Role of conservative residue Cys158 in the formation of an active photoprotein complex of obelin.
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 > CH(3) 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.
Bondar
V S
VS
Institute of Biophysics, Siberian Branch of the Russian Academy of Sciences, Krasnoyarsk, 660036 Russia. bondvs@mail.ru
Purtov
K V
KV
Malikova
N P
NP
Frank
L A
LA
Illarionov
B A
BA
D000409:Alanine / Q000235:genetics / Q000378:metabolism; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001059:Apoproteins / Q000235:genetics / Q000378:metabolism; D017124:Conserved Sequence; D003545:Cysteine / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D008163:Luminescent Measurements; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012694:Serine / Q000235:genetics / Q000378:metabolism
D001059:Apoproteins; D008164:Luminescent Proteins; D011994:Recombinant Proteins; C448311:apoobelin; C010497:obelin; D012694:Serine; D003545:Cysteine; D000409:Alanine