Role of Conservative Residue Cys158 in the Formation of an Active Photoprotein Complex of Obelin

V. S. Bondar^*, K. V. Purtov, N. P. Malikova, L. A. Frank, and B. A. Illarionov

Institute of Biophysics, Siberian Branch of the Russian Academy of Sciences, Akademgorodok, Krasnoyarsk, 660036 Russia; fax: (3912) 43-3400; E-mail: bondvs@mail.ru

^* To whom correspondence should be addressed.

Received February 14, 2001; Revision received April 20, 2001

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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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KEY WORDS: photoproteins, obelin, apoobelin mutants, bioluminescence

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