Luciferin Binding Research Articles

A circadian rhythm of the luciferin binding protein fromGonyaulax polyedra

It has previously been shown that a protein extracted fromGonyaulax polyedra strongly and specifically binds luciferin, the substrate of the bioluminescent reaction. This binding is markedly dependent on pH with tight binding at pH 8.0 and almost no binding at pH 6.5, as measured by two independent methods. A procedure for the determination of the dissociation constant (Kd) of the luciferin binding protein (LBP) is presented, and Kd is estimated to be7×10−9 M at pH 8.0, assuming an overall quantum yield of 0.1 for the bioluminescent reaction. With cells grown in a 12 h light — 12 h dark cycle, 5 to 10 times more LBP activity can be extracted from dark phase cells than from light phase cells. This rhythm persists in a circadian fashion in cultures maintained in constant dim light.

Substrate and substrate analogue binding properties of Renilla luciferase.

Luciferase from the anthozoan coelenterate Renilla reniformis catalyzes the oxidative decarboxylation of luciferin consuming 1 mol of O2 per mol of luciferin oxidized and producing 1 mol of CO2, 1 mol of oxyluciferin, and light (lambdaB, 480 nm) with a 5.5% quantum yield. In this work we have examined the binding characteristics of luciferin, luciferin analogues, and competitive inhibitors of the luciferin-luciferase reaction. The results show that luciferin binding and orientation in the single luciferin binding site of luciferase are highly specific for and dependent upon the three group substituents of the luciferin molecule while the imidazolone-pyrazine nucleus of luciferin is not directly involved in binding. Anaerobic luciferin binding promotes a rapid concentration-dependent aggregation of luciferase which results in irreversible inactivation of the enzyme. This aggregation phenomenon is not observed upon binding of oxyluciferin, luciferyl sulfate, or luciferin analogues in which the substituent at the 2 position of the imidazolone-pyrazine ring has been substantially altered.

Dinoflagellate bioluminescence: a comparative study of invitro components.

In vitro bioluminescence components of the dinoflagellates Gonyaulax polyedra, G. tamarensis, Dissodinium lunual, and Pyrocystis noctiluca were studied. The luciferases and luciferins of the four species cross-react in all combinations. All of these species possess high-molecular weight luciferases (200,000-400,000 daltons) with similar pH activity profiles. The active single chains of luciferases from the Gonyaulax species have a MW of 130,000 while those from P. noctiluca and D. lunula have a MW of 60,000. Extractable luciferase activity varies with time of day in the two Gonyaulax species, but not in the other two. A luciferin binding protein (LBP) can easily be extracted from the two Gonyaulax species (MW approximately 120,000 daltons), but none could be detected in extracts of either D. lunula or P. noctiluca. Scintillons are extractable from all four species, but they vary in density and the degree to which activity can be increased by added luciferin. Although the biochemistry of bioluminescence in these dinoflagellates is generally similar, the observations that D. lunula and P. noctiluca apparently lack LBP and have luciferases with low MW single chains require further clarification.

Isolation and partial characterization of a peptide derived from the luciferin binding site of firefly luciferase

2-Cyano-6-chlorobenzothiazole (CCB), a substrate analog of firefly luciferase, inactivates this enzyme slowly at pH 8 to about 20% of original activity without affecting free sulfhydryl groups. The inactivated luciferase contained 1.5–2.0 moles of CCB per 100,000 daltons of luciferase. It is believed that the benzothiazole derivatives are incorporated at the luciferin binding sites. Tryptic digest of the inactivated luciferase and subsequent electrophoresis yielded a fluorescent peptide containing benzothiazole derivative. The peptide was found to contain pyroglutamic acid at the N-terminal end. The sequence of the peptide was established tentatively to be: pyroglu-X-gly-ala-val-(asp)-ile-leu where X is an amino acid (possibly tyr) to which the benzothiazole derivative is attached.

Substrate-binding properties of firefly luciferase: I. Luciferin-binding site

Various techniques were used for characterization of the luciferin-binding site of the firefly luciferase. Both equilibrium-dialysis and fluorescence-titration techniques revealed the existence of two identical, noninteracting binding sites for dehydroluciferin, which is a potent competitive inhibitor. Kinetically obtained dissociation constants of various luciferin analogues and other competitive inhibitors revealed that most of the binding energies of these compounds reside in the backbone ring structure and that various substituents did not influence the binding to any significant extent. Exceptions to this are the methyl group at the 6-position of the benzothiazole ring and the carboxylic acid group. The binding energy of the luciferin backbone structure to the luciferin-binding site is 7.5 kcal/mole. Two parts of the luciferin molecule, the benzothiazole ring portion and the thiazoline ring portion, contribute 6.0 kcal/mole and 1.5 kcal/mole respectively to the binding energy. Comparison of the binding energies of several heterocyclic compounds structurally related to benzothiazole suggests that the nitrogen atom of the benzothiazole ring may be important in orienting the compound to a fixed position in the luciferinbinding site. The binding studies with N-ethylmaleimide-inactivated luciferase gave further evidence that the two sulfhydryl groups essential for the luciferase activity are located at the luciferin binding sites, most likely one at each binding site. Excitation spectra of luciferase-bound dehydroluciferin indicated that the phenolic group of dehydroluciferin is in unionized form on the enzyme even in a medium of high pH. This effect and a large enhancement of the blue fluorescence peak at 440 mμ suggest the environment of luciferin-binding site to be quite hydrophobic in agreement with earlier observations.