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435 — Interaction of FAD and NAD with aromatic amino acids
569
Bioelectrachemishy and Biaenergetics. 8 (1981) 569-574 A section of J. ElectmanuI. Chem.. and constituting Vol. 128 (1981) Rlsevier Sequoia S.A., Lausanne - E’rinted in The Netherlands
435-
EPo’gERAClTON
OF FAD AND NAD
WITH AROMATIC
AMKNO ACIDS
C_L.CHATi'ERJEE National Institute of Arthritis, Bethesda. MD 20205 (U.S.A.)
Metabolic
and Digestive
Diseases,
National
Itzstitute
of Health,
A. TORSTENSSON Analytical
Chemistry.
Chemical
Center,
University
of Lund,
S-220
Oi, Lund
(Sweden)
C.K. MITRA * Irstitute of Biomokcular NY 12222 (U.S.A.)
Stereodynamics.
State
University
of PJew York at Albany.
Albany.
(Revised manuscript received May 19th 1981)
The interaction of FAD and NAD (both in the oxidized forms) has been studied by U-V.viz.spectroscopy as well as by p.m.r. studies. The interaction of these two coenzymes with the four aromatic ammo acids phenylalanine, histidine, tyrosine and tryptophan has been studied in dilute aqueous solutions as a function of temperature. It appears that the interaction of NAD with these four aromatic amino acids is insignificant under the experimental conditions_ On the other hand, it appears that the interaction of FAD with tryptophan is small but significant. The effects due to self-aggregation and intermolecular stacking seem small for FAD and negligible for NAD under the experimental conditions involved. The small but significant chemical shifts observed for the FAD-tryptophan system suggest a geometric arrangement in which the two aromatic bases overlap each other. The interaction is probably a charge transfer type, but the effect is too small to be conclusively decided from the U.V.vi.sspectra_ It is probable that similar interaction is present for other systems but is too weak to be detected under the experimental conditions employed. The strength of the interaction is probably directly related to the effectiveness of the overlap of the two bases involved.
INTRODUCTION
The coenzymes Nicotinamide Adenine Dinucleotide (NAD, oxidized form) and Flavin Adenine Dintzcleotide (FAD, oxidized form) act as electron transfer mediators in many biological redox processes. The redox enzyme is essentially a passive catalyst and the substrate and the coenzyme undergo a coupled synergic oxidation-reduction. Therefore it is both important and interesting to study the electron transfer from the coenzyme and the sub&ate and vice versa. The electron transpo& process has not been completeely understiod, but a possibility exists that the substrate and the coenzyme bind close to each other at the active site on the e?zyme. If such a scheme is realistic, then the enzyme plays Little l
To Fphom correspondence should
03024598/81/0000-0000/$02.75
be addressed. @ L98L
Eketier
Sequoia
S.A.
role in the electron transfer except to bring the substrate and the coenzyme together with a favorable geometry. However, the electron transfer will be most
efficient only when the substrate and the coenzyme are very close to each other, becalusesuch interaction is like!y to fall off very fast with increasing distance. From a quantum mechanical point of view, electron transport across a longer distance is still possible if a suitable low-lying vacant molecular orbital is available at the active site and coupled to both the substrate and the coenzyme. It is not possible at present to test this hypothesis but once an accurate X-ray structure of the coenzyme-enzyme-substrate becomes available, this conjecture can be readily put to test. NAD and F_4D are both unusual dinucleotides in the sense that they contain 5’-5’ pyrophosphate linkage. Further the sugar attached to the flavin moiety in FAD is a ribitol, an open chain sugar. A possible reason for the latter fact may well lie in steric considerations, e.g., there may be too much of steric interference
between the fiavin base and a ribose.
It has already been demonstrated that NAD and FAD have flexible conformations in aqueous solutions ]1,2]. Thus it is highly probable that the coenzyme may adopt a completely different conformation upon binding to the enzyme. Binding of NAD” to malate dehydrogenase has been studied in some detail 133. It is interesting to note that in the X-ray analysis, the nicot~~ide ring is sxurroundedby histidine, arginine and tyrosine. Although these are tentative assignments, the authors claim that the NAD binds with an open conformation and the adenine is too far away to influence significantly the redox electron transfer process. Our earlier work also suggests that in dilute solution the interaction between the two bases in NAD and FAD is too smalI to be significant ]4,5]. Perahia et al. have also concluded from theoretical considemtions that NAD prefers an extended structure, although various folded conformations are also probable [ 6] _ The theoretical details of the charge transfer interaction between molecules were developed by Mill&en around 1950 [‘7]. Although quite elaborati theories are now available, we have restricted ourselves to a qualitative analysis mainly due to a lack of detailed knowledge of the conformations of the interacting systems and their complexities. We have studied the interaction of FAD and NAD with four aromatic aminoacids: phenylanahne, tyrosine, histidine and tryptophan. The U.V.-vis spectra were recorded to study the charge transfer interaction, if any. The n.m.r. study was made to study in detail the interaction. The n.m.r. chemical shifts are extremely sensitive to the intermoleeuku interaction. Since the interaction will be a function of temperature, temperature variation was employed to delineate the interaction between the coenzyme and the amino acids.
All the chemicals were obtained from Sigma Chemical Co. and were used without further purification. The ultraviolet--visible spectra (U.V_-vis, 200 nm
to 600 nm) were recorded on a Beckmann AC’I’AC111 spectrophotometes with thermostatted cuvettes. The n.m.r_ spectra were recorded on a JEOL FCXl.00 n.m.r. spectrometer in the FT mode (8K data points, 2000/2 spectral width)_
571
For the U.V.-vis study, 0.1 n&I solutions of FAD, NAD’, phenylalanine (Phe)! tyrosine (Tyr), histidine (His) and tryptophan (Trp) were made in 0.1 M phosphate buffer at pH 7.0. The concentrations of NAD’ and FAD were determined spectrophotometrically using the standard values of extinction coefficients [E&9] _ For other solutions made under similar conditions, concentrations were obtained gravimetricahy. The mixtures of coenzymes and amino acids, viz. NAD + Phe, NAD f Tyr, NAD f His, NAD f Trp, FAD f Phe, FAD + Tyr, FAD f His and FAD C Trp were also recorded in addition to NAD and FAD alone. All these spectra were recorded with a blank phosphate buffer in the second optical path. Two sets of spectra were obtained, one at 10°C and the other at 60°C. For the n.m.r_ study, 5 m&f solutions in 0.1 12/1phosphate buffer at pH 7.0 were used to enhance the signal strength_ All the samples were lyophilized three times to eliminate residual water. All the concentrations were obtained gravimetrically and therefore are not exact. However, this does not interfere with our general conclusions. DSS (2,2dimethyl-2-silapentane-5-sulphonate, sodium salt) was used as an internal standard. Two sets of spectra were recorded, one at 23°C and the other at 65”C, except for FAD f Trp system, where the spectra were recorded from LO to 60°C in steps of 5°C increments. RESULTS
The U.V.-vis spectra of NAD and NAD l amino acids are shown in Fig. la, at 10°C and at 60°C. The spectra clearly suggest that the temperature has virtually no perceptible effect on the interaction, if any, of the coenzyme and ammo acids under the experimental conditions. However, it is still possible that an interaction might be observed at a lower temperature and at 10°C the interaction is too weak compared to the thermal effects. At lower temperatures, the thermal effect subsides and other interactions will predominate. This was not attempted because of experimental difficulties in recording spectra at too low a temperature. The spectra for FAD and FAD f amino acids are shown in Fig. lb. The essential conclusions are the same, except that for the FAD f Trp system a small but significant change could be observed. This prompted us to study the FAD f Trp system in much greater detail. The difference is quite small and can be seen only by overlaying the two spectra on each other. A possible alternative would have been to use different spectroscopy, but this was not unde_&aken because of experimental difficulties. The n.m.r_ is a quite powerful tool compared to the U.V.4.s spectroscopy and is capable of providing more detailed information. The interaction between the coenzyme and the amino acid is a function of the degree of overlap between the two aromatic bases. Therefore it is expected intuitively that flavin will interact more strongly with a given amino acid than NAD. Similarly, FAD is expected to interact more strongly with an amino acid with a longer base. Bearing this in mind, we can really explain why FAD and Trp interaction is clearly observable whereas others are not. Further, since chemical shifts in n.m.r. are far more sensitive than the electronic transitions in U.V.vis spectroscopy to these charge transfer interactions, more accurate and meaningful results can be deduced from the n.m.r. spectral data.
NACl+Trp
NAC+Tyr
Fig. 1. The U.V.-vis spectra of FAD (a) and NAD (b) alone and in the presence of various amino acids. The lower curves represent spectra taken at 10” C and the upper curves are the spectra at 6O’C. No large significant changes can be seen in these cures although a careful comparison of the FAD + Trp system reveals 2 smal! shift towards the lower frequency_
In Table 1, we have presented the chemical shifts of NAD and FAD protons at the two experimental temperatures in the presence of the three aromatic amino acids Phe, His and Trp. The base protons of the aromatic amino acid residues have not been listed mainly because of difficulty in comparison. However, they also do not show appreciable shifts in the presence of the coenzyme. A careful examination of Table 1 reveals that NAD’ protons do not show any intrinsic temperature dependence. This suggests that at the concentration employed and under the experimental conditions, self-aggregation and column formation does not take place to any appreciable extent. Therefore, shift changes observed with NAD’ t amino acids must be ascribed to charge transfer type interactions in which the nicotinamide residue of NAD’ and the aromatic residue of the bases stack against each other. It is interesting to note at this point that such stacking has been observed in single crystal studies [IO]. The error in our experimental chemical shift is approximately ~0.001 p-p-m. and thus it can be concluded that NAD+ also interacts with Trp in a manner similar to FAD, but to a smaller extent. The interaction is of the similar magnitude for His and Phe. -4s expected, the shift values approach the values for free coenzymes at high temperatures, suggesting that the interaction between the coenzyme and the amino acids break down at high temperatures due to the thermal effects. Although the solutions used for n.m.r. were quite dilute (-5 n&f in coenzyme
573
TABLE
I.
Effect on the aromatic coenzyme protons by various amino acids chemical shifts are in p.p.m. with respett to DSS)
(All
CC)
HCSF
HCSF
20 65
7.57 7.77
7.53 7.64
20 65
7.68 7.78
7.56 7.60
Coenzyme + His
20 65
7.68 7.78
7.57 7.63
Coenzyme + Tq
20 65
7.62 7.78
7.56 7.63
Coenzyme, alone Coenzyme + Phe
Nicotinamide protons in NAD
Flavin protons in F,4D a
Temp.
6
HC2N
HC4N
HCSN
HCGN
9.34
8.88 8.8s
8.23 8.23
9.18
9.33
8.83
9.33
8.86
8.18 8.23
9.15 9.17
9.33 9.34
8.83 8.88
8.18 8.24
9.l.5 9.19
9.3’1 9.32
8.8X 5.86
8.17 8.22
9.13
9.34
9.18
9.17
a The
assignments are from Ref. 11. b The assignments are from Ref. L2.
f 5 m&I in amino acids), it can be seen that the flavin proton still shows appreciable temperature dependence. This suggests that some degree of intermolecular stacking is still present for FAD at 5 mM concentration. In fact, this temperature dependence is larger thzn when amino acids are present together with FAD. This needs some correction and the difference in chemical shift when the amino acid (tryptophan) is present and absent, are presented in Table 2, from 5 to 65°C. The values in the Easttwo rows show these differences for HC5 and HC8 of flavin. The results suggest that there is some small but significant interaction present between the coenzyme (FAD) and amino acids (tryptophan). However, we cannot discount the interaction between the other coenzyme (NAD) or other amino acids (Phe, His).
TABLE
2
The temperature dependence of the flavin aromatic protons in FAD in presence and absence of kyptophzn (All chemical shifts are in p.p.m. with respect to DSS) Proton
5OC
10°C
15°C
20” c
25°C
3o*c
60” C
65°C
FAD &one
HC8F HC5H
7.48 7.48
7.50 7.50
7.54 7.51
7.57 7.53
7.60 7.53
7.64 7.56
7.77 7.64
7.77 7.64
FAD f tryPtoPh=
HCBF HC5F
7.54 7.54
7.57 7.57
7.58 7.56
7.62 7.56
7.66 7.57
7.67 7.58
7.79 7.63
7.78 7.63
Diff a
HCSF HC5F
0.06 0.06
0.07 0.07
0.04 0.05
0.05 0.03
0.06 0.04
0.03 0.02
0.02 -0.01
0.01 0.01
a The difference corresponds to the same proton chemical shift at the same temperature in presence and absence of the amino acid.
574 CONCLUSIONS
Weber [ 131 concluded from fluorescence studies that the charge transfer between the two aromatic bases in NADH is possible, However, from our studies on U-V.-vis spectra we could not observe significant interaction between the two bases in NAD’. Weber’s conclusions are supported by Shifrin [14f under various conditions of solvent and pH. However, the mode of interaction observed in a fluorescence study and in the U-V.-vis spectra are quite different. Sarma and Kaplan [15f have concluded that NADH maintains its conformation on binding to lactate dehydrogenase. However, the intimate detailed geometry of tile binding site is not available due to poor X-ray crystal resolution. We have demonstrated in this study unequivocally that FAD interacts with tryptophan in aqueous solution. The interaction of NAD’ with tryptophan has been demonstrated in single crystal studies [IO] and our results suggest that such an interaction is possible. Although U.V.-vis specti do not show Zargeshift towards lower wavelength for any of the systems, n.n.r. studies show clearly small but significant interaction between the coenzyme and the amino acids_ However, it is likely that under more favorable conditions we shall be able to observe a larger chemical shift and thereby will be able to predict unambiguously a possible stacking geometry.
The authors are thankfuf to the referees for their constructive criticisms. REFERENCES 1 Lt. Ktinosbo and Y. Kyogoku. Biochemistry. 11 (1972) 741. 2 R.H. Sarma ad X.0. Kaplan. Biochemistry. 9 <1970) 557. 3 L..J.B a-asak end L.E. Webb in Structure and Conformation of ;*;iucieic Acids and Pmteirr Nucleic Acid Interactions. X1. Sundaralingam and S.T. R20 (Editors). University Park Press. Baltimore, 1975. p_ 3i3. 4 C. Mitm and A. Torstenson. Bioelectmcbem. Bioenerg.. 5 (1978) 601. 5 C. Mitra sod h. Torstenson. Bioelechochem. Bioeners.. 7 (1980) 7iS. 6 D. Per&i% 3. Pand A. S-. in Stmc:ure and Conformation oL Nucleic Acids aad Protein Nucleic Acid Interactions. Xl. Sundaralingam and S.T. Rao (Editors). University Park Press, Baltimore. 1975. P. 685. 7 R.S. Mu&ken. 3. Am. Chem. Sot.. 74 (1952) 811. 8 C.R.C. Hmdbook of Chemistry and Phudcs. C.R.C. Press, 53rd e&x.. P. C-37. 9 H. Beinert. in P.D. Bayer. H. Lardy and K. _MyrbZck (Editors). The Enzymes. 2nd edn.. Vol. II. Part It. Academic Press. New York and London. 1960. p. 339. LO J.R. Heniott. A_ Cam-an and D.A. Derdnieau.J. Am. Chem. Sot.. 96 (1974) LS85. 11 G. Kotowyer. N. Ten& h1.P. Klein and hl. Calvin. J. BioL Chem,. 244 (1969) 5656. 12 R.H. Suma and R.J. &Xynott. J. Am. Chem. Sot.. 95 ClS’Y3) ?-%70. 13 G. Weber. Nature (London). 180 (1957) 1409. 14 S. SbiFrin and N-0. Kaplan. Nature (Londoc). 182 (1959) 1529. 15 R.X. Sarma and N-0. ECrplan, Pmt. NatL Acad. Sd.. 67 (1970) 1375.
Bioelectrachemishy and Biaenergetics. 8 (1981) 569-574 A section of J. ElectmanuI. Chem.. and constituting Vol. 128 (1981) Rlsevier Sequoia S.A., Lausanne - E’rinted in The Netherlands
435-
EPo’gERAClTON
OF FAD AND NAD
WITH AROMATIC
AMKNO ACIDS
C_L.CHATi'ERJEE National Institute of Arthritis, Bethesda. MD 20205 (U.S.A.)
Metabolic
and Digestive
Diseases,
National
Itzstitute
of Health,
A. TORSTENSSON Analytical
Chemistry.
Chemical
Center,
University
of Lund,
S-220
Oi, Lund
(Sweden)
C.K. MITRA * Irstitute of Biomokcular NY 12222 (U.S.A.)
Stereodynamics.
State
University
of PJew York at Albany.
Albany.
(Revised manuscript received May 19th 1981)
The interaction of FAD and NAD (both in the oxidized forms) has been studied by U-V.viz.spectroscopy as well as by p.m.r. studies. The interaction of these two coenzymes with the four aromatic ammo acids phenylalanine, histidine, tyrosine and tryptophan has been studied in dilute aqueous solutions as a function of temperature. It appears that the interaction of NAD with these four aromatic amino acids is insignificant under the experimental conditions_ On the other hand, it appears that the interaction of FAD with tryptophan is small but significant. The effects due to self-aggregation and intermolecular stacking seem small for FAD and negligible for NAD under the experimental conditions involved. The small but significant chemical shifts observed for the FAD-tryptophan system suggest a geometric arrangement in which the two aromatic bases overlap each other. The interaction is probably a charge transfer type, but the effect is too small to be conclusively decided from the U.V.vi.sspectra_ It is probable that similar interaction is present for other systems but is too weak to be detected under the experimental conditions employed. The strength of the interaction is probably directly related to the effectiveness of the overlap of the two bases involved.
INTRODUCTION
The coenzymes Nicotinamide Adenine Dinucleotide (NAD, oxidized form) and Flavin Adenine Dintzcleotide (FAD, oxidized form) act as electron transfer mediators in many biological redox processes. The redox enzyme is essentially a passive catalyst and the substrate and the coenzyme undergo a coupled synergic oxidation-reduction. Therefore it is both important and interesting to study the electron transfer from the coenzyme and the sub&ate and vice versa. The electron transpo& process has not been completeely understiod, but a possibility exists that the substrate and the coenzyme bind close to each other at the active site on the e?zyme. If such a scheme is realistic, then the enzyme plays Little l
To Fphom correspondence should
03024598/81/0000-0000/$02.75
be addressed. @ L98L
Eketier
Sequoia
S.A.
role in the electron transfer except to bring the substrate and the coenzyme together with a favorable geometry. However, the electron transfer will be most
efficient only when the substrate and the coenzyme are very close to each other, becalusesuch interaction is like!y to fall off very fast with increasing distance. From a quantum mechanical point of view, electron transport across a longer distance is still possible if a suitable low-lying vacant molecular orbital is available at the active site and coupled to both the substrate and the coenzyme. It is not possible at present to test this hypothesis but once an accurate X-ray structure of the coenzyme-enzyme-substrate becomes available, this conjecture can be readily put to test. NAD and F_4D are both unusual dinucleotides in the sense that they contain 5’-5’ pyrophosphate linkage. Further the sugar attached to the flavin moiety in FAD is a ribitol, an open chain sugar. A possible reason for the latter fact may well lie in steric considerations, e.g., there may be too much of steric interference
between the fiavin base and a ribose.
It has already been demonstrated that NAD and FAD have flexible conformations in aqueous solutions ]1,2]. Thus it is highly probable that the coenzyme may adopt a completely different conformation upon binding to the enzyme. Binding of NAD” to malate dehydrogenase has been studied in some detail 133. It is interesting to note that in the X-ray analysis, the nicot~~ide ring is sxurroundedby histidine, arginine and tyrosine. Although these are tentative assignments, the authors claim that the NAD binds with an open conformation and the adenine is too far away to influence significantly the redox electron transfer process. Our earlier work also suggests that in dilute solution the interaction between the two bases in NAD and FAD is too smalI to be significant ]4,5]. Perahia et al. have also concluded from theoretical considemtions that NAD prefers an extended structure, although various folded conformations are also probable [ 6] _ The theoretical details of the charge transfer interaction between molecules were developed by Mill&en around 1950 [‘7]. Although quite elaborati theories are now available, we have restricted ourselves to a qualitative analysis mainly due to a lack of detailed knowledge of the conformations of the interacting systems and their complexities. We have studied the interaction of FAD and NAD with four aromatic aminoacids: phenylanahne, tyrosine, histidine and tryptophan. The U.V.-vis spectra were recorded to study the charge transfer interaction, if any. The n.m.r. study was made to study in detail the interaction. The n.m.r. chemical shifts are extremely sensitive to the intermoleeuku interaction. Since the interaction will be a function of temperature, temperature variation was employed to delineate the interaction between the coenzyme and the amino acids.
All the chemicals were obtained from Sigma Chemical Co. and were used without further purification. The ultraviolet--visible spectra (U.V_-vis, 200 nm
to 600 nm) were recorded on a Beckmann AC’I’AC111 spectrophotometes with thermostatted cuvettes. The n.m.r_ spectra were recorded on a JEOL FCXl.00 n.m.r. spectrometer in the FT mode (8K data points, 2000/2 spectral width)_
571
For the U.V.-vis study, 0.1 n&I solutions of FAD, NAD’, phenylalanine (Phe)! tyrosine (Tyr), histidine (His) and tryptophan (Trp) were made in 0.1 M phosphate buffer at pH 7.0. The concentrations of NAD’ and FAD were determined spectrophotometrically using the standard values of extinction coefficients [E&9] _ For other solutions made under similar conditions, concentrations were obtained gravimetricahy. The mixtures of coenzymes and amino acids, viz. NAD + Phe, NAD f Tyr, NAD f His, NAD f Trp, FAD f Phe, FAD + Tyr, FAD f His and FAD C Trp were also recorded in addition to NAD and FAD alone. All these spectra were recorded with a blank phosphate buffer in the second optical path. Two sets of spectra were obtained, one at 10°C and the other at 60°C. For the n.m.r_ study, 5 m&f solutions in 0.1 12/1phosphate buffer at pH 7.0 were used to enhance the signal strength_ All the samples were lyophilized three times to eliminate residual water. All the concentrations were obtained gravimetrically and therefore are not exact. However, this does not interfere with our general conclusions. DSS (2,2dimethyl-2-silapentane-5-sulphonate, sodium salt) was used as an internal standard. Two sets of spectra were recorded, one at 23°C and the other at 65”C, except for FAD f Trp system, where the spectra were recorded from LO to 60°C in steps of 5°C increments. RESULTS
The U.V.-vis spectra of NAD and NAD l amino acids are shown in Fig. la, at 10°C and at 60°C. The spectra clearly suggest that the temperature has virtually no perceptible effect on the interaction, if any, of the coenzyme and ammo acids under the experimental conditions. However, it is still possible that an interaction might be observed at a lower temperature and at 10°C the interaction is too weak compared to the thermal effects. At lower temperatures, the thermal effect subsides and other interactions will predominate. This was not attempted because of experimental difficulties in recording spectra at too low a temperature. The spectra for FAD and FAD f amino acids are shown in Fig. lb. The essential conclusions are the same, except that for the FAD f Trp system a small but significant change could be observed. This prompted us to study the FAD f Trp system in much greater detail. The difference is quite small and can be seen only by overlaying the two spectra on each other. A possible alternative would have been to use different spectroscopy, but this was not unde_&aken because of experimental difficulties. The n.m.r_ is a quite powerful tool compared to the U.V.4.s spectroscopy and is capable of providing more detailed information. The interaction between the coenzyme and the amino acid is a function of the degree of overlap between the two aromatic bases. Therefore it is expected intuitively that flavin will interact more strongly with a given amino acid than NAD. Similarly, FAD is expected to interact more strongly with an amino acid with a longer base. Bearing this in mind, we can really explain why FAD and Trp interaction is clearly observable whereas others are not. Further, since chemical shifts in n.m.r. are far more sensitive than the electronic transitions in U.V.vis spectroscopy to these charge transfer interactions, more accurate and meaningful results can be deduced from the n.m.r. spectral data.
NACl+Trp
NAC+Tyr
Fig. 1. The U.V.-vis spectra of FAD (a) and NAD (b) alone and in the presence of various amino acids. The lower curves represent spectra taken at 10” C and the upper curves are the spectra at 6O’C. No large significant changes can be seen in these cures although a careful comparison of the FAD + Trp system reveals 2 smal! shift towards the lower frequency_
In Table 1, we have presented the chemical shifts of NAD and FAD protons at the two experimental temperatures in the presence of the three aromatic amino acids Phe, His and Trp. The base protons of the aromatic amino acid residues have not been listed mainly because of difficulty in comparison. However, they also do not show appreciable shifts in the presence of the coenzyme. A careful examination of Table 1 reveals that NAD’ protons do not show any intrinsic temperature dependence. This suggests that at the concentration employed and under the experimental conditions, self-aggregation and column formation does not take place to any appreciable extent. Therefore, shift changes observed with NAD’ t amino acids must be ascribed to charge transfer type interactions in which the nicotinamide residue of NAD’ and the aromatic residue of the bases stack against each other. It is interesting to note at this point that such stacking has been observed in single crystal studies [IO]. The error in our experimental chemical shift is approximately ~0.001 p-p-m. and thus it can be concluded that NAD+ also interacts with Trp in a manner similar to FAD, but to a smaller extent. The interaction is of the similar magnitude for His and Phe. -4s expected, the shift values approach the values for free coenzymes at high temperatures, suggesting that the interaction between the coenzyme and the amino acids break down at high temperatures due to the thermal effects. Although the solutions used for n.m.r. were quite dilute (-5 n&f in coenzyme
573
TABLE
I.
Effect on the aromatic coenzyme protons by various amino acids chemical shifts are in p.p.m. with respett to DSS)
(All
CC)
HCSF
HCSF
20 65
7.57 7.77
7.53 7.64
20 65
7.68 7.78
7.56 7.60
Coenzyme + His
20 65
7.68 7.78
7.57 7.63
Coenzyme + Tq
20 65
7.62 7.78
7.56 7.63
Coenzyme, alone Coenzyme + Phe
Nicotinamide protons in NAD
Flavin protons in F,4D a
Temp.
6
HC2N
HC4N
HCSN
HCGN
9.34
8.88 8.8s
8.23 8.23
9.18
9.33
8.83
9.33
8.86
8.18 8.23
9.15 9.17
9.33 9.34
8.83 8.88
8.18 8.24
9.l.5 9.19
9.3’1 9.32
8.8X 5.86
8.17 8.22
9.13
9.34
9.18
9.17
a The
assignments are from Ref. 11. b The assignments are from Ref. L2.
f 5 m&I in amino acids), it can be seen that the flavin proton still shows appreciable temperature dependence. This suggests that some degree of intermolecular stacking is still present for FAD at 5 mM concentration. In fact, this temperature dependence is larger thzn when amino acids are present together with FAD. This needs some correction and the difference in chemical shift when the amino acid (tryptophan) is present and absent, are presented in Table 2, from 5 to 65°C. The values in the Easttwo rows show these differences for HC5 and HC8 of flavin. The results suggest that there is some small but significant interaction present between the coenzyme (FAD) and amino acids (tryptophan). However, we cannot discount the interaction between the other coenzyme (NAD) or other amino acids (Phe, His).
TABLE
2
The temperature dependence of the flavin aromatic protons in FAD in presence and absence of kyptophzn (All chemical shifts are in p.p.m. with respect to DSS) Proton
5OC
10°C
15°C
20” c
25°C
3o*c
60” C
65°C
FAD &one
HC8F HC5H
7.48 7.48
7.50 7.50
7.54 7.51
7.57 7.53
7.60 7.53
7.64 7.56
7.77 7.64
7.77 7.64
FAD f tryPtoPh=
HCBF HC5F
7.54 7.54
7.57 7.57
7.58 7.56
7.62 7.56
7.66 7.57
7.67 7.58
7.79 7.63
7.78 7.63
Diff a
HCSF HC5F
0.06 0.06
0.07 0.07
0.04 0.05
0.05 0.03
0.06 0.04
0.03 0.02
0.02 -0.01
0.01 0.01
a The difference corresponds to the same proton chemical shift at the same temperature in presence and absence of the amino acid.
574 CONCLUSIONS
Weber [ 131 concluded from fluorescence studies that the charge transfer between the two aromatic bases in NADH is possible, However, from our studies on U-V.-vis spectra we could not observe significant interaction between the two bases in NAD’. Weber’s conclusions are supported by Shifrin [14f under various conditions of solvent and pH. However, the mode of interaction observed in a fluorescence study and in the U-V.-vis spectra are quite different. Sarma and Kaplan [15f have concluded that NADH maintains its conformation on binding to lactate dehydrogenase. However, the intimate detailed geometry of tile binding site is not available due to poor X-ray crystal resolution. We have demonstrated in this study unequivocally that FAD interacts with tryptophan in aqueous solution. The interaction of NAD’ with tryptophan has been demonstrated in single crystal studies [IO] and our results suggest that such an interaction is possible. Although U.V.-vis specti do not show Zargeshift towards lower wavelength for any of the systems, n.n.r. studies show clearly small but significant interaction between the coenzyme and the amino acids_ However, it is likely that under more favorable conditions we shall be able to observe a larger chemical shift and thereby will be able to predict unambiguously a possible stacking geometry.
The authors are thankfuf to the referees for their constructive criticisms. REFERENCES 1 Lt. Ktinosbo and Y. Kyogoku. Biochemistry. 11 (1972) 741. 2 R.H. Sarma ad X.0. Kaplan. Biochemistry. 9 <1970) 557. 3 L..J.B a-asak end L.E. Webb in Structure and Conformation of ;*;iucieic Acids and Pmteirr Nucleic Acid Interactions. X1. Sundaralingam and S.T. R20 (Editors). University Park Press. Baltimore, 1975. p_ 3i3. 4 C. Mitm and A. Torstenson. Bioelectmcbem. Bioenerg.. 5 (1978) 601. 5 C. Mitra sod h. Torstenson. Bioelechochem. Bioeners.. 7 (1980) 7iS. 6 D. Per&i% 3. Pand A. S-. in Stmc:ure and Conformation oL Nucleic Acids aad Protein Nucleic Acid Interactions. Xl. Sundaralingam and S.T. Rao (Editors). University Park Press, Baltimore. 1975. P. 685. 7 R.S. Mu&ken. 3. Am. Chem. Sot.. 74 (1952) 811. 8 C.R.C. Hmdbook of Chemistry and Phudcs. C.R.C. Press, 53rd e&x.. P. C-37. 9 H. Beinert. in P.D. Bayer. H. Lardy and K. _MyrbZck (Editors). The Enzymes. 2nd edn.. Vol. II. Part It. Academic Press. New York and London. 1960. p. 339. LO J.R. Heniott. A_ Cam-an and D.A. Derdnieau.J. Am. Chem. Sot.. 96 (1974) LS85. 11 G. Kotowyer. N. Ten& h1.P. Klein and hl. Calvin. J. BioL Chem,. 244 (1969) 5656. 12 R.H. Suma and R.J. &Xynott. J. Am. Chem. Sot.. 95 ClS’Y3) ?-%70. 13 G. Weber. Nature (London). 180 (1957) 1409. 14 S. SbiFrin and N-0. Kaplan. Nature (Londoc). 182 (1959) 1529. 15 R.X. Sarma and N-0. ECrplan, Pmt. NatL Acad. Sd.. 67 (1970) 1375.