Abstract
Chemical modifications with water-soluble carbodiimides (EDC and CMC) were performed to elucidate whether some carboxyl residues are involved in the catalytic activity of membrane-bound pyrophosphatase ofRhodospirillum rubrum. EDC and CMC cause a loss of hydrolytic activity following pseudo-first-order kinetics up to 10 min of reaction. The enzyme was completely protected against EDC inhibition by PPi or Mg2+, whereas PPi or Mg2+ gave partial protection against CMC inactivation. Mg-PPi protected completely against the inhibition caused by both carbodiimides. These data suggest that the carboxyl moiety modified by EDC is at the active site. At longer times of inactivation with both carbodiimides, we could not observe a linear relationship in semilogarithmic plots of residual activity versus time, indicating that at least two carboxyls are involved in the inactivation, which correlates with the partial protection against CMC inactivation by PPi. We found that the activator site for Mg2+ is apparently at or near the active site of the enzyme. This is supported by the fact that PPi protects completely the activator effect of this divalent cation.
Similar content being viewed by others
References
Baccarini-Melandri, A., and Melandri, B. A. (1978). InThe Photosynthetic Bacteria (Clayton, R. K., and Sistrom, W. R., eds.), Plenum Press, New York and London, pp. 615–628.
Baltscheffsky, H., Von Stedingk, L. V., Heldt, H. W., and Klingenberg, M. (1966).Science 153, 1120–1121.
Baltscheffsky, M. (1978). InThe Photosynthetic Bacteria (Clayton, R. K., and Sistrom, W. R., eds.), Plenum Press, New York and London, pp. 598–613.
Baltscheffsky, M., Baltscheffsky, H., and Boork, J. (1982). InTopics in Photosynthesis. Vol. 4 (Barber, J., ed), Elsevier, pp. 249–272.
Baykov, A. A., Dubnova, E. B., Pashkov, A. Yu, and Avaeva, S. M. (1987).Biol. Membr. 10, 1019–1025.
Bond, M. W., Chiu, N. Y., and Cooperman, B. S. (1980).Biochemistry 19, 94–102.
Cacesi, M. S., and Cacheris, W. P. (1984).Byte, May, pp. 340–362.
Celis, H., Romero, I., and Gómez-Puyou, A. (1985).Arch. Biochem. Biophys 236, 766–774.
Celis, H., and Romero, I. (1987).J. Bioenerg. Biomembr. 19, 255–272.
Church, F. C., Lundblad, R. L., and Noyes, C. M. (1985).J. Biol. Chem.,260, 4936–4940.
Cohen-Bazire, G., Sistrom, W. R., and Stainer, R. Y. (1957).J. Cell. Comp. Physiol. 49, 25–68.
Cooperman, B. S., and Chiu, N. Y. (1973).Biochemistry 12, 1676–1682.
Gilles, M. A., Hudson, A. Q., and Borders, C. J., Jr. (1990).Anal. Biochem. 184, 244–248.
Gomi, T., and Fujioka, M. (1983).Biochemistry 22, 137–143.
González M., and Cooperman, B. S. (1986).Biochemistry 25, 7179–7185.
Guillory, R. J., and Fisher, R. R. (1972).Biochem. J. 129, 471–481.
Hoare, D. G., and Koshland, D. E. (1967).J. Biol. Chem. 242, 2447–2453.
Komissarov, A. A., Sklyankina, V. A., and Avaeva, S. M. (1987).Bioorg. Khim. 13, 599–605.
Kuranova, I. P., Terzyan, S. S., Voronova, A. A., and Hansen, G. (1983).Bioorg. Khim. 9, 1611–1619.
Lahti, R. (1983).Microbiol. Rev. 47, 169–179.
Lahti, R., Pohjanoksa, K., Pitkaranta, T., Heikinheimo, P., Salminen, T., Meyer, P., and Heinonen, J. (1990).Biochemistry 29, 5761–5766.
Lahti, R., Salminen, T., Latonen, P., Heikinheimo, P., Pohjanoksa, K., and Heinonen, J. (1991).Eur. J. Biochem. 198, 293–297.
Lowry, O. H., Rosebrough, N. J., Farr, A. L., and Randal, R. J. (1951).J. Biol. Chem. 193, 265–275.
Morjana, N. A., and Scarborough, G. A. (1989).Biochim. Biophys. Acta 985, 19–25.
Nyrén, P., Nore, B. F., and Strid, Å. (1991).Biochemistry 30, 2883–2887.
Randahl, H. (1979).Eur. J. Biochem. 102, 251–256.
Ray, W. J., Jr., and Koshland, D. E., Jr. (1961).J. Biol. Chem. 236, 1973–1979.
Renosto, F., Seubert, P. A., Knudson, P., and Segel, I. H. (1985).J. Biol. Chem. 260, 11903–11913.
Sebald, W., and Hoppe, J. (1981).Curr. Top. Bioenerg. 12, 1–64.
Sosa, A., Ordaz, H., Romero, I., and Celis, H. (1992).Biochem. J. 283, 561–566.
Sumner, J. B. (1944).Science 100, 413–415.
Terzyan, S. S., Voronova, A. A., Smirnova, E., Kuranova, I. P., Nekrasov, Y. V., Arutyunyun, E. G., Vainshtein, B. K., Höhne, W. E., and Hansen, G. (1984).Bioorg. Khim. 10, 1469–1482.
Verstappen, R., and Rausch, T. (1988).FEBS Lett. 236, 420–424.
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Romero, I., Celis, H. Evidence of an essential carboxyl residue in membrane-bound pyrophosphatase ofRhodospirillum rubrum . J Bioenerg Biomembr 24, 617–624 (1992). https://doi.org/10.1007/BF00762354
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF00762354