Share Email Print
cover

Proceedings Paper

Localization of the active site of an enzyme, bacterial luciferase, using two-quantum affinity modification
Author(s): L. Z. Benimetskaya; I. I. Gitelzon; Andrew L. Kozionov; S. Yu. Novozhilov; V. N. Petushkov; N. S. Rodionova; Mark I. Stockman
Format Member Price Non-Member Price
PDF $14.40 $18.00

Paper 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 +/- 10 and 120 +/- 10 amino acid residues; the regions 50 +/- 10 and 215 +/- 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) 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.

Paper Details

Date Published: 1 November 1991
PDF: 4 pages
Proc. SPIE 1525, Future Trends in Biomedical Applications of Lasers, (1 November 1991); doi: 10.1117/12.48227
Show Author Affiliations
L. Z. Benimetskaya, Institute of Automation and Electrometry (Russia)
I. I. Gitelzon, Institute of Biophysics (Russia)
Andrew L. Kozionov, Institute of Automation and Electrometry (Russia)
S. Yu. Novozhilov, Institute of Automation and Electrometry (Russia)
V. N. Petushkov, Institute of Biophysics (Russia)
N. S. Rodionova, Institute of Biophysics (Russia)
Mark I. Stockman, Institute of Automation and Electrometry (Russia)


Published in SPIE Proceedings Vol. 1525:
Future Trends in Biomedical Applications of Lasers

© SPIE. Terms of Use
Back to Top