simulation of the spectrum is good if both phosphorus nuclei are included in the LAOCOON type calculation with a 0.01 ppm shift difference. When the cuenzyme is oxidized, the ~tp shift

* Numbered A's and N's indicate the atoms (hydrogens and carbons) or the adenine nucleotide and those o1' nicotinamide necleolide unit. Tespectivety. Unspecified numbers indicate atoms of beth units.

51

TABLE 1

tH ¢l,¢mlcal shift values for NADP +, NADPH and direct X i

Measured in ppm (,~) relative to external DSS: estimated aeeurac~ :k0.005 ppm. unless specified.

NADP + NADPH XI (a) (b) (c) (v)

A2 8_130 8.214 8.230 8.198 A8 8.=~(I 8.452 8,453 8.427 N2 9.280 6.913 6.919 7,091

2.815 d Z853 d N4 8.809 2.903

2,728 ~ 2.719 ~ N5 8.180 4.80 r 4.79 r 4.4.56 N6 9.105 5.943 5.893 6.099 AI ' 6.093 6.183 6,194 6.167 A2" 4.055 4.915 4.933 4.920 A3' 4.594 4.~65 4.577 4_586 A4' 4.363 4.354 4.375 4.372 AS' 4.283 4.277 4.286 2.272 AS" 4.187 4.172 4.194 4.209 N ] ' 6.023 4.77 r 4.?6"] 4.85 N2' 4.456 4.146 4.256 4.302 N3' 4.403 4.197 4.~8 4.300 N4' 4A93 4.045 4.028 4.044 N5" 4.311 4.020 4.091 4.125 N5" 4.204 4.0t? 4.015 4.042

DsO solution (pH 7.2)~ D~O/NH s solution (pH 9.3).

c 0.I5 M sodium tetraborate buffer in 020 (pH 9.3)- e.© The 8eminat protons at C.-4 are diastereotopic; (d) N4a, (e) N4b. f Par6ally overlapped by HOD signal: accuracy 4-0.01 ppra.

H CONH2 H H

4,4 d i m e r s

TABLE II

H,H coupling constant values/or NADP ÷, NADPII and direct X i

J vaktes are measured in Hz and are I~ivco without tlif~: estimalfd accuracy 4-0.1 Hz, unless Slz:cified.

j NADP+ a NADPH b X[ b

N2,N4 1-7 0.7 ¢ 0.5 c.d 0.6 N2,N6 1.5 1.7 IA N2,N5 0.3 d 0.5 = N4,N4 18.2 f 2.3 * N4,N5 8.1 3,3 ~ 3.7 e 4.8 N4.N6 1.3 1.9 = 1.7 ~ 0.7 NS,N6 6,3 8.2 8.0 NI',N2 ~ 5,5 7.5 h 7.0 h N2',N3' 5.0 5.4 h $,5 h NY,N4' 2.7 2,1 h 2.3 h N4",N5' 2.5 3.5 3.7 N4*,NS" 2.3 5.0 5.0 NS",N5" 12.0 11.8 12.0 AI',A2' 5.0 4.7 5.0 A2',A3' 5.3 4.9 5.0 A3',A4' 4.6 5.1 4.8 A4',AS" %6 2.7 3.0 A4',AS" 4.6 4A 4.6 AS',AS" 11.8 1[.8 11.8

= DzO solution (pH 7.2). b 0.t5 M sodium tetraborat¢ buffer iu I~O (pH 9.3). The atmlysis in

DzO/NH J solution (pH 9.3) gave the same results; for XI, see Table II of Ref. l; for NADPH, erdy J(A5' ,A$") changes to 11.6 Hz.

¢ Coupling constants with the geminal protons, N4a (left) and N4b (tight), respectively (see Table I)

d E~timated from the linevddth. = Not mcmsttred., but obtained f,om th~ calculated ~p~-ttum. f Geminal coupling constant. a Coupling coeslant between protons at the ring-ring junction. h Accuracy *0.2 Hz.

di f fe rence is 0,35 pp~a, a n d t he p r o t o n s p e c t r u m (Fig, 2) is nearly f i r s t -order ;rod can b e unambiguously analysed.

For both NAD?H and dimor XI, this degeneracy

was r e m o v e d b y t~sing s o d i u m t e t r a b o r a t e b u f f e r iR D 2 0

and l eav ing the p H v a l u e o f 9.3 u n c h a n g e d , In this

so lu t ion the chemica l shif t d i f f e rence b e t w e e n the phos -

pho rus nuclei c f the cha in increases to 0 . 2 - 0 . 3 F p m a n d

CONH2 H H

R B CONH= ' - 3

6 - - lk

4,6 d l m e r s

Scheme 1.4,4.- and 4,6 -lirff~l NADP dimers [ll. R = adenosine (2% phosph at e)-.diphospha t =-ribo.~.

TABLE Ill 3 / + P chemical '.~i~ifr values for NADP , NA DPH and dimer Xi

In ppm ft~m eatemal 85% H~PO, t, estimated accuracy :t:0.01 ppm. Negative numbers denote upfie[d shifts.

NADP + NADPH X|

(~) (b) (c) (b) (c)

P-2'A 3.477 4.44)9 4.434 4.26" 4.288 P-5'A -- 10.$44 -- 10.392 -- 10.534

- ]0.474 - 10,505 P-5'N - 10.886 -10.170 - 10.308

" D:O solution (pH 7.2). b DzO/NH 3 solution {p~ 9.3).

0.]5 M sodium tctraboratc buffer in D~O (pH 9.3).

N 3' N2"

N4"* NS' hiS"

As*'

AS" A4'

52

I

4 . 4

I ' t I I I

4.3 4,2 4.1 4.0

Fig. 1. tH-NMR spectrum (300 MHz, D20/NH 3 {pH 9,2)) of NADPH. region from 40 to 4.4 6.

ppm

leads to a small de.shielding of 0.05-0.1 ppm for pro- tons N2", N3" and NS'. Although the dbose protons of adenine [ragments are not significantly affected (0.01 ppm), the shift difference belweea 3~p nuclei induces

variations in the patterns of the coupled protons A4' and AS' (Fig, 3); the analysis is thus easier, since the P,H coupling constant values can be cross-checked with those obtained from the 31p spectrum. In addilion, the

N2" N3"

4 I

1 I I I

4.5 4.4 4.3 4.2 ~ ppm Fig. 2. 'H-NMR spectrum (500 MHz. D20/NH ~ (pH 7.2)) of NADP ~'. re,on from 4.1 to 4.5 &

N2'

N4"

(b)

A5" A4' . J l ,,

A 5 1 N 5 t/ N 5

4 . 4 0 4 30 4 20 4 1 0 4 0 0 /i ppm Fig. 3. (a), I H-NMR SlXa:tram (300 MHz. DzO/~etr~horate (pH 9.3)) of NADPH, region from 3.9 to 4.4 6: (b), cal¢ulaled spectrum.

slightly Iowfield shift of NS' decreases the overlapping with NS", allowing a complete analysis also for the nicotinamide fragment. All H,H aad P,H coupling con- stants were obtained.

~lp a#d ~SC ~pectra of NADPH, NADP + and of lhe 4, 4-dimer XI

The phosphorus at C-2' is always a doublet of ~bout 7 Hz for all compounds, while the two phosphate sig- nals of the chain vary. NADP + shows a well-resolved patteru (Fig. 4), whereas both NADPH and dimer XI give a degenerate singlct in D20/NH3, even at 121.4 MHz. This is in agreement with the results of Feeney et aL [20,21[ who found the same degeneracy in the 31p

spectrum of the reduced coenzyme at pH 7.9 (KCI, Tris buffer, EDTA). With sodium tetraborate buffer in D20, P-5'N moves to lowfield, 0.3 ppm for NADPH and 0.2 ppm for X! (Fig, 5). The shift difference between the phosphates of the chain allowed to obtain all P,H and P,P coupling constartts (Table IV), thus confirming the results from the proton spectra, The assignment of adenosine-5'-phosphate, P-5'A, and nicotinamide ribo- side-5'-phosphat¢, P~5'N, can now be made with cer- tainty (Table I11): for NADP + at pH 7,2, P-5'A is at low field and P-f 'N at high field, while, for both NADPH and direct Xl at pH 9,3, P-5'N is deshielded with respect to the partner. The relative attribution of the two phosphates remains the same when NADPH is

' ;4

P-S'A I P - 5 ' N

i I I I i I I

- 1 0 . 4 - 1 0 . 6 - 1 0 . 8 - 1 1 . 0

Fig. 4. :qP-NMR spec~Jum (121.4 MHz, D 2 0 / N H 3 (pH 7.2)) of the pyrophosphate fragment in NADP +.

p p m

bound to the enzyme dih3,drofolate reductase [20,21], but for NADP + the shift appears to be reversed with respect to the enzyme-NADP*-folate complex [21].

]JC spectra were performed in the same conditions used for proton and phosphorus spectra. The signal assignments (Tab]e V), obtained by heterormclear two-

P - 5 " N P - S ' A

ppm - 10.1 - 10.2 - 10 .3 - 10.4 31 Fig. 5. P-NMR spectrum (121.4 MHz, D~O/tetraborate (pH 9.3}1 of the py~phosphate ffagmel~t in NADPH.

I - 1 0 . 5

55

TABLE IV

P,H P,C and P,P coupling eonstunl values for NADP +, NADPH wrd dimer XI

J values are measured in Hz from IH, 3lp uod I~C spectra and are given without sign:, estimated accuracy ±0.1 Hz unlegs specified,

NADP+ a NADPH b XI h

J(P.HI P2',A2' 7.1 6.8 6.8 PS",A4" 2.0 2.0 2,0 Ps",As' 5.0 S.2 5.0 PS",AS" 4.9 4.8 5.0 PS'.N4' 3.0 1.7 1.7 PS',N5' 4.2 5.0 5.0 PS',N~" 5.0 s.6 5.2

/(p.c) P2'.hl" 5.5 8.0 8.5 ~ P2'.A2' 5.0 4,2 4.2 ¢ P2'.A3' 4.5 2.0 PS",A4' 8.5 9.0 8.5 c PS',A5" 5.0 5.0 PS',Na' 8.4 8.2 8.5 ~ PS',N5 ~ 5.2 6-0

J(P,P} 20.5 ~r0.0 20.0

a Dz O solution (pH 7.2}. 0.15 M sodium tctraborate buffer in D~O (pH 9.31. The coupling coaslanls for adeaine-nbose fragment and J(P5'N4"; ~',¢,,. also measured in D20 /NH ~ solution fpH 9.3) and show similar values. These cv.plings were obtained onty [rum D~O/NH~ solution.

Conformation of ribose rings An inspection of Tables i -V shows that coupling

constants and chemical shifts of dimer X! are equal to those of NADPH, except for the nuclei involved in the ring-ring junction; in addition, the coupling constant values are similar Io those of the corresponding mono- nuelcotides 5'-AMP and NMN [3,7,23,24].

The ribose rings in these mononucleotides have been reported to exist as an equilibrium mixture of C(2" )-undo (S-type) and C(Y).endo IN-type) conformers; the same conformations have also been suggested for the coen- zymes in solution I3,23,24,25]. We have calculated, fol- lowing tLe method of Altona et al. [26-281, the relative population of the S-type vs. N-type conformers, for dimer XI, NADP +, NADPH and, by using the coupling constant values of the literature [23,24], for the mono- nuclcotides 5'-AMP, NMN +, NMNH (Table VI). A large number of X-ray data give averaged values of the pseudorotational angles PN = 9 ° and Ps -~ 162% Calculated J values from model geometries of PN = 9* and Ps ~ 153" are in agreement with our experimental values of J (HI ' ,H2 ' )+J (H3 ' ,H4") (9.6-9.8 Hi 0. This sum for NMN* and for the nicotinamide fragment of the oxidized coenzyme is 7,8 and 8,2 Hz, respectively, because the substituent effect of the positively charged nitrogen atom directly attached to C-I ' decreases the

dimensional correlation, are in agreement with those reported [4,22] for the coenzymes; only NS' vs. AS' are reversed for NADPH. When tetraborate solution is used, NADPH and dimer XI display dcshielding effects on NI' , N2' and NY of 3.5-6.5 ppm, which are signifi- cantly larger than those found for the corresponding protons and for phosphorus. At present we do not yet co have a definite interpretation of the effect being studied, A6 but from preliminary results it appears that it is due to A2 the borate ion mid not to the counter-ions Na + or A4 A8 NH~, An investigation on the effect of these and other hi2 counter-ions is in progress. N6

Several carbon-phosphorus coupling constants for AS ribose nuclei were also measured (Table IV). Of partieu- N~ lar interest are the three-bond interactions of A4' and N3

NI ' N4' with the vieinal phosphorus atom of the diphos- AI' phate chain never determined before for the coenzymes. A4" The coupling constant values were calculated from the N4' experimental a3C spectra, since there are second order A2'

N2' effects. For instance A4" and N4' patterns of NADPH N3" and dimer XI are triplets of 4.0 Hz. The same signals A3' for NADP + arc doublets of 9.0 Hz. The strong degener- NS' acy in the former cases is again a consequence of the AS' coincident shifts of P-5'N and P-5"A. The three corn- N4 pounds show similar values for these couplings (8.2-9.0 Hz).

TABLE V

~'C chemical shift values for NADPH and dimee XI

In ppm (8) from exlemal DSS~ accuracy _+0.01 ppm The assign- ments were performed by heterouuduar shift-correlated twoMimen- ~ioual experiments.

NADPH XI

(a) (h) (a) (h)

175.33 175.55 175.61 174.67 158,16 158.29 157.98 158. I ', 155,35 155.39 15524 155.37 151,62 151.59 15t.54 151.63 142.75 142.82 142.61 142.78 140.79 141.13 13"].47 137.98 126.61 127.13 131.55 132.13 121.33 121.47 121,18 121.(13 107.83 107.80 105.71 105.96 102.73 102.1.'1 t05.61 105.60 97.58 101.07 97.14 100.61 89.49 89.67 89.16 8%28 85.48 85.63 85.52 85.62 84.93 85.57 84.75 85.62 78,99 79.31 78.98 79.18 73,36 80.22 74,64 81÷12 73.10 78.69 72.95 78.52 72.65 72,76 72.76 72.86 68.33 68.33 68.38 69.02 67.88 67.94 68~03 68.07 24,48 24.58 41 ~63 41.1~4

' DzO/NH J solulion (pH 9.3), h 0,15 M sodium tgtrgbQrate buffer in D20 (pH 9.3).

56

TABLE V1

P~putaOuns (%) olcon/ormees for NADP ", NADPH~ dimer XI and for Ihe corresponding monunurleot~des

The populations were ohtained from the experimental coupling constants of Table il and Ill; for the monucleotides 5'-AMP, NMN + and NMNH, the experimental values of the literature [23.24] were used. The calculations were performed by solving the 0ppropriate equation (see Materials and Methods). Estimated actor :1: 5~ unless specified.

Compound Fragmenl Ribose a Around C(5')-O(5') h Around C(4') C(5') ~ H(4')-C(4')~C(5') ~O-(5")-P d

S #' P' #" #- C v' v- w ~ P'-Y"

NADP + A .58 80 75 12 13 62 36 2 54 N 85 79 76 15 9 88 It l 81

NADPH A 52 87 75 12 13 63 34 3 54 N 94 76 72 15 13 50 37 13 46

Dimer XI A 56 ~ 75 13 13 59 34 7 54 N 87 80 ")4 14 13 48 36 15 46

5'-AMP e 73 ~ 78 ~ I t 11 70 ~ 28 2 NMIq * f 93 8t l0 9 91 K 7 2 NMMH t 90 78 1l II 57 s 24 h l~ ~

47 67 47 36 44 36

' Population calculated following the proceduxe of Altona el aL [26-28l and assuming pseadorotatioaal angles Ps 153° and P~ 9 ° and puckering angle ~= 37 0.

b The ~8 t population in the first column was calculated from J(C4'-PS') hy using Eqa. 2 and J values [31] of 10.0 Hz and 2.5 Hz for angles of 180 ° and 60". respectively. The populations of the.6' conformers in the other colursns were calcolal~ by using Eqn, 3 and J values [31] of 23.0 Ha and 2.4 Hz for angl~s of 180 ° and 60*~ resp~:tivdy. The populations of the T conformers were ¢-alcuiutod by using Eqn. 3 and the "non~:lassicar model angles [29.301 y+ 53 °, ¥' IS2* and Y- 292 °.

d Percemage or w conformation [33,34] for this fragment. Obtained from 'J(H4'-IPS"), assuming a maximum coupling og 3.7 Hz [30] and zero coupling for non-W geometry.

r Calculated from the ¢xperaneatal coupling constants at pH 8,0-8.3 123.24]. In agreement with the reported values [23.24l: 67, 85, 65. 93 and 505. respectively.

h These values derive from estimated coupling constants [23].

value o,*" J ( H I ' , H 2 ' ) . For these compounds, the calcula- tions were thus performed omit t ing this coupling.

The puckering angle ¢,~, assumed to be equal for N- and S-type conformation, was est imated from J (H2 ' ,H3 ' ) . The high values (4.9-5.5 Hz) of this cou- pl ing indicate a low puckering ampli tude of about 37 o, which is in agreement with the majori ty of the X-ray da ta (¢b m = 36-38 ° ) [26]. Thus, with the values Pr~ = 9 ° , Ps = 153° and em = 37¢, we can calculate the molar fraction of the S-type puckering mode. Actually with a Ps value of 153 ° the S conformations are intermediate between the C(2')-eado =E and the twist ~T. The results are reported in Table VI

The eonformational equil ibr ium for the adenine- ribose ring results a 50% mixture of the two puckering modes (except for 5 ' -AMP with a 75% of S), whereas for the ribose adjacent to the pyridine moiety the popu- lation of the S conformer increases to 85-95%.

Conformation about bonds C{5')-0(5') and C{4')-C(5') The proton-proton and proton-phosphorus coupling

constants invohdng H-4', H-5' , H-5" and P atoms have never been measured for these coenzymes [5,29-31]. The P,H three-bond couplings for dinucleotides were generally est imated from the line width of phosphorus signals [7,20,2I], which are always very close to each other, and oflen have short T z relaxation times, leading to a considerable broadening [20]. Actually, the eonfor-

mational flexibility about these bonds makes the s tudy of the pyrophosphat¢ chain very difficult in solution. In the s~lid phase, the conformat ions of the backbone chain for N A D + [16,18] and for several coenzym¢ pro- tein complexes [19] have been determined. The orienta-

tion about C ( 5 ' ) - O ( 5 ' ) and C ( 4 ' ) - C ( 5 ' ) lies in the trans (,/] t) and gauche-gauche (7 + ) range, respectively, for both A and N unils, conformat ion which is usually considered as preferred for 5 '-nuelcotidas [19]. A similar

"ON~ : ( b r i d g e )

O s ' o I

-~-- C$' _ base

Scheme IL Mononucleofide fragment,

57

conformation has also been suggested for the coen- zymes in solution 17].

The complete set of couplings for the nuclei of the pyrophosphate chain is given in Tables II and lIl. From the values of J(P, HS'), J(P, HS") and J(P,C4') we derived the relative populations of the three main con- formers around C(5'}-O(5') (j8 conformers), and from J(H4',H5'), 3(H4",HS") and aJ(P.H4") those aronnd C(4')-C(5') (7 conformers). The assignment of H-5' at low field with respect to H-5" was based on the results of stereospecifie partial deuteration of adenosine and its derivatives [32]. With this assignment, we have calcu- lated from the experimental coupling constants that conformer 7 is always very little populated, for both A and N fragments. This result is in agreement with the finding, from a large number of crystal structures, that this rotamer is rare [19].

For the relationship between angles and P,H vie'real couplings we have used the Karplus/Altona equation reported in [29]. The relative populations of the three conformers Bt(trans), ~+(gauche) and #-(gauche) were then calculated following usual procedures (see Materials and Methods) and by means of the limiting coupling constants values Jl~ = 23.0 l-k. and J~ = 2.4 Hz, which are supported by m~ny experimental data 1311. The populations of the minor conformers (#+ and /~-) could be obtained, as the individual coupling con- stants of phosphorus with H-5' and H-5" were both available.

The population of the preferred conformation, ,8', was independently deduced from an other parameter: the carbon-phosphorus coupling J(P, C4'), which is par- ticularly significant, since it is dose (8.2-9.0 Hz), for

H ~ H s . H ~ C 4 + C ' H~'

I C~' Hs' Hs"

[$t ( I r , m n * ) l a p l i - [o=uche)(-=© I p* {g.uuc=he}l',~.c=l

- - ~ r - - - - - ~ / - - - - - ' 3

HS' H4' Hs" Os' k14' Hs' Hs" 1~14, Os'

7 + (gll)[*==l ,ft {mr)lap] ¥-(qu} l -*©l Scheme IIL Newman projeclions along the backbone angles ,0= C(4")-C..(5")-O(5')-P ,~ad ',t=CA3')-C(4")-CO')=O(5'). Atomic nernbering conforms with the IUPAC-[UB convention, as ~[erenced

by Altona [30],

both coenzyrnes tad dimer Xi, to the maximum value (10 Hz) observ+;c for truss interactions i . . u d ~ i d ¢ ~ [311. The population P(/]~) was calculated by using the limiting coupling constant values J, . . . . --10 Hz and J , . . c ~ ~ 2.5 Hz [311.

As shown in Table V], both adenine and nicotin- amide fragments display a preference of 75-80% for conformer ~t. and the results from proton and carbon data are in agreement, The minor ,.onformers/]÷ and . ~ are equally populated, except for the N fragment of NADP +, where ,8+ is twice/~-.

The conformation about C(4')-C(5') bonds was studied by using the 'non classical' model for the three main conformers, as suggested by Altona et al, [29,30], i.e., 7 +_ 53 °, -i, t = 182 ° and 7 - = 292 °.

The relative population of the thcee conformers were thus obtained (Table VI) and compared with those similarly calculated from the experimental data [2324] of mononucleotides 5'-AMP and NMN. The adenine fragment showz a constant behaviour, with a preference for conformer ~,+ (59-63%) and -r t (34-36%}. Con- former "t- is pratically absent or shows a very low population, in agreement with the expectation [191. The nicotinamide fragment instead varies from a high pref- erence for isomer 7* in NADP + (88%) to lower values (about 48-505) in NADPH and dimer XI, with a consequent larger participation of the minor conformers 7 ~ and y-. The values of mononueleotides are very similar: y+ 70% (5'-AMPL 90% (NMN +) and 57% (NMNH).

These results are also confirmed by the values of the long-range couplings (J(P, H4'). The maximum value of this interaction, corresponding to a W geometry for the fragment H(4 ' ) -C(4 ' ) -C(5 ' ) -P [33,34], has been taken equal to 3.7 Hz, with an average of experimental co-- piing constants of 3.3 Hz [30]. Among the compounds studied, NADP ÷ shows the largest values, with a 3.0 Hz coupling for the nicotinarnide fragment, in agreement with the population found for ¥+ and .B ~ conlormers. The lowest value (1.7 Hz} occurs for the same fragment of NADPH. Assuming n maximum coupling of 3.7 Hz and a zero coupling for all "non-W' geometries, we have calculated the 'percentage of W conformation" (Table VI), which are in line with the product p t ~+ obtained from the vicinal couplings.

Conformation of the dihydronicotinamide ring in NADPH and in the 4,4- and 4,6-dimers

A conformational analysis of the tetrahydrobipyd- dine system of the 4,4- and 4,6-dimers must consider the puckering of the dihydronicotinamide ring and the rotation about the C(4)-C(4) and C(4)-C(6) bond, re- spectively, at the ring-ring junction.

In the case of 4,4-dimers, the proton-proton interne- lions J(N4,NS) and J(N4,N6), are diagnostic for the ring puckering, whereas the rotational process affects

58

',he coupling at the junction site, J(N4,N4). It is inter- esting to note that "~J(N4,Nr) differs greatly from di- merle to monomeric ~pecies, being 1.7 and 1.9 Hz for NADPH and 0.5-0.7 Hz for XI and the other two diastercoisomers [1]. As a transoid aUylic coupling shows its maximum value (3.0-3.5 Hz) when the dihedral angle ~,= H(4)-C(5)-C(6)-H(6) is about 90 °, and a very small magnitude for angles near 0 ° [33], we can deduce that the N4 protons of the dimers are predomi- nantly in pseudo-equatorial orientation, with z, angles of not more than 200-30 °. This corresponds lo the boat Bi. 4 and lAB conformations for the 4R and 4S configuration, respectively. For axially oriented N4 pro- tons, • angles measure 100", which should lead 1o a coupling of about 3 Hz. A planar structure of the 1,4-dihydropyridine rings, or flexible forms inlercon- verling between two limiting boat conformations, are e~pected to give allylic coupling of 1.5-2.0 Hz. Conse- quently, the values of 1.7 and 1.9 Hz found for the geminal N4 protons of NADPH are in agreement with a neatly planar ring, or with a flexible structure (averaged couplings) rapidly flipping between two boat forms. The energy difference between planar and boat confor- mation has been reported to amount to only 2.7 kJ. m J [35]. Thus, the co~,clusions drawn by Kaplan etal . [8] that the dihydropyridine ring of the reduced coenzyme NADH is locked in a puckered conformation, are not consistent with our results. These authors utilized a 0.:5-0.8 Hz difference in coupling cone:ants, which in addition were obtained from not enough resolved spec- tra of deuterated materials. An examination of the vicinal coupling J(N4,NS) confirms, for 4,4-rimers, the equatorial preference of N4 protons, since values of 5.0-5.5 Hz exclude ~.ngles of 90-1(10 °. The geminal N4 protons of NADPH show coupling with N5 of 3,1 and 3.7 Hz: the small difference again cannot be taken as evidence for a preferred ring puckered conformation [8]. The non-equivalence of N4 protons is a consequence of the presence of chiral centers, which makes the two nuclei diastereotopic.

The c, lher ;we |oug-range couplings are not very informative; J(N2,N6) shows for the coenzymes and 4,4-directs similar values, as expected, which are in agreement with a W geometry [33]. J(N2,N4) is not a pure allylic interactio,l, because the conjugative effect of CONH2 group increases t h e , contribution to the cou- pling mechanism with respect to the ~r contribution. The vlnylogous amide delocalization, as shown in the structure of Scheme 4, is considered to be dominant, with respect to the amide delocalizalion, in NADH [36] and in the 1,4-dihydronicotinamides [371. In addition, the effect of the electron-withdrawing substituent, at the central atom of the coupling pathway, is strongly de- pendent on the orientation of the substituent. The dif- ferent values found for the three dimers [1], ranging I¥om 0.5 to 0.9 Hz, clearly retlect different steric situa-

NH2

[ C

[ R

Scheme IV. Coplanar tr,,nsoid eonformaticm of lA-dihydronicolina- mides, with vinylogc~Js amide delocalization,

lions, which however are difficult to evaluate. Actually the problem of the preferred conformation of the amide group for NADPH in aqueous solution is still an open question [36,371. A coplanar t r an~ id conformation as shown in Scheme IV has been established for N-sub- stituted 1,4-dihydronicolinamides in solid phase [17]. The same conformation has also been suggested [37] for NADH and NMNH in solution, on the basis of ultra- violet absorption and fluorescence data, and of the chemical shift difference between N2 and N6 protons in model compounds. The deshielding (about 1.0 ppm) of N2 vs. N6 for NAPDH, as well as for the three 4,4-di- mers, might indicate that the oxygen atom of the amide is preferenlially oriented towards N2.

Molecular mechanics calculations showed that the rotation around bond N4,N4 is easy, when the dihydro- pyridine rings of 4,4-rimers assume the boat conforma- tion with H-4 equatorially oriented becanse the two rings remain approximately parallel. On the other hand the rotation becomes hindered in the ease of axial orientation. Preliminary results of these calculations also showed that the conformations of the rings are preferentially boat, instead of planar, and the H-4 pro- tons are equatorial, in agreement with experimental data. The small values of the vicinal coupling at the ring-ring junction, J(N4,N4), are in favour, for the three isomers, of gauche conformations, the dihedral angles • = H(4)-C(4)-C(4)-H(4) having values of about 60 °. Significant populations of other forms with u an- gles between 120 ° and 180" can be excluded; the eclipsed forms with o = 120 o although compatible with NMR data, are energetically disfavoured. Thus a dy- namic mixture of gauche conformations, or a single gauche, with boat forms of the rings, are the most preferred. This may lead to the stacking of the two dihydropyridine systems, enough to, allow possible z, orbital interactions between them. The relative popula- tion of the two gauche forms (Scheme V) can vary in the three 4,4-directs, and this accounts for the larger difference in chemical shifts found [1] for N4 than for the other protons.

59

H-

c ; ' ~ c s :

['14 . . - C a ~ . g'°

i c, '~- ' -c~

4S,4R dimers

g,

cZY'C~ !

H4

r . . . ' C ~ H4

4S,4S dirnere

H4 H4

N. ~ '" "~"~N--

48,6H dimeri Scheme V. Newman projection ~f the gauche forms for some diraers,

along the junction bond betv,ee. 'the two dihydtop~fidine ziags~

The 4,6-rimers, described in the preceding paper [1], contain both the 1,4-dihydro and tl3e 1,6-dihydro- pyridine systems. The couplitlg constants for the former are very similar to those found (or Xl and its di- astereoisomers, thus indicating a boa[ conformation with H-4 equatorially oriented. The J values are symmetri- cally comparable to those of the partner 1,6-dihydro- pyridine ring (see Table V of Ref. 1); in particular, the nearly zero values of the ailyLic interaction 4j(4B,6B), the low values (2,3--3.8 Hz) of the coupling constant at the ring-ring junction, and the 5.4-5.6 Hz of the three- bond coupling (J(SB,6B). This shows that the confor- mation of 1,6-dihydropyridine rings is a twist-boat, with N6 protons equatorially oriented, and that the rotamers about N(4)-N(6) bond at the ring-ring junction are preferentially gauche (Scheme V).

Coaclnsiens

The preferred conformation of the dihydronico- tinamide rings in solution, for the dimeri¢ species studied, (NADP)2, is a puckered structure; i.e., a boat form for the 1,4-dhhydro- and a twist-boat for the 1,6-dihydro-rings, with equatorial orientation of both protons at the ring-rlng junction. The rotation about

the b~-'td at the junction site is not hindered, but gauche co~:ormations are preferred. This allows some stacking of the two dihydropyridine units.

In the reduced coenzyme NADPH, the dihydronico- tinamide ring is planar or rapidly flipping between two boat forms. Recently [1"/] the crystal structure of N-sub- stituted dihydronicotinamides, models fat the ~e~- zymes, was reported to be practically planar. Since the energy difference between planar and boat form amounts only to 2.7 kJ-mo1-1 [35], a highly flexible conformation appears the most favoured in solution. Although the NMR data cannot distinguish between planar and interconverting boat structures, the puck- ered conformation given for NADPH by Kaplan et at. [8] and generally accepted [5,15,17], can definitely be excluded.

The reduced coenzyme NADPH and the 4,4-dimer examined~ atthough different in the form of the dihy- dronicoPmamide ring, are similar in the ribose struclure and in the conformation about the C(4')-C(5") and C(5')-O(~') bonds.

The conformation of the ribose rings consists of an equilibrium mixture of C(2').endo(S-type) and C(3")- eado (N-type) families of conformers, as suggested by Sarma et al. [3,23]. The two puckering modes have approximately equal populations for the adenine-ribose in NADP +, NADPH and in the 4,&dimer (S 52-585), Whereas the nicotinamide-ribose shows a significant in- crease (85-94~) of the S-type. The mononucleofides NMN ÷ and NMIqH behave in a manner analogous to the corresponding units in the coenzymes, whereas 5'- AMP shows 75~ of S. The increase of C(2')-endo puck- ering does not appear to be correlated with the positive charge on the aicotinamide nitrogen, but rather it should be related to the geometry at the glycosidic bond and to the nature of the attached base. This correlation has mainly been postulated on the basis of X-ray data and energy calculations [19]; if this is true also in solution, we should expect a larger population of syn conformer for the pvridyl than for the adenosyl moietyl. Actually, the syn and anti conformers were found to be equally populated for the pyridyl fragment of HAD + in solu- tion [12,13], while definite and unambiguous evidence of a presumed [24] anti preference for the adenosyl moiety, is still lackin& An attempt to delermine the X angles by NOESY technique was unsuccessful because the correlation time of dimers in water lies in the region where the NOE effect is close to the zero-crossing point between positive and negative NOE.

As concerns the rotation about the C(5')-O(5') ester bond, all the compounds studied display the same high preference for copra .,~..,~/~+ (75-80%), in agreement with the X-ray analyses of HAD + [16,18] and of the majority of nucleotides [19|, where angle # is largely limited to the ap (trims) range. However, the minor gauche conformers ~+ and ~- at : present in solution

60

and are equally populated, except for the nicot inamide fragmenl of N A D P +, where [/+ is about twice/~- .

The rotamers about C14 ' ) -C15 ' ) are distr ibuted be- tween -r + (48-69%) and yt (28 37~), with a preference for the gauche conformation. The preference increases to about 90% for the nicol inamide fragment of N A D P + and for the corresponding monomer N M N +. This is no1 related to the ribose puckering, since N A D P H and N M N H , with the same puckering mode. show ealy 50~ of conformer 7++ Actually, the 3' angle does not seem to be correlated with any other angle 119], contrary to what

has been previously suggested [24t; rather, the type of base seems to play a role in stabil izing the ~,+ form, expecially when electron-wilhdlawing groups are pre- sent on the base [19]. These results confirm the above predictions: the posil ive charge on 1he nicot inamide ring appears to be responsible of ~he stabilization of the 7 + conformer, possibly inducing electrostatic attractive

interactions between the base and lhe phosphate group, also mediated by water molecules.

That these interactions occur, has also been pos- tulated [40] on the ba i l s of the atp npfield ~hift gener- ally observed in the oxidized dinueleotides and mono- nucleotides, wi th respect to the reduced forms. Now the assignment of ~lp signals es(ablishes that the most af-

fected of the two is indeed the phosphate in the nicotin- amide unit. However, i t is known [311 that strong changes in 31p chemical shift are due to variat ion in the a torsion angles about the P-O{5 ' ) ester bonds, as welt as to variat ion in the bond angles at 1he phosphorus atoms; variat ions that might also be induced by electro- static interactions. Several data are available from X-ray analyses [19], but in solution the only information on these angles comes from chemical shift values.

In the pyrophosphale chain, an addit ional and im- por tant factor are the torsion angles a t the P O(bridge); these angles have been considered to be the main source of conformational flexibility for these coenzymes, in

which the two nudco t ide units have always been seen as rigid entities 119]. This model should probably be re- vised as well as the old que,',tion of whether these coenzymes exist in a folded or extended form, and if so to what extent, After the papers of Ellis et al. [12331 this problem has no longer been tackled, and is still wait ing for an unambiguous answer.

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