Phosphorus31 Nuclear Magnetic Resonance Spectral Assignments of Phosphorus Compounds in Soil NaOH–EDTA Extracts

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PHOSPHORUS-31 NUCLEAR MAGNETIC RESONANCE SPECTROSCOPY REVEALS TWO CONFORMATIONAL FORMS OF CHLOROACETOL PHOSPHATE-BOUND TRIOSEPHOSPHATE ISOMERASE Klans D. SchnackerzL T. K. Kuan2, Warren J. Goux2 and Robert W. Gracy3* 1Institute of Physiological Chemistry, University of Wiirzburg, D-8700 Wiirzburg, F.R.G. 2Department of Chemistry, University of Texas at Dallas, Dallas, Texas 3Department of Biochemistry and Molecular Biology, University of North Texas/Texas Coll. of Osteop. Med., Fort Worth, Texas 76107 Received October 23, 1990

SUMMARY. Chloroacetol phosphate covalently reacts with Glu-165 in the catalytic center of triosephosphate isomerase. Reaction of the enzyme with the substrate analogue results in two 31p resonances at 6.8 and 5.5 ppm. Dissociation with guanidinium chloride results in a single resonance at 4.5 ppm. Reassociation and redimerization of the triosephosphate isomerasechloroacetolphosphate complex restores only the resonance at 5.5 ppm. The two 31p resonances appear to represent different conformations of the enzyme which are trapped upon reaction with the affinity label. ~ 1990 AcademicP..... Inc.

Triosephosphate isomerase (EC 5.3.1.1) is the prototype of eight-stranded ct/~ barrel enzymes. Kinetic and thermodynamic studies have shown that the enzyme has evolved to a state of near "perfect" catalytic efficiency (1), and structural studies from several species have characterized the highly conservative housekeeping enzyme. In addition, the enzyme undergoes specific postsynthetic modifications (deamidation in mammals and oxidation in avian species) which appear to trigger its degradation in vivo (2-4). These modified forms of TPI accumulate in aging cells and tissues, and thus the enzyme has become a model to investigate the molecular basis for modified proteins in aging. We have observed that the binding of substrates enhances the deamidation of the enzyme suggesting a mechanism whereby the enzyme may "wear out" (5, 6). Physical studies on the

To whom correspondence should be addressed. Abbreviations: BME, 2-mercaptoethanol; CAP, chloroacetol phosphate; EDTA, ethylenediamine tetraacetic acid; GmHCI, guanidinium hydrochloride; HPLC, high performance liquid chromatography; NMR, nuclear magnetic resonance; PAGE, polyacrylamide gel electrophoresis; SDS, sodium dodecyl sulfate; TPI, triosephosphate isomerase (EC 5.3.1.1).

0006-291X/90 $1.50 Copyright © 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.

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enzyme, both in solution (7, 8) and in the crystalline state (9), have indicated that binding of substrate moves a "flexible loop" (residues 167-176) approximately 7/k to close over the active site. We have initiated a series of detailed studies to probe interactions between the catalytic center and the modification sites. One such approach is to utilize 31P-NMR probes of the catalytic center as reporters. We demonstrate here 3'P-NMR spectra of the affinity label chloroacetol phosphate and the CAP-labeled TPI. Reaction of the enzyme with CAP appears to trap two different conformational states of the CAP-enzyme complex. MATERIALS AND METHODS Chemicals: Chloroacetol phosphate was synthesized as the dimethylketal and hydrolyzed prior to use according to Hartman (10). Substrates, coenzymes, enzymes and all reagents were analytical grade or better and were purchased from Sigma. Enzyme; TPI was isolated from chicken muscle by the HPLC method of Jacobson et al. (11) and was homogeneous by both non-denaturing and SDS PAGE. The catalytic activity and protein concentrations were assayed as described previously (12). Reaction of CAP with TPI: The reaction of CAP with TPI (52 mg in 10.0 ml) was conducted essentially as described by Hartman (13) with a 3.5 mole excess of CAP per catalytic center. The reaction was carried out at pH 7.5, in 50 mM HEPES buffer containing 1 mM EDTA. After reaction at 4" C for 1 rnin., 1.5 mg of NaBH, was added for 30 min. to reduce the carbonyl, followed by the addition of 2-mercaptoethanol (70 mM) to stop the reaction and exhaustive dialysis against 50 mM HEPES, 1 mM EDTA, 70 mM BME. Under these conditions the enzyme was inactivated 97.5 to 99.8%. NMR techniques: Fourier transform 31P-NMR spectra were recorded at 72.86 MHz on a Bruker WH-180 wide-bore superconducting spectrometer. Sample volumes of 10 mi in 20 mm diameter tubes were used. A concentric 5 mm NMR tube containing 2H20 was employed as field/frequency lock. All spectra were recorded with broadband proton decoupling (0.4 W). In general, a 1200 Hz spectral width was acquired in 4096 data points with 60* pulse angle and 1.7-s repetition time. The exponential line broadening used prior to Fourier transformation was 10 Hz. Continuous air flow through the spectrometer probe head kept the temperature at 20°+1"C. Titration curves were obtained by iterative computer analysis (14, 15). RESULTS AND DISCUSSION Free chloroacetol phosphate exhibited a major (5.05 ppm) and three minor (4.27, 3.58, and 2.52 ppm) 31P-NMR resonances at pH 7.28. The major signal titrated with a pK of 6.02 (Fig. 1), whereas the signal furthest upfield exhibited a pK of 6.87 and probably represents ingrganic phosphate. The two smaller signals (4.27 and 3.58) were not discernible below pH 6. Since the CAP in aqueous solution exists as a mixture of the ketose and its hydrate, it is to be expected that the major resonance represents the hydrate. The smaller signals may reprsent the keto form and incompletely hydrolyzed dimethylketal. Fig 2A shows the 31P-NMR spectrum after reaction of TPI with CAP. Two 31p signals were observed at 6.79 ppm (30-35%) and 5.46 ppm (65-70%), respectively. The chemical shifts of 737

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& m

3

pH

Figure 1. pH dependence of the 3~p chemical shift of chloroacetol phosphate. Free cNomacetolphosphate (9.3raM) in 50 mM HEPES buffer containing 1 mM EDTA was titrated and the ~lp spectra recorded (O). Chemical shifts in ppm are downfield changes relative to 85% phosphoric acid. The solid titration line was obtained by iterative computer analysis of the experimental data. For details see Materials and Methods.

these resonances were unchanged over the pH range in which the enzyme is stable (pH 6-8). Several possibilities for the occurrence of the two signals were considered, including covalent or noncovalent binding of CAP at a second site on the protein, or two stable conformations of the CAP-TPI complex. In order to determine the nature of the two resonances, CAP-TPI was subjected to denaturation titrations with increasing concentrations of guanidinium chloride. After each addition of GmHC1 a 31P-NMR spectrum was recorded. In the presence of 0.2 and 0.4 M GmHC1 no differences in the spectra were observed, but at 0.6M GmHC1 the resonance at 6.8 ppm began to disappear and a new resonance at 4.6 ppm began to emerge (Fig 2B). Addition of further GmHC1 (0.8-4.0 M) resulted in single resonance at 4.6 ppm (Fig 2C). In all cases no additional 31p signals were observed. When the GmHCI was removed and the protein allowed to refold and redimerize, only the resonance at 5.5 ppm was observed (Fig 2D). Under these conditions the enzyme was reactivated. These data can be compared with previous denaturation titrations of TPI which showed a sharp sigrnoidal decrease in catalytic activity coincident with a transition of the s20,w values from 3.6 S to 1.7 S occurring between 0.7 and 0.9 M GmHC1 (16). These studies and the refolding experiments of Waley (17) established that the primary action of GmHC1 with TPI is disruption of the intrasubunit bonds rather than complete unfolding. Although Coulson et al. (18) originally reported that CAP reacted with a tyrosine in chicken TPI, this was shown to be incorrect, and that the site of reaction is solely Glu-165 (19). Hartman reacted TPI with CAP followed by reduction of the carbonyl with borohydride stabilizing the

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Figure 2. 31P_NMRspectra of CAP bound to TPI. (A) the spectrum obtained following reaction of the enzyme with CAP (see Methods for conditions). The sample had been exhaustively dialyzed against 50 mM HEPES buffer, 1 mM EDTA and 70 mM BME, pH 7.5. (B) spectrum obtained when sample A is made 0.6M in GmHCI. (C) sample A but containing 4M GmHC1. (D) sample C upon removal of the GmHC1 by dialysis against the buffer of A. The chemical shifts are relative to external 85% phosphoric acid. adjacent phosphate ester. A single peptide was isolated, sequenced and shown to contain the affinity label at Glu-165 (13). The possibility of the 31p resonance at 6.8 ppm being due to binding of CAP (either covalently or noncovalently) to a site other than Glu-165 appears to be unlikely because of the interconversion of the two resonances in GmHC1 and after reassociation. Thus, these data indicate that the two resonances observed represent two different conformations of CAP-TPI. The conformation corresponding to the resonance at (6.8 ppm) appears to be the less stable of the two, and thus far all attempts to recover this conformation from GmHC1 have been unsuccessful. The nature of the two conformations remains to beestablished, but it is tempting to 739

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speculate that in one conformation the "flexible loop" (residues 167-177) is closed over the catalytic center whereas in the other the site is open. Both conformations have been postulated from crystallographic studies (9). Finally, it should be pointed out that 31P-NMR has previously provided information on the degree of hydration and state of ionization of the substrates of TPI (20, 21). For example, the bound form of the competitive inhibitor, 2-phosphoglycolate, exhibits no change in chemical shift with pH indicating that it is bound to the enzyme as the trianion, with two negative charges on the phosphate and one negative charge on the carboxylate. These data are consistent with the pH independence of the 31p CAP-TPI resonances. All previous 31P-NMR probes of the enzyme have utilized reversibly binding substrates or substrate analogues.

The ability to trap two

conformational forms of the covalent TPI-CAP complex should provide an important new method to probe the details of the interactions of the catalytic center with other portions of the molecule. ACKNOWLEDGMENTS: These studies were supported by research grants from the Deutsche Forschungsgameinschaft (Schn 139/11-1), the National Institute on Aging (AG01274), The R. A. Welch Foundation (B0502), and The Alexander yon Humboldt Foundation. RWG is the recipient of a MERIT Award from the NIA. We thank Dr. M. Biihner for the iterative computer fit of the titration data. REFERENCES 1. Albery, W. J., and Knowles, J. R. (1977) Angew. Chem. Int. Ed. Engl. 16, 285-293. 2. Yuan, R. M., Talent, J. M., and Gracy, R. W. (1981) Mech. Ageing and Develop. 17, 151162. 3. Tang, C. Y., Yiiksel, K. 0., Jacobson, T. M., and Gracy, R. W. (1990) Arch. Biochem. Biophys. (in press). 4. Gracy, K. N., Tang, C. Y., Ytiksel, K. 0., and Gracy, R. W. (1990) Mech. Ageing and Dev. (in press). 5. Yiiksel, K. 0., and Gracy, .R.W. (1986) Arch. Biochem. Biophys. 248, 452-459. 6. Gracy, R. W., Ytiksel, K. U., Chapman, M. L., and Dimitrijevich, S. D. (1990) In Isozymes Structure and Function and Use in Biology and Medicine, Z. I. Ogita and C. L. Markert (eds) Wiley-Liss, New York pp 787-817. 7. Jones, R. B. and Waley, S. G. (1979) Biochem. J., 179, 623-630. 8. Browne, C. A., Campbell, I. D., Kiener, P. A., Phillips, D. C., Waley, S. G., and Wilson, I. A., (1976) J. Mol. Biol. 100, 319-343. 9. Lolis, E. and Petsko, G. A. (1990) Biochemistry 29, 6619-6625. 10. Hartman, F. C. (1970) Bioche.rnistry9, 1776-1782. 11. Jacobson, T. M., Yiiksel, K. U., Grant, S. R., and Gracy, R. W. (1990) Protein Express and Purif. (in press). 12. Gracy, R. W. (1975) Methods in Enzymology 41,422-447. 13. Hartman, F. C. (1971)Biochemistry 10, 146-154. 14. G/Sbber, F. and Lachmann, H. (1978) Z. Physiol. Chem. 359, 269. 15. Lachmann, H., and Schnackerz, K. D. (1984) Org. Magn. Reson., 22, 101-105. 16. Sawyer, T. H., and Gracy, R. W. (1975) Arch. Biochem. Biophys., 169, 51-57. 17.Waley, S. G. (1973) Biochem. J., 135, 165-172. 18.Coulson, A. F. W., Knowles, J. R., Priddle, J. D., and Offord, R. E. (1970) Nature, 227, 180. 19.Waley, S. G. Miller, J. C., Rose, I. A., O'Connell, E. L. (1970) Nature, 227, 181. 20. Webb, M. R., Standring, D. N., and Knowles, J. R. (1977) Biochemistry 16, 2738-2741. 21. Campbell, I. D., Jones, R. B., Kiener, P. A., and Waley, S. G. (1979) Biochem. J. 179, 607-621. 740