Binary and ternary complexes of metal ions, nucleoside 5′-monophosphates, and amino acids

.I morg nucl ('hem, Vol, 42, pp. 785-792 © Pergamon Press Lid, I980 Printed in Great Britain

0022-1902]801050141785,'$02.00/0

BINARY AND TERNARY COMPLEXES OF METAL

IONS, NUCLEOSIDE 5'-MONOPHOSPHATES, AND AMINO ACIDSt JAMES B. ORENBERG,L BEDA E. FISCHER and HELMUT SIGEL Institute of Inorganic Chemistry, University of Basel, Spitalstrasse 51. CH-4056 Basel, Switzerland

(Received 17 July 1979; received for publication 10 September 1979) Abstract--The aromatic-ring stacking interactions between the indole moiety of L-tryptophan and the purine or, pyrimidine moieties of AMP 2 or CMP ~'+, respectively, were studied by ~H NMR spectroscopy. Under the influence of increasing concentrations of _L-tryptophan the resonances of H-2, H-8, and H-I' of AMP 2- and of lq-5 and 14-6 of CMP 2- are shifted upfield. Computer curve fitting of the shift data gave the stability constants '~'~P' Tro~--6.83 M I and K(CMPIIH (CMP) Trp) =0.77±0.35 M w(in D,O: K~,,up~m 27°C; I =03 M) which show that the ± 0.81 _ purine moiety forms the more stable stacks. Repetition of the experiments under conditions where the amino acid exists as the anion, i.e. as tryptophanate, gave KIAMP~TroI=2.24±0.29M-I AMp CMP _ + and KICMPI~Trp~-0.14-0.05M (in D,O; 27°C: I = 0.14).15 M). Hence, it is evident that the formation of an ionic bridge between the negatively charged phosphate moiety of the nucleotides and the positively charged ammonium group of _L-tryptophan favors the formation of the stack between the nucleic bases and the indole residue. In the case with AMP 2 the stability constant of this ion pair formation could be estimated, i.e. Kw=0.6 M ~ (in D20; 27°C; I =0.14).15 M), a value which compares well with Kw = 1.35 ±0.76 M ~ for the (CH3hN+/Trp - interaction, lntramolecular equilibrium constants for the ion pair interaction within the stacked complex and for the stacking interaction within the ion pair complex are also estimated and discussed. Stability constants of metal ion complexes could only be determined with Ni 2+ as a precipitate formed with Cu 2+ and Zn -'+. The stability constants of the binary systems, Ni2*/AMP 2 , Ni2+/CMP 2 , Ni2+/Trp , and Ni2+/Ala -, were determined by potentiometric pH-titrations. The somewhat enhanced stability of Ni(Trp)2 compared with Ni(Ala)2, and of Ni(AMP)~ compared with Ni(CMP)~ (for which no evidence of formation was observed) is explained by self-stacking within these binary 1:2 complexes. Based on the data of the binary systems, the following ternary systems were studied and the corresponding stability constants determined: Ni-'+/AMP -" /Trp , Ni2+/AMP 2 /Ala-, Ni2+/CMP2-/Trp , and Ni2+/CMp2-/Ala . A most likely intramolecular purine/indole stack in the ternary Ni(AMP)(Trp)- complex is not significantly reflected in the measured stability constants. This observation and the implications of stacking with regard to the stability of ternary complexes, as well as the general importance of "bridged" stacking adducts for biological systems are briefly discussed. INTRODUCTION Specificity leading to selective and preferred interactions is obviously important in biological systems. As far as species containing a metal ion of the 3d series are concerned, it is now well established that among mixedligand complexes those composed of the rr-accepting imidazole group of the histidyl residue and an O donor are preferably formed [4, 5]. Certainly another possibility favoring specificity and selectivity are stacking interactions between aromatic residues [2].

+This is Part 33 of the series "Fernary Complexes in Solution" (published by H. S.); for Parts 31 and 32 see [I] and [2], respectively. Simultaneously it is Part 2 of a series (published by JB.O., who was in Basel until the end of 1978 on sabbatical leave from the Chemistry Department of San Francisco State University) on "Coordinating Systems in Biochemical Evolution"; for Part I see [3]. :]:Permanent address: J. B. Orenberg, Department of Chemistry, San Francisco State University, 1600 Holloway Avenue, San Francisco, CA 94132, U.S.A ~Abbreviations: AA, amino acid; Ala, _L-alaninate; AMP, adenosine 5'-monophosphate; ATP, adenosine 5'-triphosphate; CMP, cytidine 5'-monophosphate; En, ethylenediamine; L, general ligand; M, general metal ion; NMP, nucleoside 5'-monophosphate, i.e. AMP and CMP; NMR, nuclear magnetic resonance; L-Irp, tryptophanate. Hit may be added that glycinate seems less suitable as a reference because its complexes show a somewhat exceptional stability 1141.

As one may expect that specific interactions, for example between nucleotides and amino acids, played a role during the first steps of biochemical evolution, we felt that it would be of interest to study the stability of simple ternary metal ion/nucleotide/amino acid complexes in order to see if certain ligand combinations are favored and if any selectivity is achieved. The occurrence of stacking between the indole moiety of tryptophan or other indole derivatives and the nucleic bases of nucleosides or nucleotides in aqueous solution is well-known [6-11]. In fact, such interactions are expected to manifest themselves in certain ternary metal ion/nucleotide/amino acid complexes in the formation of intramolecular ligand-ligand "bonds". Indeed, it has already been observed that these intramolecular stacking interactions in mixed-ligand complexes may enhance the', stability of such species[12], e.g. M(ATP)(Trp) 3 complexes [13].§ We thought it worthwhile, therefore, to study the,. stability of the ternary metal ion complexes formed in the mixed-ligand systems consisting of AMP 2 or CMP 2 , and tryptophanate or alaninate. Alanine was selected as a reference amino acid because very little if any ligand-ligand interaction in the ternary nucleotide complex is expected.ll As a pre-condition for our objective to study ternary complexes, a detailed knowledge of' the binary systems was necessary, i.e. Trp/AMP, Trp/CMP, metal ion/nucleotide and metal ion/amino acid, which were therefore also investigated. The stability of the metal ion containing complexes was deter785

J.B. ORENBERG et al.

786

mined by potentiometric pH titration, while the pure ligand systems were studied by JH NMR.

EXPERIMENTAL PROCEDURES

Materials. The metal nitrates (zur Analyse) and .L-tryptophan (fiir biochem. Zwecke) were obtained from Merck AG, Darmstadt, Germany; the concentrations of the metal stock solutions were determined by titration with ethylenediamine-N, N, N', N'-tetraacetate. The disodium salt of AMP (pentahydrate; puriss.) and _L-alanine (puriss.) were from Huka AG, Buchs, Switzerland, and the disodium salt of CMP (reinst) was from Serva Feinbiochemica GMBH, Heidelberg, Germany. tH NMR measurements. The tH NMR spectra were recorded on D20 solutions With a Bruker WH-90 FF spectrometer (90.025 MHz) at 27°C, using the center peak (triplet) of the tetramethylammonium ion resonance for measurements at pD 8.4 as internal reference; all these chemical shifts were converted to a trimethylsilylpropanesulfonate reference by adding 3.188 ppm [12]. For the measurements at pD 12.4 the resonance of dioxane was used as an internal standard, because the resonances of (CH3)4N+ were shifted upfield under the influence of tryptophanate; again all chemical shifts measured were converted to a tfimethylsilylpropanesulfonate reference by adding 3.764 ppm. The pD of the solutions was obtained by adding 0.40 to the pH meter (Metrohm potentiometer E 353 using a Metrohm glass electrode EA 125) reading [15]. The pD adjusted in the solutions was selected by taking into account the isotope effect observed for the deprotonation of H2PO4- and H(ATP) 3- [16]. All the other experimental details are given in the legend to Fig. 1. The experimental results were analyzed using a Hewlett-Packard 9821A calculator connected to a HP 9862A plotter.

Determination of equilibrium constants by potentiometric pH titrations. The titrations were carried out with a Metrohm Potentiograph E 536 using Metrohm EA 121 glass electrodes (25°C). The acidity constants of H2(NMP) and H(NMPy, i.e. KH~NMP)H and KHtNMP;, were determined by titrating 50ml of aqueous 1.8x10 -J M HNO3 and KNO3 (I=0.1 M) in the presence and absence of NMP 2- (I.2 x I0 -3 M) under N2 with 1 ml of 0.1 M NaOH. The differences between such a pair of titration curves were measured and the two overlapping acidity

0.15 ppm upfietd

A

/AM,P I+2 /

0.10 005

7/~ / /

/AM,PFFf

, ~ A M , P H-8

_~,,,: ~, _oo, T . . . . .

u0

constants were calculated from the relation between pH and neutralization degree. The conditions for the determination of the stability constants K~i(NMP)were the same as for the acidity constants, but a part of KNO3 was replaced by Ni(NO3)2 with the ratios of NF+: NMP = 2:1, 4:1, 8:1 and 12:1. The stability constants K~ItNM,) of the binary complexes were computed by taking into account the species H +, H2(NMP), H(NMP)-, NMP 2 , Ni 2+, and Ni(NMP). For CMP as ligand, the stability constants that were calculated on this basis did not show a dependence on pH, i.e. the complex Ni(CMP)~- does not form under the present conditions in observable concentrations which is in accordance with earlier investigations[17]. For AMP as ligand, the additional species Ni(AMP)~- was considered by using the value log K Ni Ni(AMP } -Ni AMP _ log KNilAMPI20.18 from the literature [17]; then the calculated stability constant for Ni(AMP) showed no dependence on pH. It should be noted that the concentration of Ni(AMP)~- was always less than 12% of [Ni(AMP)] and less than 5% of [AMP]tot. The acidity constants K~tAAI of the amino acids were determined by titrating 50 ml of aqueous 2 x 10 4 M HNO3 and KNO3 (I = 0.I M) in the presence and absence of H(AA) (1.2 x l0 -3 M) as described above. Again the difference between such a pair of titration curves was used for the calculation. The conditions of measurement for the determination of the stability constants, Ni KNitAA), were the same as for the acidity constants, but KNO3 was partly replaced by Ni(NO3)2 to give ratios of Ni2+: AA = 1: l, 2:1 and 3: 1. Titrations of solutions without ligand were used as a basis for the evaluation. The calculation of KNitAA) N~ was done by taking into account the species H +, H2(AA)+, H(AA), AA-, Ni2÷, Ni(AA) +, Ni(AA)2 and Ni(AA)3-. To enable this calculation procedure, previously titrations had been carried out with ligand in excess, i.e. Ni(NO3)2 was 6 x 10 4 M and the ratios of Ni2+: AA were 1:5, 1 : 10, 1:20 and I: 40. From these data K Ni(AA),NIxlZ i NiIAA)Ni(AA)2 and KNi}AAI3 Ni AA2 were calculated in the usual way. The stability constants given for Ni(AA) +, i.e. l o g KNi(AA), Ni are the average values obtained from those titrations where Ni 2+ was in excess, as well as from those titrations where the amino acid was in excess. The conditions used for the titrations of the mixed-ligand systems were similar to the binary ones: [HNO~] = 2 × 10-4 M and [Ni(NO3h] = 1.2 × 10-3 M. The ratios of the reactants were

0.05 [H(Trp)]

~ 0.05 [Trp-]

AM,P

14-2

AM ,PH-I'

AMP,H-e CM,PH-5

O.lOM

Fig. !. Upfield shifts of the resonances of H-I' (0), H-2 (ID) and H-8 (~) of AMP2 (0.01 M full lines) and of the resonances of H-5 ((I)) and H-6 (O) of CMP 2- (0.01 M dotted lines) which show a dependence on increasing concentrations of .L-tryptophan, compared with the resonance positions in the same nucleotide concentration alone at pD 8.4 (A/I = 0.1 M, KNO3) or pD 12.4 (B]I = 0.1-0.15 IV[,KNO3) in D20 (90.025 MHz; 27°C). The curves shown are the computer calculated best fits of the experimental data with the parameters given in Table I. In case of the CMP2- systems the calculation was carried out by using an average limiting shift-difference 8o- 8~ of 0.318, i.e. of (0.413 + 0.223)/2 (see Table 1).

78"7

Ternary nucleotide/amino acid/metal ion complexes Ni:+ :AA:AMP : I: 1:1, 1:1:2 and 3:1:3, and N F ÷ : A A : C M P = I : I : 2 , 1:1:3, 2:1:2, 1:1:4 and 2:1:4. The overall stability constants,/3N,tyM~')~AA~, . Yi of the ternary complexes Ni(NMP)(AAy were computed [18] by taking into account the species H +, H2(NMP), H(NMP)-, NMP 2-, Ni(NMP), H2(AA)+ [19,20], HIAA), A A , Ni(AAY, Ni(AA)2, Ni 2+ and Ni(NMP) (AA) ; in the AMP-containing systems, Ni(AMP)~ was also considered. The differences in consumed NaOH used in the calculations were those between litrations of a solution containing only HNO~ and one containing also the ternary system.

and (CMP)(H • Trp) 2 adducts according to eqn (1) N M P 2- + A A ~ ( N M P ) ( A A ) 2K(NMP)

,NM,',,AA) = [(NMP)(AA) 2 ]/([NMP2-][AA]).

The method of choice for such a study is ~H NMR spectroscopy as it allows a definite conclusion about the'. occurrence of aromatic ring stacking in a given system [2, 12, 13]. Indeed, the purine protons H-2 and H-8 and the ribose proton H - I ' of AMP z-- are shifted upfield in D20 solution at pD 8.4 in the presence of _L-tryptophan, which is clear evidence for the formation of a stacked adduct. The observed 'H NMR spectra are'. very similar to those of the A T W - / T r p system [2]. The.. variation of the upfield shifts of H - I ' , H-2, and H-8 of AMP 2- with increasing _L-tryptophan concentration is, shown in Fig. la. Computer fitting of these data, i.e. of the shifts of H-I', H-2, and H-8, and of the reference'.

RESULTS AND DISCUSSION

Aromatic-ring stacking in the binary tryptophan/nucleotide systems To obtain a firm basis about the strength of a possible intramolecular ligand-ligand interaction in the ternary MtAMP)(Trp) and M(CMP) (Trp) complexes we studied first the metal ion free systems. The main purpose was to characterize the stability of the (AMP)(H • Trp) 2

Fable I. ~H NMR chemical shifts (in ppm) observed for the stacking between the purine moiety of AMP2- or the pyrimidine moiety of CMP 2 and the indole residue of H(Trp) or Trio , together with the stability constants (NMP) K~Mp~A.~ [see eqn (1)] of the corresponding binary aromatic-ring stacked adducts a System

AMp2-/H(Trp) pD

= 8.4

CMp2-/H(Trp)

Proton of NMP

60

U p f i e l d shift 60 - 6 ~

~

.(NMP) ~(NMP)(AA)

H-2

8.249

7.836~O.052

O.413

6.91!1.21

H-8

8.610

0.223

H-I'

6.137

8.387+0.052 -5.88910.057

0.248

{,.20+1.96 _ 6.23~1.95

(H-8) - (H-2) c O.361

O.553~0.031

0.192

7.65~1.76

H-5

pD = 8.4 H-6

6.139

5.916

0.223

~.24 d

6.139

5.726

O.413

0.65 d

8.114

7.891

0.223

O.78 d

8.114

7.701

0.413

0.41 d

AMp2-/Trp -

H-2

8.251

7.772~O.179

0.479

2.07~0.93q

pD = 12;4

H-8

8.615

8.392

0.223

1.87 e

H-I'

6.127

5.879

0.248

1.94 e

0.563~0.O34

0.198

].09~0.69

CMp2-/Trp pD

H-5

no shift

6.141

5.918

0.223

O.18 d

6.141

5.728

O.413

0.IO d

H-6

8.119

7.896

0.223

O.17 d

H-I'

there

= 12.4

8.119

7.706 seems

6.83~O.81 b

J

there

is p r a c t i c a l l y

KAverage

O.77+O.35 d --

H-I'

(H-8) - (H-2) c 0 . 3 6 5

(1)

O.413 to be a slight

2.24+0.29 e --

J

O.14+0.O5 d --

O.O9 d downfield

J

shift

aThe measurements (lAMP2 ] or [CMP 2-] = 0.01 M; see also Fig. 1) were carried out in I~O at 27°C. The chemical shifts were measured relative to internal (CH3)4N+ at pD 8.4 (I = 0.1 M, KNO0 or to internal dioxane at pD 12.4 (1 = 0.1 0.15 M. KNO~) and converted to values downfield from sodium trimethylsilylpropane sulfonate by adding 3.188 ppm or 3.764 ppm. respectively. bWeighted mean and standard error of the mean value. ~Differences in the chemical shifts between H ~ and H-2. JFor the CMP 2 systems no 6~ could be computed, as the experimentally measured shifts which are dependent on [H(Trp)] or [Trp ] show practically no curvature (see Fig. l). Therefore a stability constant K was estimated by using the upper and the lower limits of the AMP 2- systems, i.e. 80-8~ 0.413 and 0.223 ppm respectively. The "true" value of K is certainly within the given limits, which are a conservative estimate. eFor H-8 and H-I' of AMP2- at pD 12.4 the scatter of the data is too large and the curvature too small (see Fig. Ib) to get reasonable results for &. Therefore the 6o- 6~ values from the corresponding results obtained at pD 8.4 were also used at pD 12.4; this procedure is justified as the results for 6o- 6~ at pD 12.4 and pD 8.4 for H-2, and especially for the standard independent difference (H-8)-(H-2) are within the error limits identical. The value given for K is the average with its standard error.

788

J. B. ORENBERGet al.

independent shift difference 6(H-8)-6(H-2), gives a stability constant for (AMP)(H • Trp) 2of (AMP) -+ M- I KtAMPXrrTq,)--6.83--0.81 (D20; I=0.1M, 27°C; Table l). This constant agrees well with earlier determinations in similar systems[6,9] and it is within experimental error identical with the one measured recently for (ATP)(H • Trp) 4- [2]. It should be noted that under the conditions of the present *H NMR experiment ([AMP2-] = 0.01 M) the amount of self-stacked AMP2- is certainly very small: assuming that the constant for self-stacking of AMP2- corresponds to the one of ATI~-[21], which is certainly true in a first approximation [9], then less than 2% of AMP2- is selfstacked. No evidence could be found [2] in the *H NMR spectrum for self-stacking of _L-tryptophan; this result corresponds to the observations with tryptamine at pD 7.9, where only an insignificant self-association was found [9]. The corresponding experiments in D20 solution at pD 8.4 with the CMp2-/L-tryptophan system give evidence that there is still some stacking in this system. However, the chemical shifts observed under the influence of increasing concentrations of tryptophan for H-5 and H--6 of the pyrimidine moiety and of H-I' of the ribose moiety of CMP 2- are very small (see Fig. la). Computer curve fitting of the data gives a stability constant for (CMP)(H'Trp) 2- of K tcuP)(CMa){l-I-vw~=0.77-+0.35 M-I (D20; I =0.1 M, 27°C; Table 1). The rather small chemical shifts in the CMp2-/_Ltryptophan system and the low stability of the corresponding binary adduct, if compared with the results obtained for AMP2-/_L-tryptophan, correspond with the observations made for the UTW-/2, 2'-bipyridyl [2, 16] and the ATW-/2, 2'-bipyridyl[2, 12] systems. In other words, as one would expect, the larger stability of the purine adducts, compared with the pyrimidine adducts, reflects the decreasing size of the aromatic-ring systems forming the stacks. Hence, in aqueous solutions with a pH of about 7-8 and with [NMP2-] = [H. Trp] = 0.01 M, about 6% of AMP2- exist as (AMP)(H • Trp) 2-, while with CMP 2- the adduct is formed only to about 0.8%. At a pH of about 8 the nucleoside 5'-monophosphates exist as dianions, i.e. the phosphate moiety is completely deprotonated, while _L-tryptophan is under these conditions present in its zwitterionic form. Therefore, one could imagine an ionic interaction resulting between the negatively charged phosphate moiety of the nucleotide and the positively charged ammonium group of _L-tryptophan, and that this interaction influences the stability of the aromatic-ring stacks. To verify this assumption, an additional series of 'H NMR experiments was carried out in i)20 solutions at pl~ 12.4 (see Fig. lb), where the ammonium group of L-tryptophan is deprotonated, i.e. tryptophanate exists in solution, and hence no ionic pair between the nucleotides and the amino acid can be formed. Here it should be mentioned that in these experiments at pD 12.4 dioxane had to be used as internal reference, because the proton resonances of (CH3)4N+ are shifted upfield in the presence of increasing amounts of Trp . We attribute this observation to the formation of an ionic pair between (CH3),,N+ and the carboxylate group of _L-tryptophanate in D20 at pD 12.4. Computer fitting of the corresponding shift data gives a stability constant for (CH3)4N+/Trp- of Kw = 1.35-+0.76 M-' (D20; 27°C; I= 0.1--0.15 M, KNO3). The results of the NMP2-/Trp - experiments at pD 12.4

are summarized in Table 1. A comparison of the shift data for AMp2-/Trp at pD 8.4 and 12.4 shows that the maximal shifts, i.e, 6o- 8~, are for all corresponding protons about the same at both pH values. However, the computed stability constant of (AMP)(Trp) 3- is KtAMP)
Stability of the binary metal ion complexes of nucleotides and amino acids Before the stability of ternary metal ion complexes can be determined it is necessary first to study the corresponding binary metal ion systems. The acidity constants of the ligands and the stability constants of the binary metal ion complexes formed with the nucleotides or the amino acids were determined from potentiometric pH-titrations. The definitions of the corresponding constants are given by eqns (2)-(4), where L = NMP 2- or AA- and n = l, 2, or 3: H2LZ+~HL+ + H + K H2L = [HL+][H+]/[H2L2+]

(2)

H L + ~ L + H + K".L = [L][H+I/IHL+]

(3)

2+ ML~.-I~ +

L~ML~ + K ME. ML¢"

2+ -- [ML.2 + ]/([ML¢.-,d[L]). (4)

i} - -

Our attempt to carry out the measurements with Cu2+ and Zn2+ failed, because under such experimental conditions where a reasonable amount of the mixed-ligand complexes should exist, these metal ions form precipitates. This is probably due to the rather pronounced tendency for hydrolysis of these metal ions [22, 23] or the insolubility of their M(NMP) complexes. Therefore, we had to restrict our study to Ni2+, which seems biologically less meaningful, but which still allows us to evaluate the influence of metal ions on nucleotide/amino acid interactions. In this connection it should be added, that it is known that Ni 2+ does occur naturally in conjunction with RNA from various sources [24]. Its roles are not clearly established, but the possibility of the metal cation being involved in nucleic acid metabolism does exist [25]. Nickel is now also recognized as an essential trace element in higher animals [26]. In addition, it should be mentioned here that jack beans urease [27, 28] is a Ni2+ metalloenzyme. The values of the potentiometrically determined equilibrium constants of the Ni 2+ complexes are listed in Table 2; they are in reasonable agreement with those given in the literature [29-31]. It may be pointed out that the earlier observation of Frey and Stuehr [17, 32] about the lower stability of Ni(CMP), compared with the stability of Ni(AMP), is confirmed by our own results: the stability difference is a factor of about 4 (corresponding to 0.6 log unit). This difference in stability between Ni(CMP) and Ni(AMP) has lead to the conclusion [17, 32] that there is a Ni2+/N-7 interaction in Ni(AMP), which is responsible for the enhanced stability

789

Ternary nucleotide/amino acid/metal ion complexes of this complex [33, 34]. Another interesting feature of the data of Table 2 is the different stability of the Ni(Ala)2 and Ni(Trp)2 complexes. From the following comparison it is evident that the stability constant of the equilibrium, Ni(Trp)++ Trp ~Ni(Trp)2, is by a factor of about two larger (0.3 log unit), compared with the constant of the corresponding _L-alaninate system Ni K Ni,T~p~log K Ni(Trp, NiIT~p~2= 5.48 --4.92 = 0.56 log K ,ilA,,,, Ni -10g K Ni~AI~, NI~AI., -_ 5.50 -- 4.66 = 0.84.

M which was experimentally determined from potentiometric pH-titrations, is connected ,vith KMtNMP) vM~AAJ M{NMP)(AA) and 1"~ M(AA)(NMP) by eqns (8) and (9), res-. pectively.

/~M(NMP)(AA),

M2+ + NMP 2 + AA ~M(NMP)(AA) /2~MM(NMP)(AA) =

M(NMP)+ AA ~M(NMP)(AA)

log

In light of recently accumulated knowledge about the promotion of stacking interactions by metal ions [2, 12, 13, 16], we suggest that in Ni(Trph the two tryptophanates are not only coordinated to Ni ~+, but that in addition their indole moieties do stack. Furthermore, it may be noted in this connection that the stabilities of metal ion/phenylalaninate or tyrosinate (Tyr-) complexes show the same trends [14, 29, 30, 35, 36], and here again aromatlc-ring stacking is possible between the phenyl moieties within the 1:2 complexes.t This view is further supported by the observation [36] that the difference in the values of log K cc,, L - l o g Kcuc: CoL between the alaninate and the phenylalaninate or tyrosinate complexes is nearly diminished in 50% aqueous dioxane, and it is known that dioxane does indeed inhibit stacking [37]. Following the same line of reasoning one may also explain the fact that a Ni(AMP)~ species is formed, while in the Ni:+/CMP: system no 1:2 complex was found [17]. As we have already seen, the stacking tendency of the purine moiety is much larger than the one of the pyrimidine residue, and this also holds for selfstacking [21]. Therefore, we believe that possibly aside from a M:+/N-7 interaction [33, 34] which may also play a role, stacking is the main reason for the formation of a Ni(AMPI~ species.

The ternary metal ion/nucleotide/amino acid systems The stability constants of a ternary system are defined by eqns (5}-(7). The overall stability constant ~Similarly, the stability of several ternary M(Phe)(Tyr) complexes is also somewhat increased [35], which is probably due to intramolecular stacking.

[MtNMPJ(AA)]/([MZ+][NMP 2 ][AA ])(5)

M(NMP) KM{NMP){A~ =

[M(NMP)(AA) ]/([M(NMP)][AA ])

M(AA) ~ + NMP2-~M(NMP)(AA) K MtAA~

M~AA),NMP,= [M(NMP)(AA) ]/([M(AA)+][NMP: I) (7)

M(N Mp) M = Iog/3M~NMp~,AA~--Iog KM,NMP, log ][..r ~MtNMP~,AA~ M

(81

M(AA) -l o g K M ( A A ) t N M P ) --

(9)

M

log f l M ( N M P ) ( A A ) -- log KMMMtAA~.

One way to quantify the stability of ternary complexes [5, 38, 39] relative to binary complexes is according to eqn (10), AIog KM = ,og I vI~.M(NMP)(AA M(NMP) ) -- log KMtAA) =log K M(AA)(NMP) M(AA) -- log KMtNMP~ (10) i.e. by comparing the difference in stability for example for the reaction between M(NMP) or M(aqf + and AA-. The value of A log Ku is the logarithm of the constant due to equilibrium (11); this value also may be calculated according to eqn (12). M(NMP)+M(AA)~M(NMP)(AA)

+ M 2~

(L)

H PKH2 L

H PKHL 9.85+0.02

(11)

log KM = Iog/3~,NMP~AA~--(Iog KMtNMP~ M + log KM, AA0. (12) In general, negative values for m log K~ are expected. since usually for binary systems K~L>K~2129-31].. This is in accordance with the statistical value[5] obtained for the coordination of two different bidentate ligands to a regular octahedral coordination sphere, i.e. log Ko,,2/z~=-(I.38. For the coordination of a monodentate and then a bidentate ligand, or vice versa.. log Ko,~,¢2~= - 0.18. Hence, the statistical expectation

Fable 2. Acidity constants of the ligands and stability constants of the binary Ni2~/amino acid and Ni2+/nucleotide systemsa Ligand

(61'

Ni KNi L

log K NiL NiL2

log K NiL 2 NiL3

5.50+0.04

4.66+0.03

3.29+0.08

4.92+0.03

3.83+0.05

log

Ala-

2.26 b

Trp-

2.39 c

9.47+O.01

5.48+0.03

CMP 2-

4.36+O.O1

6.30+0.03

2.00+0.06

AMP 2-

3.84+O.O1

6.30+0.04

2.61+O.O5

2.43 d

"The constants were determined from potentiometric pH-titrations of aqueous solutions at 25°C and I = 0.1 M (KNO3). For the definitions of the constants see eqns (2) through (4). Given are the mean values of two completely independent sets of titrations, together with the standard errors of the mean values (see also text). bRef. [20]. ~Ref. [19]. dSee experimental section.

790

J. B. ORENBERG et al. Table 3. Stability constants of the ternary Ni2+/nucleotide/amino acid systemsa 1

^Ni °g~Ni (NMP) (AA)

. . Ni (NMP) lOgKNi (NMP) (AA)

. . Ni (AA) IOg~Ni (AA) (NMP)

&IogKN i

N M P 2-

AA-

AMP 2-

Ala-

8.10 _+ O.21

5.49

2.60

-O.O1

AMP 2-

Trp-

8.45 _+ 0.08

5.84

2.97

0.36

CMP 2-

Ala-

7.75 _+ 0.04

5.75

2.25

0.25

CMP 2-

Trp-

7.78 + 0 . 0 8

5.78

2.30

0.30

aThe constants were determined from potentiometric pH-titrations of aqueous solutions at 25°C and I = 0.1 M (KNO3) by taking into account the results listed in Table 2. For the definitions of the constants and several calculations see eqns (5) through (12). Given are the mean values of two completely independent sets of titrations, together with the standard errors of the mean values (see also text).).

for A log KM for the present Ni2÷/nucleotide/amino acid systems is somewhere between the given values.t As already mentioned, only the ternary Ni2+/nucleo tide/amino acid systems could be studied, since with Cu 2+ and Zn 2+ a precipitate formed. The stability constant computed for the ternary Ni 2+ complexes was Ni log/~N~e~P×AA) which corresponds to the overall equilibrium (5). This constant and related stability constants of the ternary complexes are listed in Table 3 together with the values calculated for A log KM from eqn (12). The standard errors of the mean values listed in Table 3 appear to be rather large; one of the main problems is that the formation degree of the ternary complexes is not very high. In addition, all the given data (Tables 2 and 3) are average values which resulted from titrations carried out independently by two workers; within the complete data set obtained by each worker, the values were consistent and the range of error was much smaller. It should be emphasized that the positive values of A log KNI which are listed in Table 3 do not allow the firm conclusion that the corresponding ternary complexes are significantly more stable than is expected on a statistical basis and that equilibrium (11) is shifted to the right. In general, for such a conclusion to be drawn we feel it necessary that the values differ by more than three times the standard error of the mean [1, 4, 5, 12]. That the stability of these ternary complexes is not especially favored may also be shown for the AMP 2- containing systems (for which the stability of Ni(AMP) 2- is also known) in another way. This other approach, which differs from the one using A log KM and which is also commonly used [5,40,41] to quantify the stability of a ternary complex is based on the equilibrium constant, X, as defined by eqn (13); log X may be calculated according to eqn (14): M(NMP)~- + M ( A A h ~ 2 M(NMP)(AA)X = [M(NMP)(AA)-]2/([M(NMp)2-][M(AAh]) (13)

tThe structures of Ni(CMP) and Ni(AMP), which influence the size of the statistical values for A log KM somewhat, shall not be discussed here, as this was done in detail very recently by Martin and Mariam [33, 34]. But it may be mentioned that especially in the case of AMP2- the situation is complicated, as this nucleotide may coordinate also over N-7 to metal ions. ¢This difference can be calculated as the unknown constant Ni CMP KNi/CMP{2 cancels in this calculation.

log X = 2 log f l MM( N M P ) ( A A ) M

M

- (log/3 M<)qMv)2+ log/3 M(AA)2 ) M(NMP) = (10g KMtNMP)(AA)

--

M(AA) log V ~ M(AA)2 !

+ (log vi xMM(AA)(NMP) (AA) I~. VM(r~MP) ~ -- lug; Ix M(NlViP)21.

(14)

The statistical value for log X is the same for all geometries of the coordination sphere of a metal ion and it is 0.6 [41,42]. With eqn (14) and the data of Tables 1 and 2 one may calculate log X N i ( A M P ) ( T r p ) = 1 . 4 6 and log X N i ( A M P X A I a ) = 1.00 for the Ni2+/AMP2-/Trp - and Ni2+/AMp2-/AIa systems, respectively. These values are hardly different from the statistical value, and more important, they are of the same order as 1OgXNitE,×Ser)=l.18 and 1ogXNitGlyXAla)= 1.29 of the ternary Ni2+/ethylenediamine/serinate [43] and Ni2+/glycinate/alaninate [44] systems, respectively, and the ternary complexes of these systems are certainly not considered as being of a remarkable stability [45]; such a conclusion would be true for ternary complexes with values of logX between 2 and 6 [1,5,45]. Another important comparison which must be made is that within the data listed in Table 3 for the four mixedligand systems. That the pyrimidine/indole interaction does not contribute significantly to the stability of the ternary Ni(CMP)(Trp)- complex was to be expected from the results described by the 'H NMR study. Hence, it is not surprising that the A logKNi value of the Ni(CMP)(AIa)- complex, in which no stacking interaction is possible, is practically identical with the corresponding value for Ni(CMP)(Trp)-. The same conclusion results from the log X approach (eqns 13 and 14), i.e. from the difference~: 1OgXNi(CMP)Crrp)-log XNi
791

Ternary nucleotide/aminoacid/metal ion complexes log X as well, goes in the expected direction, i.e. it appears that in the equilibria ill) and (13) Ni(AMP) (Trp) may b e slightly favored compared to the corresponding equilibria with Ni(AMP)(Ala) . With this result in mind it seems justified to ask the following question: how large might a stability increase be that results from an intramolecular stacking in Ni(AMP)(Trp) ? The values of the experimentally Ni determined constant /3N~(AMr',~T,~, as well as the derived Ni{AMP) constant KNi(AMP)(Trp), depend upon the total formation of the Ni(AMP)(Trp) species. The concentration of Ni(AMP)(Trp) includes two isomeric species, one in which an intramolecular stacking occurs, and one in which the two ligands are simply coordinated to Ni 2". Therefore we can define the intramolecular equilibrium ~15)

/

/

Wro

Ni -~

=

///

--

Ni-'+ /

AMP ~"

AMP2-

K,.;*, : [Ni(AMP)(Trp).~,~ke,~]/[Ni(AMP)(Trp)o,,~.I.

(15) The corresponding equilibrium constant K*s, is dimensionless and hence independent of the absolute concentration of Ni/AMP)(Trp) . If one defines in addition a constant, Koo~., which quantifies the equilibrium between Ni(AMP) and Trp for the none-stacked (i.e. open) isomer of Ni(AMP)(Trp) , one arrives, b,¢ combining all related equilibrium constants, at eqn (16)t Ni(AMP) KNi(AMP)tTrp) = Kope,(l + K*st).

(16)

Obviously, in a system where no stacking is possible at all, e.g. in Ni(AMP)(Ala) , Ks*t=0 and hence Koch, = Ni(AMP) KN,AMr'~A,.I. However. if one assumes that about 50% of Ni(AMP)(Tro)- exist as the stacked isomer, an assumption which appears to be in the right order based on the results obtained for the Zn/ATp4-/Trp system [2], one obtains Ks* = I, and hence • Ni(AMP) KNitAMP)tTrp) = 2 X

mope..

This result means,

that if 50%

of NiIAMP)(Trp) should exist in the stacked isomeric form, A log KN~ for the N i 2 " / A M P 2 - / T r p - system should be 0.3 log units larger than the value of A log KN~ for the Ni/AMP 2 /Ala system. This change is rather small and not easy to determine in an unequivocal way. Therefore, it is rather surprising to note that this difference in the A IogK,~ values is actually observed for these systems #This reasoning is analogous to the one used recently by Mariam and Martin/34] for the problem of macrochelate formation in MZ+/nucleotidesystems. ~These dimensionless constants K~* or K*p for the intramolecular stack or ion pair formation should not be confused with the equilibrium constants Kst (M 1) or Ku, (M t) for an intermolecular stack or ion pair formation. AMP rq,I = 6.83 M-I §The calculation was done with Ktot= KIAMP~H. and the A6 values for H-2 and for (H-8)-(H-2) (see Table I); one obtains K~p= 0.94 M ~ and 0.21 M i, respectively,and as the average/he value given above.

(Table 3), although it cannot be considered as significant due to the large range of error. In addition it should be noted that the formation of only 20% of the stacked isomer would correspond to K*s, = 0.25, and this wouht result in a difference of only 0.1 log units between the A log KN~ values, a difference which could hardly be detected unequivocally between equilibrium constants which were determined by potentiometric pH-titrations. GENERAL CONSIDERATIONS In principle, it is quite clear that the concentration of weak stacked adducts may be increased by an additional polar interaction (Fig. 1). In fact, the described differences in stability between (AMP)(H. Trp) 2 and (AMP)(Trp)3 prove this unequivocally: the formation of an ion pair between the phosphate moiety and the ammonium group in (AMP)(H • Trp) 2 is responsible for an increase in stability by a factor of about three. This result is also in very good accordance with a study by Dimicoli and H61~ne[6] on the adduct formation of AMP 2 with the tryptamine cation or indole acetate, where the association constants, Ks,, are 6.2 M-' and 1.8 M ~ respqctively. The ion pair formed between AMP 2 and the ammonium group of tryptamine enhances again the stability of the adduct. In this connection it is worthwhile to discuss the connection between the three possible isomeric species of (AMP)(H. Trp) 2- somewhat more in detail. With a reasoning similar to the one used for the derivation of eqn (16), one may now deduce eqn (17) Ktot = Ks, + KIp + KIp" Ks*

= Ks, + KIp + Kst • K*p = Kst + Kw(l + K*,) = KIp + Kst(l + K'p).

(17)

In this equation Ks, is the formation constant of the stack and Kip of the ion pair, while K*st and K*p are the corresponding intramolecular (and hence dimensionless) constants.:[: Besides the overall constant Kto,=K~AMJ'I~AMp~, •Trp~ (determined at pD 8.4; Table 1), only Ks, which equals K~AMP~¢T,~,~ ~AMPt is known, because this latter constant was determined at pD 12.4, i.e. under condition,; where no ionic interactions are possible. However, assuming that the orientation of the aromatic rings in the', stacks at pD 12.4 and 8.4 is the same, the shift difference', A6 = ~5o- S~ should be larger at pD 12.4 than at pD 8.4. where a part of (AMP)(H. Trp) 2 may exist as an ion pair, (AMP)(H • Trp)~g, which will not contribute to the' upfield shift; hence eqn (18) holds A(~(pD 12.4) -- A(~(pD 8.4) _

A~(pD 12.4)

--

(AMP)(H • Trp)~p _ K w (AMP)(H • Trp),2o,- ~ " (18)

From the results in Table 1 it is immediately obvious that the values of A8 = 3o- 6= at pD 12.4 and pD 8.4 are within the error limits identical for the shifts of H-2 and the shift difference (H-8)-(H-2) of the AMP protons; indeed we have already made use of this fact (see footnote e in Table 1). This result must mean of course that the value for KIP is rather small: if one calculates, despite the indicated limitations, with eqn (18)§ the stability constant for the (AMP)(H • Trp)~r; ion pair one obtains Kw = 0.6 M-'. This value is also in accordance with the CMp2-/(H • Trp) system for which the upper limit of its stability constant may be calculated from eqn

792

J.B. ORENBERG etal.

(17) and the constants given in Table 1, i.e. for this system K~p~<0.6 M -z. Both these results agree also surprisingly well with Kjr, = 1,35 +0,76 M -z, obtained for (CH3)4N+/Trp -. We are now, of course, also in the position to calculate with,eqn (17~ the values for the intramolecular constants Kst and K~p: one obtains 6.7 and 1.8, respectively. This means, e g for the dimensionless constant Kst = 6.7, which corresponds to the one defined in eqn (15) (but now with H ÷ instead of Ni2÷ as the ionic bridge), that about 90% of the ion pair are also associated over a stack. In this connection it is worthwhile to mention that the N(I)-methylnicotinamide ion binds weakly to tryptophan-62 of lysozyme forming a stacked adduct [46]. Moreover, in a related isonicotinylium glycoside, a polar interaction of the sugar with the enzyme occurs in addition to the stacking, and this combination of two weak interactions leads to an increased stability constant [47]. We believe that in vivo the formation of stacking adducts can also be enhanced by bridging two suitable ligands with a metal ion; that this is possible in vitro has already been shown [2, 12, 13, 16]. The following reasoning demonstrates that such a promotion is even possible under rather unfavorable conditions as they are observed in this study. In the physiological pH range the Ni(AMP)(Trp)- complex is formed in a 10-2 M solution of each of the reagents to, in total, about 45%; a conservative estimate of 33% for the stacked isomer of this complex leaves us with 15% ternary complex with an intramolecular stack, based on total reagent concentrations. This amount should be compared with the 2% of stack formed in solution (also 10-2 M for each reagent) in the absence of an ion pair or metal ion promotion. As the concentrations of Ni(AMP)(Trp)- and Ni(AMP)(Ala)- do not differ very much in the physiological pH region (pH = 7.5) in a l0 -2 M reagent solution (the formation degree is 48 and 33%, respectively), we believe and the data of this study suggest that evolutionary selectivity in nucleotide/metal ion/amino acid systems is probably not so much achieved, at least for a given metal ion, by differences in complex stabilities, but rather by the ability to form specific and distinct structures as is possible for example by intramolecular stacking interactions. .

•

.

Acknowledgements--We thank Mr. K. Aegerter of the Institute of Organic Chemistry for recording the 90 MHz ~H-NMRspectra and Ms. R. Baumbusch for skilful assistance with a part of the potentiometric titrations, and the Swiss National Science Foundation for a research grant. The computer (Univac 1108) was made available by the Rechenzentrum der Universit/it Basel; this support is also gratefully acknowledged. REFERENCES

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