Localization of the active site of an enzyme bacterial luciferase using two-quantum affinity modification

摘要

Abstract: For the first time the method of two-quantum affinity modification has been employed to probe the structure of an enzyme, bacterial luciferase. Position of the flavin-binding site of this enzyme, which was previously unknown, has been established. The obtained data indicate that the flavin site is positioned on the $alpha@-subunit. The closest contact of the protein chain of the enzyme with the chromophoric group of the flavin takes place near 80 $POM 10 and 120 $POM 10 amino acid residues; the regions 50 $POM 10 and 215 $POM 10 are also close to the flavin. The established localization does not contradict suggestions on positions of the flavin and phosphate sites of the bacterial luciferase, which had earlier been made from the data on evolutionary stability of various luciferases. The present method can, in principle, be applied to a great number of enzymes, including all flavin-dependent enzymes. Enzymatic catalysis has high speed and specificity. Creation of a method of determination of the elements of the primary structure of a protein, making up the active site (in which substratum conversion occurs), could be a significant advance in clearing up mechanisms of enzymatic catalysis. It was proposed to localize active sites of the enzymes, whose substrata are chromophores, using this method of two-quantum affinity modification. An enzyme- substratum complex is irradiated with laser light of sufficiently long wavelength ($lambda $GRT 300 nm) which is not directly absorbed by the enzyme. Two-quantum quasiresonant excitation of the substratum activates it to the state with energy 5-7 eV, which is then radiativelessly transferred to neighboring protein groups. This energy exceeds the energy of activation of peptide bond breakage. Therefore, the enzyme will be disrupted in the vicinity of its active site. In the present paper the above approach has been implemented for the first time. Information has been obtained about the position of the flavin-binding site of bacterial luciferase. In these experiments an enzyme from luminous bacteria photobacterium leiognathi was used. It consists of two subunits having molecular weight of 41,000 and 38,000 correspondingly. The enzyme catalyzes the reaction of oxidizing aldehyde and reduces the flavin (FMN). The enzyme specimen was prepared by the multistep clearing technique. Protein concentration was measured.!8