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Ternary systems of Zn2+ and Cd2+, 2-(α-hydroxyethyl)thiamin pyrophosphate (HETPP) and the pentapeptide Asp-Asp-Asn-Lys-Ile.
Inorganica Chimica Acta 349 (2003) 279 /283 www.elsevier.com/locate/ica
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Ternary systems of Zn2 and Cd2, 2-(a-hydroxyethyl)thiamin pyrophosphate (HETPP) and the pentapeptide Asp-Asp-Asn-Lys-Ile. Implications for the mechanism of thiamin enzymes Gerasimos Malandrinos, Maria Louloudi *, Nick Hadjiliadis * Department of Chemistry, Laboratory of Inorganic and General Chemistry, University of Ioannina, 45110 Ioannina, Greece Received 27 September 2002; accepted 20 December 2002
Abstract To obtain structural information on the active-site of thiamin-dependent enzymes in solution, the interaction of Zn2 and Cd2 ions with the pentapeptide Asp-Asp-Asn-Lys-Ile surrounding the thiamin pyrophosphate moiety in the transketolase enzyme, and the tertiary Zn2 /or Cd2 /pentapeptide /HETPP systems have been studied by NMR spectroscopy in aqueous solutions at physiological pH. The HETPP, 2-(a-hydroxyethyl)thiamin pyrophosphate, represents an active intermediate of thiamin catalytic cycle formed after the addition of pyruvate substrate on thiamin molecule. The present data show the existence of the tertiary metal /[pentapeptide] /[HETPP] complexes at physiological pH, where the metal coordination sphere is completed by both peptide backbone and HETPP molecule. The metal coordinated HETPP molecule adopts the so-called S conformation in solution. The importance of the present findings correlated with previous results is discussed and possible functional implications are suggested. # 2003 Elsevier Science B.V. All rights reserved. Keywords: Thiamin complexes; Metal complexes; Thiamin catalysis
1. Introduction The main enzymic reaction catalyzed by pyruvate decarboxylase (PDC; EC 4.1.1.1) is the non-oxidative decarboxylation of pyruvate to acetaldehyde [1,2]. The generally agreed reaction mechanism concerns the PDCmediated decarboxylation of pyruvate proposed by Breslow [3]. Deprotonation of TPP generates the ylid which attacks pyruvate to give the lactyl-TPP. This undergoes decarboxylation to form the enamine/carbanion species that is protonated to give hydroxyethylTPP known as ‘active aldehyde’ intermediate. Release of acetaldehyde regenerates the ylid form of TPP [4 /6]. In terms of the relative orientation of the thiazolium and pyrimidine ring, the TPP molecule can adopt three different conformations, the F, S and V [7]. In all crystal structures of TPP-dependent enzymes, TPP adopts the V * Corresponding authors. Tel.: /30-651-98420/98419; fax: /30651-44831/98419. E-mail addresses: [email protected] (M. Louloudi), [email protected] (N. Hadjiliadis).
conformation which brings 4?-NH2 close to C(2) [8 /12]. On the other hand, all the C(2)-substituted thiamin intermediates which have been either isolated from enzymic systems or synthesized in vitro adopt without exception the S conformation [13]. In addition, in all analyzed crystal structures of thiamin-dependent holoenzymes [8 /12], the metal ions (Ca2, Mg2) that are also cofactors, bridge the protein environment with the TPP molecule via two oxygen atoms of its phosphate group. The side chains of an Asp and Asn, the main chain oxygen of another residue and a water molecule complete the metal coordination sphere [8 /12]. Recently, we have studied the tertiary system [Cu2] / [pentapeptide] /[hydroxyethyl-TPP], where the synthesized Asp-Asp-Asn-Lys-Ile pentapeptide modeled the metal binding site Asp185-Asp186-Asn187-Lys188Ile189 of transketolase [14,15]. It was found that: (i) the metal is bound to HETPP at N(1?) and the pyrophosphate group and to the peptide through the a-NH2 amide nitrogen and carboxylic oxygen atoms; (ii) the S conformation of HETPP was retained in the
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ternary complexes. Continuing our studies we report here the NMR studies on the ternary systems [Zn2/or Cd2]/[pentapeptide] /[hydroxyethyl-TPP]. The results substantiate our previous ones with Cu2, strongly suggesting a mechanism of action of thiamin enzymes, emphasizing the importance of S conformation.
2. Experimental All materials used were purchased from Sigma Chemical Co. and CBL Chemicals Ltd. The synthesis of the pentapeptide DDNKI (Scheme 1) and the ligand HETPP have been reported previously [16,18]. The NMR techniques used include one-dimensional 1H, 13 C, 31P NMR and two-dimensional 1H /1H NOESY, ROESY, 1H/1H TOCSY, 1H/13C HMQC and 1H /13C HMBC experiments. All spectra were recorded in Bruker 400/600 MHz spectrometers at 298 and 273 K. Details of the methods have previously been reported [15].
3. Results and discussion 3.1. Interaction of Zn(II) and Cd(II) with DDNKI The assignments of the most affected upon coordination 13C chemical shifts at 298 and 273 K concerning the systems Zn(II) / and Cd(II) /DDNKI as well as the free peptide are given in Table 1. The interpretation of the chemical shifts was achieved based on two-dimensional 1 H/1H TOCSY, 1H/13C HMQC and 1H /13C HMBC spectra. The most striking effect is seen for the C /O group of Asp1 residue in Zn(II)/DDNKI system at 273 K, which shifts 4.94 ppm downfield compared to the free peptide at the same temperature. The signal assigned to the same group of Asp1 in Zn(II)/DDNKI at room temperature, could not be identified. However, a Asp1-C /O /Zn(II) coordination can be proposed, based on the large change taking place in the Ca resonance of the Asp1 residue.
Scheme 1. The pentapeptide Asp-Asp-Asn-Lys-Ile.
In the Cd(II)/DDNKI system at 298 K, a downfield shift of 0.58 ppm observed for the Asp1-COO group indicates an interaction of the carboxylate group with Cd(II). Supporting evidence for this Asp1-COO / Cd(II) interaction comes from the Cb resonance of Asp1 residue, which shifts 1.72 ppm upfield. The 13C chemical shift of the Asp1-COO atom in the range of 1.54 ppm, in the Cd(II) /DDNKI system at low temperature, clearly suggests Cd(II) coordination to Asp1-COO group. For the C /O group of Asp1 residue, a weaker Cd(II) /O /C interaction is also observed. Based on spectral data, we propose, in all cases, metal/DDNKI species formation involving coordination of the terminal amino group (Asp1-NH2) and of b carboxylate of Asp1 residue (Asp1-COO ). The coordination environment is completed by a C /O peptide group of Asp1 (Scheme 2). In this way, the amino group which can form a six-membered chelate ring with the metal ions and the OOCb-Asp1, anchors the metal ion supported by peptide C /O (Asp1) binding stabilizing a five-membered chelate ring. 3.2. Interaction of K2 {[Zn(HETPPH)Cl2]2} and K2 {[Cd(HETPPH)Cl2]2} with DDNKI 3.2.1. 13C NMR spectra Selected 13C chemical shifts of the tertiary systems [Zn2]/ and [Cd2]/[pentapeptide] /[hydroxyethylTPP] at both 298 and 273 K for the peptide moiety are shown in Table 2. The chemical shifts have been assigned by using two-dimensional 1H/1H TOCSY, 1 H /13C HMQC and 1H /13C HMBC spectra. At 298 K, the spectral data clearly suggest metal ion / peptide interaction. The mode of Zn2 and Cd2 coordination persists with H2N-Asp1, OOCb-Asp1 and O /C-Asp1 binding (Scheme 3). At low temperature, a tridentate coordination of peptide in Zn2 /DDNKI / HETPP system, involves binding of the same functional groups of the Asp1 residue as at room temperature (Scheme 3). In the Cd2 /DDNKI /HETPP system, a Cd(II) /H2N-Asp1 interaction is rather not favored, while the OOCb-Asp1 and O /C-Asp1 groups remain bound to Cd(II) (Scheme 3). The NMR spectra of the Zn2 / and Cd2 / DDNKI /HETPP systems generally suggest metalion /HETPP interaction as well. The most affected 13C resonances of the HETPP moiety, in the tertiary systems, with respect to the free HETPP molecule, are the C(2?), C(6?) and 2?-CH3 atoms (Table 3). The magnitude and the direction (downfield) of these chemical shifts at 298 K are comparable to those observed at 273 K for both zinc and cadmium systems. These indications provide strong evidence that metal complexation occurs via the N(1?) atom of the HETPP molecule.
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Table 1 Selected 13C NMR data (d , ppm) for the free peptide DDNKI and the systems Zn(II) /DDNKI and Cd(II) /DDNKI in D2O /H2O solutions (1:4 v/ v) at pH 7.0 and temperatures 298 and 273 K Asp1
Ca Cb C/O COO
DDNKI
Zn(II) /DDNKI (1:1)
Cd(II) /DDNKI (1:1)
298 K
273 K
298 K
273 K
298 K
273 K
53.78 40.77
51.04 37.98 171.00 176.46
52.85 41.68
49.93 39.00 175.94 177.94
53.38 39.05 174.12 179.71
50.60 39.62 171.77 177.95
179.13
178.08
Scheme 2. Possible structure formulae of the metal /peptide complexes.
3.2.2. 31P NMR spectra In [Cd2]/[DDNKI] /[HETPP] system at 298 K, the chemical shift of the Pb resonance of 2.8 ppm, with respect to free HETPP at the same pH, clearly indicates a Cd-binding to the Pb /O . The smaller shift of the Pa of 0.9 ppm suggests that the Pa /O position is more weakly bound (Table 4). For the same system at 273 K, the Pa and Pb resonances are shifted by 2.22 and 2.03 ppm downfield compared to those of free HETPP (Table 4). This implies an almost equivalent bidentate coordination of thiamin pyrophosphate group (Scheme 3). In [Zn2]/[DDNKI]/[HETPP] system, metal coordination to the pyrophosphate group is rather monodentate than bidentate (Scheme 3). This is supported by the observed chemical shift of 0.9 ppm at 298 K and the chemical shift of 1.29 ppm at 273 K (Table 4) of Pa resonance. Summarizing our suggestion, we propose a simultaneous binding of both peptide and thiamin molecules to metal ions. A similar structural model for tertiary [Cu2] /[pentapeptide] /[HETPP] species was also pro-
Scheme 3. Possible structures formulae of the ternary metal / HETPP /peptide complexes.
Table 2 Selected 13C NMR data (d , ppm) of the peptide moiety in the systems K2{[Zn(HETPPH)Cl2]2} /DDNKI (1:2) and K2{[Cd(HETPPH)Cl2]2} / DDNKI (1:2) at 298 and 273 K Asp1
Ca Cb C/O COO
DDNKI
K2{[Zn(HETPPH)Cl2]2} /DDNKI (1:2)
K2{[Cd(HETPPH)Cl2]2} /DDNKI (1:2)
298 K
273 K
298 K
273 K
298 K
273 K
53.78 40.77
51.04 37.98 171.00 176.46
53.21 40.56 175.39ov
50.30 36.46 172.89 178.81
53.55 40.52 174.11 179.23
50.86 36.39 173.18 177.38
179.13
ov, overlapped by other peaks.
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Table 3 Selected 13C NMR data (d , ppm) of the HETPP moiety in the systems K2{[Zn(HETPPH)Cl2]2} /DDNKI (1:2) and K2{[Cd(HETPPH)Cl2]2} / DDNKI (1:2) at 298 and 273 K
C(2?) C(6?) 2?-CH3
(HETPPH) K
K2{[Zn(HETPPH)Cl2]2} /DDNKI (1:2)
K2{[Cd(HETPPH)Cl2]2} /DDNKI (1:2)
298 K
273 K
298 K
273 K
298 K
273 K
168.7 149.7 25.7
166.2 148.2 23.3
170.0 151.1 26.9
167.2 149.4 24.2
169.8 152.5 26.5
167.4 150.1 24.5
posed involving a copper coordination to phosphate oxygens, N(1?) of pyrimidine and the functional groups of the Asp1 residue as well [14,15].
stability; (iii) in these systems, the HETPP uses the pyrophosphate group and the N(1?) atom as metal coordination sites; the metal coordination environment is completed by peptide functional groups [14,15]; (iv) the HETPP adopts always the S conformation (free ligand, binary metal/HETPP systems and tertiary metal/HETPP /petide systems) [15 /17,19]. Evaluating our data, we clarify that the existence of metal/N(1?) and metal/pyrophosphate oxygens bonds and the adopted S conformation by HETPP during the intermediate catalytic steps, at least, do not occlude the catalytic cycle. It has been proposed earlier [18], that the S conformation stabilizes all intermediates containing a hydroxyl group at C(2a)-side chain by the intramolecular electrostatic interaction S(1) O(2a) thus facilitating the O(2a) /H proton ionization. Thus, while the V conformation of thiamin is active possibly in the initial step of catalysis, we suggest here that the substrate addition during the enzymic cycle, should impose the S conformation to thiamin molecule. Therefore, the detailed mechanism may be represented as follows (Scheme 4). The metal ions most probably serve as linkers between the protein and the coenzyme through the coenzyme pyrophosphate group. On the other hand, we had previously suggested that metal ions should enter the catalytic cycle after the addition of the substrate and the subsequent formation of ‘active aldehyde’ intermediates [19]. Any further role of metal binding to N(1?) however, such as a better control of the reactivity of the 4?-amino group, except the link with the protein, needs to be further investigated.
3.2.3. 1H /1H NMR 1 H/1H NOESY spectra of the tertiary Zn2 / and 2 Cd /peptide /HETPP systems have been recorded at 273 K. The important intramolecular cross-peaks observed are those between the protons C(6?)/H/C(2a) /H, C(6?)/H/C(2a)/CH3, and C(6?)/H/C(4) /CH3 from HETPP molecule. The interaction of C(6?) /H proton with the C(2a)-side chain clearly indicates that this proton is directed to the thiazole ring. This spectroscopic feature is characteristic of thiamin derivatives adopting the S conformation [13]. The latter cross-peak between the C(6?)/H and the C(4) /CH3 suggests that thiazole ring was rotated in the same manner as in the crystal structure of HETPP [16]. Based on these, we conclude that the S conformation found in the crystal structure of HETPP and in aqueous solutions of metal / HETPP complexes, is also retained in the aqueous solutions of the tertiary M2 /peptide /HETPP systems.
4. Conclusion Our findings in this and previous works, are reviewed as follows: (i) in the binary metal/HETPP system, the metal binding sites are the pyrophosphate group and the N(1?) atom of the pyrimidine ring [14 /17]; (ii) the tertiary metal/HETPP /pentapeptide complexes around physiological pH [14,15] can be formed with wide
Table 4 31 P NMR data (d , ppm) of the HETPP moiety in the systems K2{[Zn(HETPPH)Cl2]2} /DDNKI (1:2) and K2{[Cd(HETPPH)Cl2]2} /DDNKI (1:2) at 298 and 273 K Pa
(HETPPH) K K2{[Zn(HETPPH)Cl2]2} /DDNKI (1:2) K2{[Cd(HETPPH)Cl2]2} /DDNKI (1:2) a
DdPa
Pb
a
DdPb a
2
JPaPb (Hz)
298 K
273 K
298 K
273 K
298 K
273 K
298 K
273 K
298 K
273 K
/10.6 /11.1 /9.7
/10.00 /8.71 /7.78
/8.8 /7.9 /6.0
/5.92 /5.64 /3.89
/0.5 /0.9
/1.29 /2.22
/0.9 /2.8
/0.28 /2.03
20.1 17.8 15.7
21.06 16.20 14.58
Difference in ppm of the corresponding NMR signals for the free ligand and the complexes at the same pH (Dd/dligand/dcomplex).
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Scheme 4. Proposed mechanism for thiamin catalysis.
5. Supplementary material The assignments of 1H NMR data: (i) of metal/ DDNKI recorded at 298 and 273 K; (ii) of metal / HETPP /DDNKI systems for peptide moiety at 298 and 273 K; (iii) of metal/HETPP /DDNKI systems for HETPP molecule at both 298 and 273 K. The assignments of 13C NMR data of the above systems are available from the author.
References [1] A. Schellenberg, Angew. Chem., Int. Ed. Engl. 6 (1967) 1024. [2] S. Ko¨nig, A. Schellenberger, H. Neef, G. Schneider, J. Biol. Chem. 269 (1994) 10879. [3] R. Breslow, J. Am. Chem. Soc. 80 (1958) 3719. [4] R. Kluger, Chem. Rev. 87 (1987) 863. [5] R. Kluger, Pure. Appl. Chem. 69 (1997) 1957. [6] A. Schellenberger, Biochim. Biophys. Acta 1385 (1998) 177. [7] J. Pletcher, M. Sax, G. Blank, M. Wood, J. Am. Chem. Soc. 99 (1977) 1396.
[8] Y.A. Muller, G.E. Schulz, Science 259 (1993) 965. [9] F. Dyda, W. Furey, S. Swaminathan, M. Sax, B. Farrenkopf, F. Jordan, Biochem. 32 (1993) 6165. [10] Y. Lindqvist, G. Schneider, U. Ermler, M. Sundstrom, EMBO J. 11 (1992) 2373. [11] M. Nikkola, Y. Lindqvist, G. Schneider, J. Mol. Biol. 238 (1994) 387. [12] M.S. Hasson, A. Muscate, M.J. McLeish, L.S. Polovnikova, J.A. Gerlt, G.L. Kenyon, G.A. Petsko, D. Ringe, Biochem. 37 (1998) 9918. [13] M. Louloudi, N. Hadjiliadis, Coord. Chem. Rev. 135/136 (1994) 429 (and references therein). [14] G. Malandrinos, M. Louloudi, Y. Deligiannakis, N. Hadjiliadis, J. Phys. Chem., B. 105 (2001) 7323. [15] G. Malandrinos, M. Louloudi, Y. Deligiannakis, N. Hadjiliadis, Inorg. Chem. 40 (2001) 4588. [16] G. Malandrinos, M. Louloudi, C.A. Mitsopoulou, I.S. Butler, R. Bau, N. Hadjiliadis, J. Biol. Inorg. Chem. 3 (1998) 437. [17] G. Malandrinos, M. Louloudi, A.I. Koukkou, I. Sovago, K. Drainas, N. Hadjiliadis, J. Biol. Inorg. Chem. 5 (2000) 218. [18] J. Hogg, Bioorg. Chem. 10 (1981) 233. [19] M. Louloudi, N. Hadjiliadis, J.A. Feng, S. Sukumar, R. Bau, J. Am. Chem. Soc. 112 (1990) 7233.
Note
Ternary systems of Zn2 and Cd2, 2-(a-hydroxyethyl)thiamin pyrophosphate (HETPP) and the pentapeptide Asp-Asp-Asn-Lys-Ile. Implications for the mechanism of thiamin enzymes Gerasimos Malandrinos, Maria Louloudi *, Nick Hadjiliadis * Department of Chemistry, Laboratory of Inorganic and General Chemistry, University of Ioannina, 45110 Ioannina, Greece Received 27 September 2002; accepted 20 December 2002
Abstract To obtain structural information on the active-site of thiamin-dependent enzymes in solution, the interaction of Zn2 and Cd2 ions with the pentapeptide Asp-Asp-Asn-Lys-Ile surrounding the thiamin pyrophosphate moiety in the transketolase enzyme, and the tertiary Zn2 /or Cd2 /pentapeptide /HETPP systems have been studied by NMR spectroscopy in aqueous solutions at physiological pH. The HETPP, 2-(a-hydroxyethyl)thiamin pyrophosphate, represents an active intermediate of thiamin catalytic cycle formed after the addition of pyruvate substrate on thiamin molecule. The present data show the existence of the tertiary metal /[pentapeptide] /[HETPP] complexes at physiological pH, where the metal coordination sphere is completed by both peptide backbone and HETPP molecule. The metal coordinated HETPP molecule adopts the so-called S conformation in solution. The importance of the present findings correlated with previous results is discussed and possible functional implications are suggested. # 2003 Elsevier Science B.V. All rights reserved. Keywords: Thiamin complexes; Metal complexes; Thiamin catalysis
1. Introduction The main enzymic reaction catalyzed by pyruvate decarboxylase (PDC; EC 4.1.1.1) is the non-oxidative decarboxylation of pyruvate to acetaldehyde [1,2]. The generally agreed reaction mechanism concerns the PDCmediated decarboxylation of pyruvate proposed by Breslow [3]. Deprotonation of TPP generates the ylid which attacks pyruvate to give the lactyl-TPP. This undergoes decarboxylation to form the enamine/carbanion species that is protonated to give hydroxyethylTPP known as ‘active aldehyde’ intermediate. Release of acetaldehyde regenerates the ylid form of TPP [4 /6]. In terms of the relative orientation of the thiazolium and pyrimidine ring, the TPP molecule can adopt three different conformations, the F, S and V [7]. In all crystal structures of TPP-dependent enzymes, TPP adopts the V * Corresponding authors. Tel.: /30-651-98420/98419; fax: /30651-44831/98419. E-mail addresses: [email protected] (M. Louloudi), [email protected] (N. Hadjiliadis).
conformation which brings 4?-NH2 close to C(2) [8 /12]. On the other hand, all the C(2)-substituted thiamin intermediates which have been either isolated from enzymic systems or synthesized in vitro adopt without exception the S conformation [13]. In addition, in all analyzed crystal structures of thiamin-dependent holoenzymes [8 /12], the metal ions (Ca2, Mg2) that are also cofactors, bridge the protein environment with the TPP molecule via two oxygen atoms of its phosphate group. The side chains of an Asp and Asn, the main chain oxygen of another residue and a water molecule complete the metal coordination sphere [8 /12]. Recently, we have studied the tertiary system [Cu2] / [pentapeptide] /[hydroxyethyl-TPP], where the synthesized Asp-Asp-Asn-Lys-Ile pentapeptide modeled the metal binding site Asp185-Asp186-Asn187-Lys188Ile189 of transketolase [14,15]. It was found that: (i) the metal is bound to HETPP at N(1?) and the pyrophosphate group and to the peptide through the a-NH2 amide nitrogen and carboxylic oxygen atoms; (ii) the S conformation of HETPP was retained in the
0020-1693/03/$ - see front matter # 2003 Elsevier Science B.V. All rights reserved. doi:10.1016/S0020-1693(03)00056-2
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ternary complexes. Continuing our studies we report here the NMR studies on the ternary systems [Zn2/or Cd2]/[pentapeptide] /[hydroxyethyl-TPP]. The results substantiate our previous ones with Cu2, strongly suggesting a mechanism of action of thiamin enzymes, emphasizing the importance of S conformation.
2. Experimental All materials used were purchased from Sigma Chemical Co. and CBL Chemicals Ltd. The synthesis of the pentapeptide DDNKI (Scheme 1) and the ligand HETPP have been reported previously [16,18]. The NMR techniques used include one-dimensional 1H, 13 C, 31P NMR and two-dimensional 1H /1H NOESY, ROESY, 1H/1H TOCSY, 1H/13C HMQC and 1H /13C HMBC experiments. All spectra were recorded in Bruker 400/600 MHz spectrometers at 298 and 273 K. Details of the methods have previously been reported [15].
3. Results and discussion 3.1. Interaction of Zn(II) and Cd(II) with DDNKI The assignments of the most affected upon coordination 13C chemical shifts at 298 and 273 K concerning the systems Zn(II) / and Cd(II) /DDNKI as well as the free peptide are given in Table 1. The interpretation of the chemical shifts was achieved based on two-dimensional 1 H/1H TOCSY, 1H/13C HMQC and 1H /13C HMBC spectra. The most striking effect is seen for the C /O group of Asp1 residue in Zn(II)/DDNKI system at 273 K, which shifts 4.94 ppm downfield compared to the free peptide at the same temperature. The signal assigned to the same group of Asp1 in Zn(II)/DDNKI at room temperature, could not be identified. However, a Asp1-C /O /Zn(II) coordination can be proposed, based on the large change taking place in the Ca resonance of the Asp1 residue.
Scheme 1. The pentapeptide Asp-Asp-Asn-Lys-Ile.
In the Cd(II)/DDNKI system at 298 K, a downfield shift of 0.58 ppm observed for the Asp1-COO group indicates an interaction of the carboxylate group with Cd(II). Supporting evidence for this Asp1-COO / Cd(II) interaction comes from the Cb resonance of Asp1 residue, which shifts 1.72 ppm upfield. The 13C chemical shift of the Asp1-COO atom in the range of 1.54 ppm, in the Cd(II) /DDNKI system at low temperature, clearly suggests Cd(II) coordination to Asp1-COO group. For the C /O group of Asp1 residue, a weaker Cd(II) /O /C interaction is also observed. Based on spectral data, we propose, in all cases, metal/DDNKI species formation involving coordination of the terminal amino group (Asp1-NH2) and of b carboxylate of Asp1 residue (Asp1-COO ). The coordination environment is completed by a C /O peptide group of Asp1 (Scheme 2). In this way, the amino group which can form a six-membered chelate ring with the metal ions and the OOCb-Asp1, anchors the metal ion supported by peptide C /O (Asp1) binding stabilizing a five-membered chelate ring. 3.2. Interaction of K2 {[Zn(HETPPH)Cl2]2} and K2 {[Cd(HETPPH)Cl2]2} with DDNKI 3.2.1. 13C NMR spectra Selected 13C chemical shifts of the tertiary systems [Zn2]/ and [Cd2]/[pentapeptide] /[hydroxyethylTPP] at both 298 and 273 K for the peptide moiety are shown in Table 2. The chemical shifts have been assigned by using two-dimensional 1H/1H TOCSY, 1 H /13C HMQC and 1H /13C HMBC spectra. At 298 K, the spectral data clearly suggest metal ion / peptide interaction. The mode of Zn2 and Cd2 coordination persists with H2N-Asp1, OOCb-Asp1 and O /C-Asp1 binding (Scheme 3). At low temperature, a tridentate coordination of peptide in Zn2 /DDNKI / HETPP system, involves binding of the same functional groups of the Asp1 residue as at room temperature (Scheme 3). In the Cd2 /DDNKI /HETPP system, a Cd(II) /H2N-Asp1 interaction is rather not favored, while the OOCb-Asp1 and O /C-Asp1 groups remain bound to Cd(II) (Scheme 3). The NMR spectra of the Zn2 / and Cd2 / DDNKI /HETPP systems generally suggest metalion /HETPP interaction as well. The most affected 13C resonances of the HETPP moiety, in the tertiary systems, with respect to the free HETPP molecule, are the C(2?), C(6?) and 2?-CH3 atoms (Table 3). The magnitude and the direction (downfield) of these chemical shifts at 298 K are comparable to those observed at 273 K for both zinc and cadmium systems. These indications provide strong evidence that metal complexation occurs via the N(1?) atom of the HETPP molecule.
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Table 1 Selected 13C NMR data (d , ppm) for the free peptide DDNKI and the systems Zn(II) /DDNKI and Cd(II) /DDNKI in D2O /H2O solutions (1:4 v/ v) at pH 7.0 and temperatures 298 and 273 K Asp1
Ca Cb C/O COO
DDNKI
Zn(II) /DDNKI (1:1)
Cd(II) /DDNKI (1:1)
298 K
273 K
298 K
273 K
298 K
273 K
53.78 40.77
51.04 37.98 171.00 176.46
52.85 41.68
49.93 39.00 175.94 177.94
53.38 39.05 174.12 179.71
50.60 39.62 171.77 177.95
179.13
178.08
Scheme 2. Possible structure formulae of the metal /peptide complexes.
3.2.2. 31P NMR spectra In [Cd2]/[DDNKI] /[HETPP] system at 298 K, the chemical shift of the Pb resonance of 2.8 ppm, with respect to free HETPP at the same pH, clearly indicates a Cd-binding to the Pb /O . The smaller shift of the Pa of 0.9 ppm suggests that the Pa /O position is more weakly bound (Table 4). For the same system at 273 K, the Pa and Pb resonances are shifted by 2.22 and 2.03 ppm downfield compared to those of free HETPP (Table 4). This implies an almost equivalent bidentate coordination of thiamin pyrophosphate group (Scheme 3). In [Zn2]/[DDNKI]/[HETPP] system, metal coordination to the pyrophosphate group is rather monodentate than bidentate (Scheme 3). This is supported by the observed chemical shift of 0.9 ppm at 298 K and the chemical shift of 1.29 ppm at 273 K (Table 4) of Pa resonance. Summarizing our suggestion, we propose a simultaneous binding of both peptide and thiamin molecules to metal ions. A similar structural model for tertiary [Cu2] /[pentapeptide] /[HETPP] species was also pro-
Scheme 3. Possible structures formulae of the ternary metal / HETPP /peptide complexes.
Table 2 Selected 13C NMR data (d , ppm) of the peptide moiety in the systems K2{[Zn(HETPPH)Cl2]2} /DDNKI (1:2) and K2{[Cd(HETPPH)Cl2]2} / DDNKI (1:2) at 298 and 273 K Asp1
Ca Cb C/O COO
DDNKI
K2{[Zn(HETPPH)Cl2]2} /DDNKI (1:2)
K2{[Cd(HETPPH)Cl2]2} /DDNKI (1:2)
298 K
273 K
298 K
273 K
298 K
273 K
53.78 40.77
51.04 37.98 171.00 176.46
53.21 40.56 175.39ov
50.30 36.46 172.89 178.81
53.55 40.52 174.11 179.23
50.86 36.39 173.18 177.38
179.13
ov, overlapped by other peaks.
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282
Table 3 Selected 13C NMR data (d , ppm) of the HETPP moiety in the systems K2{[Zn(HETPPH)Cl2]2} /DDNKI (1:2) and K2{[Cd(HETPPH)Cl2]2} / DDNKI (1:2) at 298 and 273 K
C(2?) C(6?) 2?-CH3
(HETPPH) K
K2{[Zn(HETPPH)Cl2]2} /DDNKI (1:2)
K2{[Cd(HETPPH)Cl2]2} /DDNKI (1:2)
298 K
273 K
298 K
273 K
298 K
273 K
168.7 149.7 25.7
166.2 148.2 23.3
170.0 151.1 26.9
167.2 149.4 24.2
169.8 152.5 26.5
167.4 150.1 24.5
posed involving a copper coordination to phosphate oxygens, N(1?) of pyrimidine and the functional groups of the Asp1 residue as well [14,15].
stability; (iii) in these systems, the HETPP uses the pyrophosphate group and the N(1?) atom as metal coordination sites; the metal coordination environment is completed by peptide functional groups [14,15]; (iv) the HETPP adopts always the S conformation (free ligand, binary metal/HETPP systems and tertiary metal/HETPP /petide systems) [15 /17,19]. Evaluating our data, we clarify that the existence of metal/N(1?) and metal/pyrophosphate oxygens bonds and the adopted S conformation by HETPP during the intermediate catalytic steps, at least, do not occlude the catalytic cycle. It has been proposed earlier [18], that the S conformation stabilizes all intermediates containing a hydroxyl group at C(2a)-side chain by the intramolecular electrostatic interaction S(1) O(2a) thus facilitating the O(2a) /H proton ionization. Thus, while the V conformation of thiamin is active possibly in the initial step of catalysis, we suggest here that the substrate addition during the enzymic cycle, should impose the S conformation to thiamin molecule. Therefore, the detailed mechanism may be represented as follows (Scheme 4). The metal ions most probably serve as linkers between the protein and the coenzyme through the coenzyme pyrophosphate group. On the other hand, we had previously suggested that metal ions should enter the catalytic cycle after the addition of the substrate and the subsequent formation of ‘active aldehyde’ intermediates [19]. Any further role of metal binding to N(1?) however, such as a better control of the reactivity of the 4?-amino group, except the link with the protein, needs to be further investigated.
3.2.3. 1H /1H NMR 1 H/1H NOESY spectra of the tertiary Zn2 / and 2 Cd /peptide /HETPP systems have been recorded at 273 K. The important intramolecular cross-peaks observed are those between the protons C(6?)/H/C(2a) /H, C(6?)/H/C(2a)/CH3, and C(6?)/H/C(4) /CH3 from HETPP molecule. The interaction of C(6?) /H proton with the C(2a)-side chain clearly indicates that this proton is directed to the thiazole ring. This spectroscopic feature is characteristic of thiamin derivatives adopting the S conformation [13]. The latter cross-peak between the C(6?)/H and the C(4) /CH3 suggests that thiazole ring was rotated in the same manner as in the crystal structure of HETPP [16]. Based on these, we conclude that the S conformation found in the crystal structure of HETPP and in aqueous solutions of metal / HETPP complexes, is also retained in the aqueous solutions of the tertiary M2 /peptide /HETPP systems.
4. Conclusion Our findings in this and previous works, are reviewed as follows: (i) in the binary metal/HETPP system, the metal binding sites are the pyrophosphate group and the N(1?) atom of the pyrimidine ring [14 /17]; (ii) the tertiary metal/HETPP /pentapeptide complexes around physiological pH [14,15] can be formed with wide
Table 4 31 P NMR data (d , ppm) of the HETPP moiety in the systems K2{[Zn(HETPPH)Cl2]2} /DDNKI (1:2) and K2{[Cd(HETPPH)Cl2]2} /DDNKI (1:2) at 298 and 273 K Pa
(HETPPH) K K2{[Zn(HETPPH)Cl2]2} /DDNKI (1:2) K2{[Cd(HETPPH)Cl2]2} /DDNKI (1:2) a
DdPa
Pb
a
DdPb a
2
JPaPb (Hz)
298 K
273 K
298 K
273 K
298 K
273 K
298 K
273 K
298 K
273 K
/10.6 /11.1 /9.7
/10.00 /8.71 /7.78
/8.8 /7.9 /6.0
/5.92 /5.64 /3.89
/0.5 /0.9
/1.29 /2.22
/0.9 /2.8
/0.28 /2.03
20.1 17.8 15.7
21.06 16.20 14.58
Difference in ppm of the corresponding NMR signals for the free ligand and the complexes at the same pH (Dd/dligand/dcomplex).
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Scheme 4. Proposed mechanism for thiamin catalysis.
5. Supplementary material The assignments of 1H NMR data: (i) of metal/ DDNKI recorded at 298 and 273 K; (ii) of metal / HETPP /DDNKI systems for peptide moiety at 298 and 273 K; (iii) of metal/HETPP /DDNKI systems for HETPP molecule at both 298 and 273 K. The assignments of 13C NMR data of the above systems are available from the author.
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