XH NMR STUDIES OF THE BINDING OF EDTA TO BOVINE PANCREATIC RIBONUCLEASE
M A N F R E D B R A U E R and F.W. BENZ §
School of Pharmacy, University of Wisconsin, Madison, Wisc. 53706 (U.S.A.)
(Received Augus t 15 th , 1977)
Summary
EDTA binds at the active site of ribonuclease causing a selective downfield shift of the C2 proton resonance of His 12 at pH* 5.5 (pH* denotes an un- corrected glass electrode pH meter reading of a 2H20 solution). A dissociation constant for EDTA binding to ribonuclease of 1.70 mM was calculated from this chemical shift change. The pKa of His 12 increased from 5.79 in ribonuclease alone to 6.73 in the RNAase • EDTA complex. Compared to these effects, the other histidine residues were not significantly affected by EDTA.
EDTA was shown to act as a competitive inhibitor of cytidine 2',3'-cyclic phosphate hydrolysis by ribonuclease with a Ki of 1.37 mM at pH 5.5, 25°C.
Molecular model building suggests that three of the four carboxyl groups of EDTA could simultaneously interact with histidine 12, lysine 41 and lysine 7. A complex of this type would account for the data described herein.
Introduction
Ethylenediaminetetraacetate {EDTA) is one of the most common metal ion chelating agents used in biochemical research. While EDTA binds strongly with a wide variety of metal ions [1], the possibility that it may also interact signifi- cantly with biological macromolecules is often overlooked. In the course of preparing iodinated derivatives of bovine pancreatic ribonuclease (RNAase) (EC 3.1.4.22) for ~H NMR analysis, we observed repeatedly that the ~H NMR spectrum of these derivatives was perturbed in the histidine C2(H) region subsequent to purification by column chromatography, whereas the un- fractionated reaction mixture showed no perturbation. This perturbation was eventually traced to the presence of EDTA in the buffers used for chromatographic separation of iodinated ribonucleases [2]. The EDTA was not removed completely by simple dialysis or gel-filtration techniques,
§ To whom requests for reprints should be addressed.
187
indicating that the binding was reasonably strong. Due to this observation and since EDTA is often added when 31p NMR studies of nucleotide binding to ribonuclease are performed [3--5], we decided to characterize the EDTA binding by ~H NMR and steady state kinetic analysis.
Materials and Methods
Bovine pancreatic ribonuclease (Worthington grade RAF, phosphate free) was used without further purification. Cytidine 2',3-cyclic monophosphoric acid (sodium salt} was obtained from Sigma Chemical Company. EDTA (ACS certified) was purchased from Fisher Scientific Company. All other reagents used were either reagent grade or the best grade available. All solutions were prepared from distilled water which had been passed through a Barnstead mixed-bed resin cartridge. All glassware (except NMR tubes and quartz cuvettes) were siliconized with Siliclad to reduce protein binding. For NMR analysis, NaO2H was obtained from Aldrich Chemical Company, and 99.8% 2H20, [2H4]acetic acid and 2HC1 (all greater than 99 atoms % 2H) were obtained from Stohler Isotope Chemicals.
To prepare ribonuclease samples for ~H NMR analysis, ribonuclease was dissolved in 0.2 M [2H]acetate buffer, pH* 5.5. We use the symbol pH* to indicate an uncorrected glass electrode pH meter reading of a 2H20 solution. The electrodes are calibrated with normal H20 buffers [6]. The pH* was measured with a Radiometer model 20 PHM 64 pH meter with a Beckman 39030 combination electrode. The final enzyme concentration was 2 mM. All the NH protons of ribonuclease were exchanged for 2H by heating the solution to 60°C for 15 min and then allowing the solution to cool to room temperature [7]. All solutions contained from 1 to 3 mM dioxane as an internal chemical shift reference. ~H NMR spectra at 270 MHz were obtained with a Bruker WH 270 NMR spectrometer. The instrument was operated in the Fourier transform mode controlled by a Nicolet 1180 computer and was field-frequency locked to 2H in the solvent. The NMR probe temperature was maintained at 34.0°C + 0.5°C. Precision-bore 5 mm NMR tubes from Wilmad Glass Company were used for all NMR samples. To adjust the pH* of the NMR sample, 1.4 M ~HC1 or 1.2 M NaO2H was added slowly with stirring until the desired pH* meter reading (±0.04 pH* units) was obtained.
For the EDTA binding curves, each EDTA solution was prepared by weighing out dried EDTA powder (acid form), dissolving it in [2H]acetate buffer and adjusting the pH* of the solution to approximately pH* 5.5 with NaO2H. Known volumes of EDTA were added to the ribonuclease solution with a Hamilton microliter syringe, and the pH* was readjusted to pH* 5.5 with 2HC1 or NaO~H if necessary. An aliquot was then transferred to an NMR tube for analysis. The sample temperature was maintained at 34°C throughout EDTA addition, pH* readjustment and ~H NMR analysis.
The pH* dependence of the chemical shift of the C2 protons of the histidine residues in ribonuclease, in the presence of 63.4 mM EDTA, was studied in a similar manner. The pH* titration data were analyzed via a modified form of the Hill equation:
5H + - - 5 o b s d __ Ka n
5H ÷ -- 5H o K a n + [H+] n
188
where 5H ÷ and 5H ° are the chemical shifts of the fully pro tonated and un. pro tonated histidine residues respectively. ~obsd is the observed chemical shift at a given pH*, K a is the dissociation constant of the histidine and n is the Hill coefficient [6]. The nonlinear regression subroutine, NREG, (Madison Academic Computing Center) was used for this analysis. Both four parametel fits (Ki, 5H ÷, 5H °, n) and three parameter fits (n fixed at 1) were obtained.
Ribonuclease activity was determined with cytidine 2',3'-cyclic monophos. phate (cyclic CMP) as substrate according to the method of Crook et al. [8], except that the assay was carried out in 0.2 M acetate buffer, pH 5.5 at 25°C. The concentrat ions of cyclic CMP solutions were determined spectrophoto. metrically based on a molar ext inct ion coefficient of 8650 at 268 nm, pH 7.1 [9]. Substrate concentrat ions were varied from 0.2 to 1.0 mM with a constanl enzyme concentrat ion of 6.36 • 10 -~ M. The enzymatic degradation of cyclic CMP was followed spectrophotometr ical ly on a Cary 118C spect rophotomete i as an increase in absorbance at 290 nm (constant slit width of 0.2 mm): a 3ifference molar ext inct ion coefficient between cyclic CMP and 3'-CMP oi 1150 (290 nm, pH 5.5) was used [10]. To characterize the enzyme inhibition caused by EDTA, the same range of cyclic CMP concentrat ions were used, and a constant concentra t ion of EDTA was added. The kinetic parameters, V and Km (with or wi thout EDTA), were obtained by a weighted least squares com- puter analysis of a Lineweaver-Burk plot [ 11].
Results
Two separate experiments were performed in which increasing amounts of EDTA were added to 2 mM ribonuclease in 0.2 M [2H]acetate buffer at pH* 5.5 with a 270 MHz 1H NMR spectrum taken after each addition. A plot of the chemical shifts of the histidyl protons versus concentrat ion of EDTA added is presented in Fig. 1. The revised assignment of the proton resonances of ribonuclease to their corresponding histidine residues is assumed throughout [12--14] . The resonances of the His 105 and His 48 C2 protons move slightly downfield with increasing EDTA concentrations. The His 12 resonance shifts dramatically downfield from 4.57 to 5.32 ppm from dioxane. The His 119 resonance moves slightly upfield as the EDTA concentra t ion is raised to 2 mM (during which His 12 has undergone more than half of its shift) and then gradually moves downfield as the EDTA concentra t ion is raised further.
It is obvious that His 12 is markedly affected by EDTA, while the other histidine residues are not altered dramatically. The fractional occupancy, r, foi EDTA binding to ribonuclease could be determined from the change in chemical shift of the His 12 C2 proton resonance as a funct ion of EDTA con- centration:
(5obsd -- 5E) r--
(5.i - 6~)
where 5E is the chemical shift of His 12 in ribonuclease alone, 5E~ is the chemical shift of His 12 in the presence of saturating amounts of EDTA, and 5obsd is the observed chemical shift of His 12 at any particular EDTA con- centration. If we assume that EDTA binding follows a classical Langmuir
189
G 5.C 2,.
i ! S 1
His t C2 , -I ~ z
His 119 C2 . ., . . ~1 ~ -" - - J
His48
4 . 0
o ~
His 105 C4
[[~a] ( - m
Fig. 1. C h e m i c a l sh i f t s o f r i bonuc lease h i s t id ine p e a k s as a f u n c t i o n of to ta l E D T A c o n c e n t r a t i o n , p H * 5.5, 34°C. The R N A a s e c o n c e n t r a t i o n was 2 raM. The circles and t r iangles r e p r e s e n t da ta f r o m t w o separa te e x p e r i m e n t s .
adsorption isotherm, the dissociation constant, KD, and number of binding sites, n, can be obtained from the equation:
[EDTA] _ KD + [EDTA]
r n n
[15]. The number of binding sites was found to be 1.02 and the K D was found
3O
,Q
IC
5.~
5.C
0 ~ 4 . 5
J 4 . 0
o ;o ~o ~o [~OTA] ,,~
~ 12
H i s 105 C 4
4.0 .~.o 6:o ~o do ~o pH
Fig. 2. The b i n d i n g of E D T A to the His 12 res idue of r ibonuc lease . The f r ac t iona l o c c u p a n c y , r, was d e t e r m i n e d by the c h e m i c a l sh i f t o f t he His 12 C2 p r o t o n r e s o n a n c e as a f u n c t i o n o f E D T A concen t r a - t ion . The line was o b t a i n e d f r o m a lineax reg ress ion ana lys i s o f the data .
Fig. 3. 1H N M R t i t r a t i o n curves o f the h i s t id ine p e a k s in the r ibonuc lease . E D T A c o m p l e x . F o r these t i t r a t i o n curves , a so lu t i on of 2 m M r ibonuc lease a n d 63 .4 m M E D T A in 0 .2 M d e u t e r o a c e t a t e b u f f e r (34°C) was used . The curves are c o m p u t e r f i ts to the m o d i f i e d Hill e q u a t i o n wi th the p a r a m e t e r s l i s ted in Table I.
19]
appreciably. The dramatic downfield shift of the His 12 resonance upon EDTA binding seen in Fig. 1 can therefore be explained to a large degree by the increase in the pKa of His 12 induced by EDTA.
The effect of EDTA on the enzymatic activity of ribonuclease was studied. It was found that 6 mM EDTA altered the Km but not the V of ribonuclease (Fig. 4}. This indicated that EDTA was acting as a competitive inhibitor ot cyclic CMP. This is not unreasonable since EDTA binds near His 12 and His 12 is one of the active-site histidines involved in catalysis. From the ratio of the Km with and without EDTA, the Ki for EDTA binding to ribonuclease was found to be 1.37 mM at 25°C, pH 5.5. This value is essentially the same as the g D determined in the NMR experiment described above.
Preliminary kinetic studies of the variation in the pKi of EDTA with pH were done with essentially the same assay, except that a more versatile buffer system of 0.05 M Tris, 0.05 M acetate and 0.10 M NaC1 was employed. EDTA inhibition was found to be competitive from pH 4.5 to pH 8.0 [17]. The inhibition was maximal in the pH range (4.5--5.0) and gradually decreased as the pH increased, reflecting, in part, the deprotonation of His 12.
Discussion
The fact that EDTA binds to ribonuclease is not surprising in that other polyanions are known to inhibit RNAase activity [18]. However, the selectivity of the effects produced by EDTA does allow us to make some comments about the nature of the complex and how it differs from other known RNAase" inhibitor complexes [ 19].
The addition of EDTA to ribonuclease causes a selective downfield shift ot the His 12 C2 proton resonance at pH* 5.5. This shift was shown to follow a classical Langmuir absorption isotherm (Fig. 2), indicating that EDTA binds at a single site close to or at His 12. The alteration in chemical shift is, for the most part, due to the increase in the pK a of His 12 from 5.79 to 6.73 induced by 63 mM EDTA. The protonated form of His 12 may be stabilized by an electrostatic interaction or hydrogen bond with one of the charged carboxyl groups of EDTA resulting in an increased pKa. The true pKa of His 12 in the saturated RNAase. EDTA complex is probably somewhat higher, since the enzyme is 100% saturated at pH 4.5 at 63 mM EDTA but is 80% saturated at pH 7.5. Fig. 3 and Table I indicate that the titration curve for His 12 in the complex can be adequately represented by the theoretical expression for a single proton association-dissociation equilibrium (i.e., Hill coefficient approx. 1). The total shift on titration of His 12 was found to be 1.49 ppm. This is in contrast to the behavior of His 12 in RNAase where the total protonation shift is approximately 1.34--1.38 ppm and the titration curve does not fit the simple Henderson-Hasselbalch relationship. Various models have been proposed to account for the complicated titration behavior of both histidine 12 and 119 in native RNAase [16,20]. A common postulate of some of these models (models A, B and C [16], model 2 [20]) is a direct or indirect perturbation of the His 12 titration curve by an acidic group with a pK a in the range of pH 4.0--5.0. Other models, however, imply that the titration curve of histidine 12 reflects a mutual interaction between histidine 12 and 119 with (model 4
1 9 2
[20]) or without (model D [16], model 3 [20]) an additional spectroscopic perturbation. Since the full titration curve of His 12 in the RNAase. EDTA complex follows the simple Henderson-Haselbalch relationship, it would appear that EDTA binding has uncoupled the interaction of His 12 with the acidic group or with histidine 119. Dianionic mononucleotide inhibitors, such as 2'- or 3'-CMP, normalize the titration curves of both His 12 and His 119 [22--23], although the monoanionic 5'-phosphonate analog of the dinucleotide UpA, namely UpCH2A-, apparently does not [33]. The RNAase inhibitor ortho- phosphate, on the other hand, accentuates the inflection in the titration curve because of its opposite effects on the pKa of His 12 and the acidic group [21].
The extra 0.11 ppm downfield shift of His 12 in the fully protonated complex is probably due to the interaction with the carboxyl group on the EDTA. Nucleotides (2'- and 3'-CMP) produce a larger downfield shift on binding (0.2--0.25 ppm), whereas orthophosphate produced a 0.1 ppm shift upfield [22].
The most dramatic difference between the effects produced by EDTA and other RNAase inhibitors, which have been studied by NMR [3--5,22--26], is that EDTA produces little, if any effect on the other histidine residues. This includes histidine 119, the other active site histidine, which is only 7 A from histidine 12 in the ribonuclease crystal [27]. Fig. 1 shows that His 119 under- goes a slight upfield shift and His 48 a slight downfield shift at low concentra- tions of EDTA while His 105 does not change appreciably. Table I indicates that the pK a values of His 105, 48 and 119 are essentially the same in the presence and absence of EDTA. In particular, the value for the Hill coefficient observed for His 119 in native RNAase remains as low, if not lower, in the EDTA complex despite the fact that His 12, which ~so had a low Hill coefficient in native RNAase, now shows a normal titration curve in the complex but with a higher pK a. The abnormal titration curves of His 12 and 119 in the native enzyme have been attributed to either mutual interactions between the two active site histidines [20] or the interactions between these residues and one or more acidic groups on the enzyme [16]. The selective effect of EDTA in normalizing the His 12 titration curve, while leaving the His 119 curve unchanged, would seem to support the latter hypothesis. However, other effects induced by very low concentrations of EDTA may provide evidence for an interaction between these active site histidines. Upon addition of less than 1 mM EDTA, the line width of His 119 C2 proton decreased, while the line width of the His 12 resonance increased. The widths of the His 105 and 48 peaks did not change. The concommitant opposing changes in both the chemi- cal shifts and line widths of His 12 and 119 may imply an interaction, direct or indirect, between these two active site histidines *
Fig. 4 shows that EDTA competitively inhibits RNAase hydrolysis of cyclic CMP. This substantiates the NMR data that EDTA binds tightly at the active site. The Ki at pH 5.5 was found to be 1.37 mM. This indicates that EDTA
* I t is a l so p o s s i b l e t h a t t h e d e c r e a s e i n t h e l ine w i d t h o f H i s 1 1 9 w a s d u e t o E D T A c h e l a t i o n o f t r a c e a m o u n t s o f p a x a m a g n e t i c i m p u r i t i e s s u c h as C u ( I I ) . I f t h i s w e r e t h e ca se , t h e n w e s h o u l d h a v e o b s e r v e d a d e c r e a s e i n t h e l ine w i d t h o f H i s 1 0 5 , s i nce b o t h X - r a y a n d N M R e v i d e n c e s u g g e s t t h a t b o t h H i s 1 1 9 a n d 1 0 5 are e s p e c i a l l y s e n s i t i v e t o c o p p e r [ 2 8 , 2 9 ] . F u r t h e r e x p e r i m e n t s wi l l be n e c e s s a r y t o c h a r a c t e r i z e t h e s e r e c i p r o c a l l i ne w i d t h c h a n g e s .
193
binds more strongly to ribonuclease than does phosphate (Ki = 4.25 mM), but less strongly than pyrophosphate (Ki = 0.115 mM) [18]. Since EDTA has four carboxyl groups with pKa values of 2.0, 2.67, 6.16 and 10.26 in free solution [30], it has, in addition to the carboxyl which interacts with His 12, one or possibly two additional charged carboxyl groups which can interact with other positively charged residues near the active site cleft. EDTA may simultaneously interact with two or three positively charged groups at the active site cleft of ribonuclease thus contributing to the overall stability of the complex.
We have built models of RNAase S [31] and EDTA with Labquip molecular models. If the EDTA backbone is constructed in a fully extended form, with the four carboxyl groups arranged to roughly maximize the distance between them, we find that two of the carboxyls can interact simultaneously with His 12 and Lys 41, and that the third carboxyl is also in position to interact with Lys 7. Thus, three of the four carboxyl groups on EDTA could interact simultaneously with three positively charged groups at the active site of RNAase. With the complex constructed in this way, His 119 is not close to any of the carboxyl groups on EDTA since the inhibitor is largely restricted to the opposite side of the active site cleft. If His 119 rotated into X-ray crystal posi- tion II or III [18], it would be closer to the fourth carboxyl, although the lack of any effect of EDTA on the NMR spectrum of His 119 argues against any direct interaction. This fact also does not support an alternate complex where one of the amino ends of the EDTA, with two carboxyls, bridges the active site cleft between His 119 and His 12 with the third carboxyl free to interact with either Lys 41 or Lys 7. Bradbury has devised a method for observing the NMR spectrum of chemically modified lysine residues [32]. Hence, it may be possible to use this method to derive more definitive information about the interaction of EDTA with specific lysine residues at the active site of RNAase.
Acknowledgements
This work was supported in part by grants from Research Corporation, UW- Graduate School and the Biomedical Research Support Grant from the National Institutes of Health. We are grateful to Sean Hehir for his assistance in the use of the 270 MHz NMR spectrometer and to G.L. Amidon and J.B. Watkins for computer programming assistance.
References
1 Schwarzenbach, G, (1957) Complexometr ic Titrations, Interscience, New York 2 Woody, R.W., Friedman, M.E. and Scheraga, H.A. (1966) Biochemistry 5, 2034--2042 3 Gorenstein, D.G. and Wyrwicz, A.M. (1973) Biochem. Biophys. Res. Commun. 54 ,976- -982 4 Gorenstein, D.G., Wyrwicz, A.M. and Bode, J. (1976) J. Am. Chem. Soc. 9S, 2308--2314 5 Haax, W., Thompson, J.C., Maurev, W. and Rilterjans, H. (1973) Eur. J. Biochem. 40, 259--266 6 Markley, J.L. (1975) Acc. Chem. Res. 8, 70--80 7 Benz, F.W., Roberts , G.C.K., Feeney, J. and Ison, R.R. (1972) Biochim. Biophys. Acta 278 ,233 - -238 8 Crook, E.M., Mathias, A.P. and Rabin, B.R. (1960) Biochem. J. 74, 234--238 9 Wiglet, P.W. (1968) in Biochemical Preparations (Lands, W.E.M., ed.), Vol. XII, pp. 107--110
14 Markley, J.L. (1975) Biochemistry 14, 3546--3553 15 Klotz, I.M. and Hunston, D.L. (1971) Biochemistry 10, 3065--3069 16 Cohen, J.S. and Shindo, H. (1975) J. Biol. Chem. 250, 8874--8881 17 Brauer, M. (1976) Masters Thesis, University of Wisconsin-Madison 18 Richards, F.M. and Wyckoff, H.W. (1971) in The Enzymes (Boyer, P.D., ed.), Vol. IV, pp. 647--806
Academic Press, New York 19 Benz, F,W. and Roberts , G.C.K. (1973) in Physico-Chemical Properties of Nucleic Acids (Duchesne
J., ed.), Vol. III, pp. 77--138 20 Markley, J.L. (1975) Biochemistry 14, 3562--3566 21 Cohen, J.S., Griffin, J.H. and A.N. Scheehter (1973) J. Biol. Chem. 248, 4305--4310 22 Meadows, D.H., Roberts, G.C.K. and Jardetzky, O. (1969) J. Mol. Biol. 45 ,491- -511 23 Rfiterjans, H. and Witzel, H. (1969) Eur. J. Biochem. 9 ,118- -127 24 Haar, W., Maurer, W. and Riiterjans, H. (1974) Eur. J. Biochem. 44 ,201- -211 25 Haffner, P.H. and Wang, J.H. (1973) Biochemistry 12, 1608--1618 26 Gorenstein, D.G. and Wyrwicz, A. (1974) Biochemistry 13, 3828--3836 27 Carlisle, C.H., Palmer, R.A., Mazumdar, S.U., Gormsky, B.A. and Yeates, D.G.R. (1974) J. Mol. Biol
85, 1--18 28 AUewell, N.M. and Wychoff, H.W. (1971) J. Biol. Chem. 246, 4657--4663 29 Patel, D.J., Woodward, C., Canuel, L.L. and Bovey, F.A. (1975) Biopolymers 14, 975--986 30 Jencks, W.P. and Regenstem (1976) in Handbook of Biochemistry and Molecular Biology (Fasman
G.D., ed.), Vol. I, 3rd edn., p. 310 31 Wyekoff, H.W., Tsernoglou, D., Henson, A.W. Knox, J.R., Lee, B. and Richards, F.M. (1970) J. Biol
Chem. 245 ,305 - -328 32 Brown, L.R. and Bradbury, J.H. (1975) Eur. J. Biochem. 54 ,219- -227 33 Griffin, J.H., Schechter, A.N. and Cohen, J.S. (1973) Ann. N.Y. Acad. Sci. 222, 693--707
