Spectral studies on two genetic forms of the human serum proteinase inhibitor, alpha-1-antitrypsin.

alpha-1-antitrypsin, the major inhibitor of proteolytic enzymes in human serum, was isolated from normal individuals (protease inhibitor type MM) and from those with an inherited deficiency (protease inhibitor type ZZ) of circulatory protein. The two proteins were compared by circular dichroism spectroscopy, and by fluorescence quenching experiments using anionic (I-), and neutral (acrylamide) probes. Both proteins share a similar secondary structure, i.e. approximately 45--50% alpha-helix and 15--20% beta-structure. Evidence was accumulated to show that the microenvironment in the vicinity of the three tryptophanyl residues is altered in Z form as compared to the M form as shown by (a) the absence of the positive dichroic band in the region 290--300 nm of the circular dichroism spectra, (b) a greater than 50% increase in quantum yield in the tryptophanyl fluorescence emission spectra, (c) an increased accessibility of tryptophan to quenching by iodide, and (d) acrylamide quenching experiments which indicate that all tryptophanyl residues in the Z protein are quenched equally or that quenching is dominated by a single residue, while in the M protein, heterogeneous quenching occurs. The potential significance of these findings in terms of alpha-1-antitrypsin deficiency state are discussed.     

Fluorescence quenching of dimeric and monomeric forms of yeast hexokinase (PII): effect of substrate binding steady-state and time-resolved fluorescence studies

Fluorescence quenching studies on the PII isoenzyme of yeast hexokinase have been performed using charged as well as polar uncharged quenchers. In both 'open' (i.e. in the absence of glucose) and 'closed' (i.e. in the presence of glucose) forms of the enzyme, bimolecular quenching rate constant (kq) for acrylamide is significantly larger than that of KI, indicating that all the tryptophans are not fully exposed to the solvent. Overall accessibility of tryptophans towards KI was greater in the presence of glucose than in the absence of glucose. At high ionic strength, the value of bimolecular quenching rate constant (kq) for KI did not change suggesting that the average environment of the accessible tryptophan residue(s) is almost neutral. Quenching by KI is dynamic in nature. Accessibility of tryptophans towards acrylamide at concentration > or = 0.2 M was more in the 'open' form of the enzyme than that observed in the 'closed' form whereas at concentration < or = 0.2 M no significant difference in the extent of quenching was observed. It is reasonable to conclude that glucose induced conformational change leads some tryptophan residue(s) to be more exposed and at the same time some tryptophan residue(s) in the hydrophobic region become more buried. Dimeric and monomeric forms of the enzyme behave similarly towards the quenching by acrylamide. In the unfolded state, the accessibility of tryptophans was considerably higher for both the quenchers. Temperature dependent study and the fluorescence lifetime data indicate that the mechanism of quenching by acrylamide is primarily dynamic in nature.     

Effect of halide anions on the binding of FAD to D-amino acid oxidase and the tryptophanyl fluorescence of the apoenzyme.

The quenching of tryptophanyl fluorescence of native and denatured D-amino acid oxidase from hog kidney was measured. About 60% of the tryptophanyl fluorescence of the native apoenzyme was quenched by iodide at pH 8.3, and 25 degrees C. All of the tryptophanyl fluorescence of the apoenzyme in 6 M guanidine hydrochloride was quenched. The tryptophanyl fluorescence quenching of the holoenzyme by 1-methyl nicotinamide chloride was low in comparison with that of the apoenzyme. These results of the quenching experiments are discussed based on the intermolecular collision quenching mechanism. By measuring the fluorescence intensities of the tryptophanyl residues and FAD of the holoenzyme solution, and the fluorescence polarization of the holoenzyme solution containing halide anions such as iodide, bromide, chloride, or fluoride, we found that FAD dissociates from the holoenzyme in the presence of iodide, bromide, or chloride, and the ability to dissociate FAD from the holoenzyme decreases in order iodide, bromide, and chloride. However, fluoride seems to enhance the association reaction of FAD with the apoenzyme. These results were consistent with the visible absorption spectra and derivative spectra of free FAD and the holoenzyme in the presence and absence of halide anions.     

Quenching of tryptophanyl fluorescence of bovine adrenal P-450C-21 and inhibition of substrate binding by acrylamide

Quenching of the tryptophanyl fluorescence of cytochrome P-450C-21 by acrylamide and its relationship to substrate binding are investigated by using steady-state and time-resolved data. The average collisional quenching constant was 0.4 M whereas the quenching constant for the total fluorescence was 10.8 +/- 0.9 M. This indicates that the quenching is essentially static. The quencher inhibited the binding of the substrate apparently competitively. The inhibition constant was 0.092 M, giving rise to an association constant of 10.9 M which is remarkably similar to the static quenching constant. It is suggested that tryptophan(s) may represent a key to the substrate-binding site in P-450C-21.     

Quenching of intrinsic tryptophan fluorescence in membranes of rat pituitary cells.

The GH4C1 strain of hormone-producing rat pituitary cells has specific receptors for the tripeptide thyrotropin-releasing hormone (TRH). Membranes prepared from GH4C1 cells show intrinsic tryptophan fluorescence which was quenched by low concentrations (10--100 nM) of TRH and Ntau-methyl TRH but not by biologically inactive analogs of TRH. Membranes from GH4C1 cells were subjected to thermal denaturation. A conformational transition was noted above 40 degrees C and an irreversible denaturation was observed at 52 degrees C. TRH-induced quenching of intrinsic fluorescence was lost completely in membranes previously incubated for 10 min at 30 degrees C while loss of [3H]-TRH binding was only about 20% at this temperature. Collisional quenching by iodide revealed that about 38% of the tryptophanyl residues in GH4C1 membranes were exposed to solvent. Quenching by TRH occurred with a shift in wavelength maximum from 336 to 342 nm suggesting that few of the tryptophanyl residues quenched by the tripeptide are totally exposed. Membranes prepared from cells preincubated with 20 nM TRH for 48 h, in which TRH receptors were decreased to 30% of control values, showed no quenching of tryptophan fluorescence in response to freshly added TRH. We conclude that the TRH-receptor interaction in GH4C1 cells is associated with a change in membrane conformation that can be measured by differential spectrofluorometry of intrinsic tryptophan fluorescence.     

Fluorescence quenching of human orosomucoid. Accessibility to drugs and small quenching agents.

The fluorescence behaviour of human orosomucoid was investigated. The intrinsic fluorescence was more accessible to acrylamide than to the slightly larger succinimide, indicating limited accessibility to part of the tryptophan population. Although I- showed almost no quenching, that of Cs+ was enhanced, and suggested a region of negative charge proximal to an emitting tryptophan residue. Removal of more than 90% of sialic acid from the glycan chains led to no change in the Cs+, I-, succinimide or acrylamide quenching, indicating that the negatively charged region originates with the protein core. Quenching as a function of pH and temperature supported this view. The binding of chlorpromazine monitored by fluorescence quenching, in the presence and in the absence of the small quenching probes (above), led to a model of its binding domain on orosomucoid that includes two tryptophan residues relatively shielded from the bulk solvent, with the third tryptophan residue being on the periphery of the domain, or affected allotopically and near the negatively charged field.     

Detection, characterization, and quenching of the intrinsic fluorescence of bovine heart cytochrome c oxidase

The intrinsic fluorescence of lauryl maltoside solubilized bovine heart cytochrome c oxidase has been determined to arise from tryptophan residues of the oxidase complex. The magnitude of the fluorescence is approximately 34% of that from n-acetyltryptophanamide (NATA). This level of fluorescence is consistent with an average heme to tryptophan distance of 30 A. The majority of the fluorescent tryptophan residues are in a hydrophobic environment as indicated by the fluorescence emission maximum at 328 nm and the differing effectiveness of the quenching agents: Cs+, I-, and acrylamide. Cesium was ineffective up to a concentration of 0.7 M, whereas quenching by the other surface quenching agent, iodide, was complex. Below 0.2 M, KI was ineffective whereas between 0.2 and 0.7 M 15% of the tryptophan fluorescence was found to be accessible to iodide. This pattern indicates that protein structural changes were induced by iodide and may be related to the chaotropic character of KI. Acrylamide was moderately effective as a quenching agent of the oxidase fluorescence with a Stern-Volmer constant of 2 M-1 compared with acrylamide quenching of NATA and the water-soluble enzyme aldolase having Stern-Volmer constants of 12 M-1 and 0.3 M-1, respectively. There was no effect of cytochrome c on the tryptophan emission intensity from cytochrome c oxidase under conditions where the two proteins form a tight, 1:1 complex, implying that the tryptophan residues near the cytochrome c binding site are already quenched by energy transfer to the homes of the oxidase. The lauryl maltoside concentration used to solubilize the enzyme did not affect the fluorescence of NATA.(ABSTRACT TRUNCATED AT 250 WORDS)     

Acrylamide and oxygen fluorescence quenching studies with liver alcohol dehydrogenase using steady-state and phase fluorometry

The fluorescence lifetime of liver alcohol dehydrogenase (LADH) has been determined by phase fluorometry at various emission wavelengths and as a function of the concentration of the quencher acrylamide. Acrylamide selectively quenches the fluorescence of the surface tryptophanyl residue Trp-15, thus allowing the fluorescence lifetime of this residue and the buried residue Trp-314 to be evaluated. Values of tau15 = 6.9 ns and tau314 = 3.6 ns are obtained, in qualitative agreement with lifetimes of these residues determined from fluorescence decay studies [Ross, J.B.A., Schmidt, C.J., & Brand, L. (1981) Biochemistry 20, 4369-4377]. The quenching of the fluorescence of LADH by oxygen has also been studied. Quenching by oxygen results in a blue shift in the fluorescence of the protein and a downward-curving Stern-Volmer plot. These data, along with oxygen quenching studies in the presence of 1 M acrylamide, are consistent with a model in which oxygen quenches the fluorescence of Trp-314 and -15 with quenching constants of 3.5 and 25 M-1, respectively. Thus, as in studies with other quenchers, Trp-314 is found to be less accessible to the quencher oxygen than is Trp-15. A lifetime Stern-Volmer plot has also been obtained for the oxygen quenching of LADH. Such a plot deviates somewhat from the intensity Stern-Volmer plot as predicted by simulations of the quenching of two-component systems.     

A fluorescence study of egg white riboflavin-binding protein

1. Denaturation of riboflavin-binding protein (RBP) by guanidine hydrochloride (Gu-HCl) was investigated by measruing the fluorescence of the protein. The denaturation-renaturation processes of RBP by Gu-HCl were fully reversible. The apo-RBP fluorescence had an emission maximum at 343 nm in the absence of Gu-HCl, and at 350 nm in the presence of 4M Gu-HCl, which completely denatured the protein. The relative fluorescence yield of apo-RBP in the presence of 4 M Gu-HCl was about 170% of that in the absence of Gu-HCl. The affinity of native apo-RBP for riboflavin was very strong, while riboflavin was not bound to the denatured form. The equilibrium system of apo-RBP and riboflavin in solutions containing Gu-HCl at various concentrations was analyzed by measuring riboflavin fluorescence. 2. The quenching of apo-RBP fluorescence, probably the fluorescence of tryptophanyl residues, by iodide anions and cesium cations was measured. The fluorescence of apo-RBP in the presence of 4 M Gu-HCl was quenched considerably by iodide and cesium, and Stern-Volmer plots were linear. However, the fluorescence of native apo-RBP was scarcely quenched by iodide or cesium. This suggested that tryptophanyl residues buried inside apo-RBP were responsible for most of the tryptophanyl fluorescence of native apo-RBP.     

Fluorescence spectroscopic studies on tryptophan at the saccharide-binding site of castor bean hemagglutinin

The environment of tryptophan in castor bean hemagglutinin (CBH) was analyzed by fluorescence spectroscopy with regard to saccharide binding. Upon binding of specific saccharides, the fluorescence maximum of 333 nm of CBH shifted to a wavelength 2 nm shorter, owing to the change in the environment of tryptophan at the saccharide-binding site. By analyzing the change in the fluorescence intensity at 320 nm as a function of concentration of saccharides, the association constants for binding of saccharides to CBH were determined. The results suggest that the saccharide-binding site on each B-chain is actually composed of a subsite with which the saccharide residue linked to galactopyranoside at the non-reducing end can interact, and another site which recognizes the galactopyranoside moiety. Quenching data indicated that five out of 22 tryptophans in CBH are surface-localized and are available for quenching with both KI and acrylamide, and three other tryptophans are buried and are available only to acrylamide. Binding of raffinose to CBH decreased by 2 the number of tryptophan residues accessible to quenchers in the CBH molecule. We speculate that raffinose binds to CBH in such a manner as to shield the tryptophan located at the subsite from quenching by KI and acrylamide. The results also suggest that the tryptophan residue at the saccharide-binding site on each B-chain is localized near the surface, and present in the positively charged environment.     

Conformational changes in pennisetin using intrinsic fluorescence spectroscopy

Fluorescence spectra of native pennisetin resulted in a single emission peak at 335 nm at excitation wavelength of 274 and 295 nm with quantum yield values for tyrosine and tryptophan as 0.086 and 0.097, respectively. These results indicate the presence of tryptophan residues in a polar environment and quenching of tyrosine residues in the native state of pennisetin. In the presence of an increasing concentration of guanidine hydrochloride (Gdn · HCl), changes such as red shift in emission peak from 335 to 344 nm, decrease in relative fluorescence intensity and increase in quantum yield value were observed, suggesting unfolding of the pennisetin molecule during denaturation. The quenching of tryptophanyl fluorescence by acrylamide and iodide further showed the presence of a single kind of tryptophanyl residue and its polar environment in pennisetin molecule.     

Spatial proximity of ATP-sensitive tryptophanyl residue(s) and Cys-697 in myosin ATPase.

The reactive thiol Cys-697 (SH2) in myosin ATPase was labeled with a fluorescent analog of maleimide, 2-(4'-maleimidylanilino)naphthalene-6-sulfonic acid (MIANS) (Hiratsuka, T. (1992) J. Biol. Chem. 267, 14941-14948). Although the tryptophan fluorescence of myosin subfragment-1 (S-1) was slightly affected by incorporation of the MIANS fluorophore, the tryptophan fluorescence of the resultant S-1 derivative (MIANS-S-1) was enhanced by ATP in a manner similar to that of unlabeled S-1. The quenching of tryptophan fluorescence of MIANS-S-1 was shown to result from a transfer of the excitation energy from tryptophanyl residue(s) to the MIANS fluorophore attached to SH2, which absorbed and fluoresced maximally at 325 and 418 nm, respectively. The energy transfer measurements were performed in the presence of acrylamide and compared to those performed in the absence of the quencher. The energy transfer efficiencies were found to be unaltered by acrylamide, indicating that the observed fluorescence energy transfer is originated exclusively from the tryptophanyl residue(s) that are not affected by acrylamide, i.e. the ATP-sensitive tryptophanyl residue(s) of S-1 (Torgerson, P. M. (1984) Biochemistry 23, 3002-3007). The distance between the tryptophanyl residue(s) and Cys-697 was calculated to be 27 A assuming a single donor-acceptor pair. Trp-510 is proposed to be one of the ATP-sensitive tryptophanyl residues.     

Quenching of intrinsic tryptophan fluorescence in membranes of rat pituitary cells

The GH₄C₁ strain of hormone-producing rat pituitary cells has specific receptors for the tripeptide thyrotropin-releasing hormone (TRH). Membranes prepared from GH₄C₁ cells show intrinsic tryptophan fluorescence which was quenched by low concentrations (10–100 nM) of TRH and N^τ-methyl TRH but not by biologically inactive analogs of TRH. Membranes from GH₄C₁ cells were subjected to thermal denaturation. A conformational transition was noted above 40°C and an irreversible denaturation was observed at 52°C. TRH-induced quenching of intrinsic fluorescence was lost completely in membranes previously incubated for 10 min at 30°C while loss of [³H]-TRH binding was only about 20% at this temperature. Collisional quenching by iodide revealed that about 38% of the tryptophanyl residues in GH₄C₁ membranes were exposed to solvent. Quenching by TRH occurred with a shift in wavelength maximum from 336 to 342 nm suggesting that few of the tryptophanyl residues quenched by the tripeptide are totally exposed. Membranes prepared from cells preincubated with 20 nM TRH for 48 h, in which TRH receptors were decreased to 30% of control values, showed no quenching of tryptophan fluorescence in response to freshly added TRH. We conclude that the TRH-receptor interaction in GH₄C₁ cells is associated with a change in membrane conformation that can be measured by differential spectrofluorometry of intrinsic tryptophan fluorescence.     

Exposure of tryptophanyl residues in proteins. Quantitative determination by fluorescence quenching studies.

Acrylamide is an efficient quencher of tryptophanyl fluorescence which we report to be very discriminating in sensing the degree of exposure of this residue in proteins. The quenching reaction involves physical contact between the quencher and an excited indole ring, and can be kinetically described in terms of a collisional and a static component. The rate constant for the collisional component is a kinetic measure of the exposure of a residue in a protein, and values ranging from 4 X 10(9) M-1 S-1 for the fully exposed tryptophan in the polypeptide, adrenocorticotropin, to less than 5 X 10(8) M-1 S-1 for the buried residue in azurin have been found. Static quenching is readily detected in proteins that are denatured, or contain only a single fluorophor. Quenching patterns for most multi-tryptophan containing proteins are difficult to analyze precisely, but qualitative information can, nevertheless, be extracted. Applications of this probing technique for monitoring protein conformational changes, such as the acid-induced expansion of human serum albumin, and inhibitor binding to enzymes, are presented. The value of this method lies in its ability to sense not only the steady-state exposure of a residue in a protein, but also its dynamic exposure.     

Fluorescence quenching studies of bovine growth hormone in several conformational states

The use of steady-state fluorescence quenching methods is reported as a probe of the accessibility of the single fluorescent tryptophan residue of bovine growth hormone (bGH, bovine somatotropin, bSt) in four solution-state conformations. Different bGH conformations were prepared by using previous knowledge of the multi-state nature of the equilibrium unfolding pathway for bGH: alterations in denaturant and protein concentration yielded different bGH conformations (native, monomeric intermediate, associated intermediate and unfolded). Because the intramolecular fluorescence quenching which occurs in the native state is reduced when the protein unfolds to any of the other conformations, steady-state fluorescence intensity measurements can be used to monitor bGH unfolding as well as the formation of the associated intermediate. These steady-state intensity changes have been confirmed with fluorescence lifetime measurements for the different conformational states of bGH. Fluorescence quenching results were obtained using the quenchers iodide (ionic), acrylamide (polar) and trichloroethanol (non-polar). Analysis of the results for native-state bGH reveals that the tryptophan environment is slightly non-polar (in agreement with the emission maximum of 335 nm) and the tryptophan is more exposed to acrylamide than most native-state tryptophan residues which have been studied. The tryptophan is most accessible to all quenchers in the unfolded state, because no steric restrictions inhibit quencher interaction with the tryptophan residue. The iodide quenching results indicate that the associated intermediate tryptophan is not accessible to iodide, probably due to negative charges inhibiting iodide penetration. The associated intermediate tryptophan is less accessible to all three quenchers than the monomeric intermediate tryptophan, due to tight packing of molecules in the associated intermediate state.     

Tryptophanyl fluorescence lifetime distribution of hyperthermophilic beta-glycosidase from molecular dynamics simulation: a comparison with the experimental data

A molecular dynamics simulation approach has been utilized to understand the unusual fluorescence emission decay observed for beta-glycosidase from the hyperthermophilic bacterium Solfolobus sulfotaricus (Sbeta gly), a tetrameric enzyme containing 17 tryptophanyl residues for each subunit. The tryptophanyl emission decay of Sbeta gly results from a bimodal distribution of fluorescence lifetimes with a short-lived component centered at 2.5 ns and a long-lived one at 7.4 ns (Bismuto E, Nucci R, Rossi M, Irace G, 1999, Proteins 27:71-79). From the examination of the trajectories of the side chains capable of causing intramolecular quenching for each tryptophan microenvironment and using a modified Stern-Volmer model for the emission quenching processes, we calculated the fluorescence lifetime for each tryptophanyl residue of Sbeta gly at two different temperatures, i.e., 300 and 365 K. The highest temperature was chosen because in this condition Sbeta gly evidences a maximum in its catalytic activity and is stable for a very long time. The calculated lifetime distributions overlap those experimentally determined. Moreover, the majority of trytptophanyl residues having longer lifetimes correspond to those originally identified by inspection of the crystallographic structure. The tryptophanyl lifetimes appear to be a complex function of several variables, such as microenvironment viscosity, solvent accessibility, the chemical structure of quencher side chains, and side-chain dynamics. The lifetime calculation by MD simulation can be used to validate a predicted structure by comparing the theoretical data with the experimental fluorescence decay results.     

Determination of the accessibility and localization of tryptophan residues in the influenza virus hemagglutinin molecule

The accessibility and localization of tryptophane residues in the influenza viral hemagglutinin molecule have been determined by measuring specific quenching of tryptophane fluorescence by neutral (acrylamide), anionic (I-) and cationic (Cs+) quenchers. It has been shown that acrylamide quenches 64% of tryptophane fluorescence in H3-hemagglutinin whereas I- and Cs+ quench only 34%. The tryptophanyl residues have been assumed to be located in the hemagglutinin molecule both in the cationic and anionic environments. 64% of tryptophanyls have been shown to be located on the surface of the protein globule.     

Conformational changes of maize and wheat NADP-malic enzyme studied by quenching of protein native fluorescence

Quenching of tryptophan fluorescence of maize and wheat NADP-malic enzyme by KI and acrylamide was studied after denaturating proteins with guanidine hydrochloride, and subjecting them to different pH values or temperatures. Protein unfolding by guanidine hydrochloride resulted in a red shift of the fluorescence spectrum, providing further support for the motion that several of the tryptophan residues evolved from an apolar to a polar environment. Protein denaturation was accompanied by an increase in the effective dynamic quenching constant values and by loss of the enzyme's activities. Thermal denaturation gave results consistent with the ones observed for chemical denaturation suggesting that a putative intermediate is involved in the denaturation process. Finally, exposure of both enzymes at various pH values allowed us to infer the number of accessible tryptophan residues in the different oligomeric conformations. The results suggest that the aggregation process seems to be different for each enzyme. Thus, as the maize enzyme associated from monomer to tetramer, one tryptophan residue would change from a polar to an apolar environment, while the association of the wheat enzyme would cause that two tryptophan residues to be excluded from quenching. Hitherto, quenching of the tryptophan fluorescence provides a good tool for studying conformational changes of proteins. The future availability of the crystal structures of plant NADP-malic enzymes will offer a good validation point for our model and the technology used.     

Tryptophan fluorescence as a probe of placental ribonuclease inhibitor binding to angiogenin

The binding of human placental ribonuclease inhibitor (PRI) to angiogenin, a human protein that induces neovascularization, occurs with a 1:1 stoichiometry and is accompanied by a 50% increase in tryptophan fluorescence. In contrast, the binding of PRI to bovine pancreatic RNase A or to angiogenin oxidized at its single tryptophan residue results in a quenching of fluorescence. These observations suggest that there is a change in the local environment of Trp-89 of angiogenin. Quenching experiments with acrylamide are consistent with the view that Trp-89 is exposed in the native protein and becomes less accessible upon formation of the complex with PRI. Stopped-flow kinetic measurements monitoring the fluorescence enhancement indicate a two-step mechanism for the binding of PRI to angiogenin. The first step involves rapid formation of an enzyme-inhibitor complex, EI, followed by a slower isomerization of EI to a tight enzyme-inhibitor complex, EI*: (Formula: see text). In 0.1 M NaCl at pH 6 and 25 degrees C, the values of K1 and K2 are 0.53 microM and 97 s-1, respectively. The apparent second-order rate constant of association at protein concentrations much less than K1 is approximated by K2/K1 and equals 1.8 X 10(8) M-1 s-1. The corresponding value for the association of PRI with RNase A is only slightly higher, 3.4 X 10(8) M-1 s-1. The effects of pH and sodium chloride concentration on the association rate of PRI with angiogenin suggest the importance of ionizable groups and ionic interactions, respectively, in the association process.(ABSTRACT TRUNCATED AT 250 WORDS)     

Fluorescence of tryptophan residues in firefly luciferases and enzyme--substrate complexes

Quenching of tryptophan fluorescence of Luciola mingrelica (single tryptophan residue, Trp-419) and Photinus pyralis (two tryptophan residues, Trp-417 and Trp-426) luciferases with different quenchers (I-, Cs+, acrylamide) was studied. The conserved Trp-417(419) residue was shown to be not accessible to charged particles, and positively and negatively charged amino acid residues are located in close vicinity to it. We found previously unreported effective energy transfer from this tryptophan to luciferin during the quenching of the tryptophan fluorescence. The distance between the luciferin molecule and Trp-417(419) was calculated: 11-15 and 12-17 A for P. pyralis and L. mingrelica luciferases, respectively. The role of the conserved Trp residue in the catalysis is discussed. ATP and AMP are also quenchers of the tryptophan fluorescence of the luciferases. In this case, an allosteric mechanism of the interaction of Trp-417(419) with an excess of ATP (AMP) is proposed.