Abstract
Using site directed mutagenesis, the conservative residue Cys158 of recombinant apoobelin was substituted for serine (C158S, S-mutant) or alanine (C158A, A-mutant). These point mutations resulted in significant changes in the apoobelin structure accompanied by slowing of photoprotein complex formation, decrease of its stability, and changing of its bioluminescence characteristics. The enzymatic properties of the photoprotein decreased in the series: wild-type protein > S-mutant > A-mutant. This is consistent with rank of nucleophilicity SH > OH > CH3 of cysteine, serine, and alanine side chain functional groups, respectively. Possible mechanisms of the involvement of the apoobelin Cys158 SH-group in the formation of the enzyme–substrate complex are considered.
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REFERENCES
Morin, J. G. (1974) in Coelenterate Biology: Reviews and Perspectives(Muscatine, L., and Lanhoff, H. M., eds.) Academic Press, N. Y., pp. 397–438.
Blinks, J. R., Prendergast, F. G., and Allen, D. G. (1976) Pharmacol.Rev., 28, 1–93.
Cormier, M. J., Prasher, D. C., Longiaru, M., and McCann, R. O. (1989) Photochem.Photobiol., 49, 509–512.
Tsuji, F. I., Ohmiya, Y., Fagan, T. F., Toh, H., and Inouye, S. (1995) Photochem.Photobiol., 62, 657–661.
Shimomura, O., and Johnson, F. H. (1975) Nature (London), 256, 236–238.
Illarionov, B. A., Frank, L. A., Illarionova, V. A., Bondar, V. S., Vysotski, E. S., and Blinks, J. R. (2000) Meth.Enzymol., 305, 223–249.
Illarionov, B. A., Bondar, V. S., Illarionova, V. A., and Vysotski, E. S. (1995) Gene, 153, 273–274.
Bondar, V. S., Frank, L. A., Malikova, N. P., Inzhevatkin, E. V., Illarionova, V. A., and Vysotski, E. S. (1999) in Bioluminescence and Chemiluminescence: Perspectives for the 21st Century (Roda, A., Pazzagli, M., Kricka, L. J., and Stanley, P. E., eds.) John Wiley and Sons, Toronto, pp. 400–403.
Marini, F., III, Naeem, A., and Lapeyre, J.-N. (1993) Nucleic Acids Res., 21, 2277–2278.
Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) in Molecular Cloning, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N. Y., p. 6.28.
Tabor, S. (1991) in Current Protocols in Molecular Biology, John Wiley and Sons, N. Y., p. 16.2.1.
Maxam, A. M., and Gilbert, W. (1980) Meth.Enzymol., 65, 499–560.
Laemmli, U. K. (1970) Nature, 227, 680–685.
Kochetov, G. A. (1971) Handbook on Enzymology [in Russian], Vysshaya Shkola, Moscow.
Hastings, J. W., and Weber, G. (1963) J.Opt.Soc.Am., 53, 1410–1415.
Bondar, V. S., Sergeev, A. G., Illarionov, B. A., Vervoort, J., and Hagen, W. R. (1995) Biochim.Biophys.Acta, 1231, 29–32.
Fagan, T. F., Ohmiya, Y., Blinks, J. R., Inouye, S., and Tsuji, F. I. (1993) FEBS Lett., 333, 301–305.
Matveev, S. V., Illarionov, B. A., Vysotski, E. S., Bondar, V. S., Markova, S. V., and Alakhov, Yu. B. (1995) Analyt.Biochem., 231, 24–39.
Bondar, V. S., Trofimov, K. P., Sandalova, T. P., and Vysotski, E. S. (1992) Biokhimiya, 57, 1039–1047.
Torchinsky, Yu. M. (1977) Sulfur in Proteins [in Russian], Nauka, Moscow.
Sandalova, T. (1996) in Protein Folds: A Distance-Based Approach (Bohr, H., and Brunak, S., eds.) CRC Press, Tokyo, pp. 308–315.
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Bondar, V.S., Purtov, K.V., Malikova, N.P. et al. Role of Conservative Residue Cys158 in the Formation of an Active Photoprotein Complex of Obelin. Biochemistry (Moscow) 66, 1014–1018 (2001). https://doi.org/10.1023/A:1012377827626
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DOI: https://doi.org/10.1023/A:1012377827626