©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Catalytically Active Forms of the Individual Subunits of Vibrio harveyi Luciferase and Their Kinetic and Binding Properties (*)

Hoon Choi (1), Chih-Kwang Tang (1), Shiao-Chun Tu (1) (2)(§)

From the (1)Departments of Biochemical and Biophysical Sciences and (2)Chemistry, University of Houston, Houston, Texas 77204-5934

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

Contradictory findings have recently been reported regarding the (in)abilities of individual subunits of the Vibrio harveyi dimeric luciferase to catalyze bioluminescence. We have produced individual and subunits separately in Escherichia coli JM109 cells by recombinant DNA techniques. Both subunits were purified to more than 90% homogeneity and found to be catalytically active, with their general catalytic properties and the specific activities similar to those reported earlier (Sinclair, J. F., Waddle, J. J., Waddill, E. F., and Baldwin, T. O.(1993) Biochemistry 32, 5036-5044). Individual subunits were significantly distinct from the native luciferase with respect to inactivations by trypsin and N-ethylmaleimide, and the stability of the flavin 4a-hydroperoxide intermediate. The active species in isolated and samples were each the predominant protein species, corresponding to a 42,000 M monomer and a 67,000 M dimer, respectively. These findings clearly indicate that the activities of the individual subunits are not due to trace contaminations of the respective counter subunits. The much reduced specific activities of the individual subunits are, in part, a consequence of diminished abilities to oxidize the aldehyde substrate. Kinetic and equilibrium measurements indicate that and each contained a reduced flavin site, an aldehyde substrate site, and an aldehyde inhibitor site. The on and off rates of the decanal inhibitor binding were substantially slower than the bindings of decanal and reduced riboflavin 5`-phosphate substrates. These findings are consistent with a scheme that the aldehyde inhibitor blocks the binding of the reduced flavin substrate.


INTRODUCTION

Bacterial luciferase, an heterodimeric flavin-dependent monooxygenase, catalyzes the light-emitting oxidation of reduced riboflavin 5`-phosphate (FMNH)()and a long-chain aliphatic aldehyde by molecular oxygen.

On-line formulae not verified for accuracy

On-line formulae not verified for accuracy

The noncovalent interaction between the and subunits is apparently very tight. However, the two subunits can be separated by ionic exchange chromatography under denaturing conditions(1, 2) . The active luciferase can be reconstituted by mixing the and followed by renaturation(1) . Renatured individual subunits also show some low activities which were, on the basis of the incomplete chromatographic separation of the two subunits, attributed to trace contaminations of the respective counter subunit(2) . Hence, the dimeric structure of luciferase has long been regarded as obligatory to the bioluminescence activity.

The dimeric luciferase utilizes one molecule each of the flavin (3) and aldehyde (4) per cycle of luminescence reaction. Earlier subunit hybridization (5, 6) and photoaffinity labeling (7) studies suggest that the single active site of luciferase may reside near the subunit interface. However, neither the exact location nor the identity of the constituent amino acid residues of the luciferase active site has been explicitly specified. Regarding the functional roles, the importance of in catalysis has been indicated by a number of studies(5, 6, 8, 9) , whereas has been implicated in maintaining luciferase in a catalytically active conformation(9) , the binding of substrates (5, 6, 7) and, recently, the expression of bioluminescence(10) . The specific functions of either subunit are, however, only poorly understood.

An important claim has recently been made that individual subunits of Vibrio harveyi luciferase are active in catalyzing light emissions with their specific activities about 5 orders of magnitude lower than that of the native dimeric enzyme(11, 12) . The low activities notwithstanding, the abilities of and to catalyze the light emission have significant implications to our understanding of the structure of luciferase active site and functionalities of the individual subunits. However, such a claim was disputed by two later studies which report that neither the individual (13, 14) nor (14) of V. harveyi luciferase is associated with any luminescence activity.

We believe that the question as to whether the individual or is catalytically active has a fundamental importance to our understanding of luciferase structure-function relationships. This work was initiated to resolve the apparent controversy and examine further the functionalities of the and subunits. We found that individual subunits are indeed active in bioluminescence. Moreover, the active subunit species have been characterized with respect to molecular weights, general catalytic and kinetic properties, sensitivities to chemical modification and proteolysis, flavin and aldehyde binding properties, stabilities of the flavin 4a-hydroperoxide intermediate, and abilities to consume the aldehyde substrate. Implications of these findings to the structure-function relationships of luciferase are discussed.


EXPERIMENTAL PROCEDURES

Materials

Octanal, decanal, and dodecanal were purchased from Aldrich, and FMN was from Sigma. DEAE Sephadex A-50 and Sephadex G-150 were both products of Pharmacia Biotech Inc. Ultro-Gel AcA 54 was obtained from Sepracor.

Construction of Plasmids Containing luxA or luxB and Expression of the Cloned Genes

A 2.53-kilobase fragment containing the complete luxA and luxB genes encoding the V. harveyi luciferase and subunits, respectively, was obtained from HindIII/KpnI digest of the recombinant phage MTX1(15) . This DNA fragment was inserted into pUC19 pretreated with the same restriction enzymes to produce the recombinant plasmid pMH2. pMH2 was digested with EcoRI to delete 1.26 kilobases for the destruction of the luxB, and was subsequently ligated to yield the plasmid pHC1 containing only the intact luxA. A luxB-containing 1.83-kilobase fragment was obtained from the KpnI/PstI digest of pMH2, and was inserted into pUC19 pretreated with the same restriction enzymes to generate the plasmid pHC2 containing only the complete luxB. Recombinant plasmids were used to transform Escherichia coli JM109, and clones were first screened for white colonies by -complementation using 5-bromo-4-chloro-3-indoyl -D-galactoside and isopropyl-1-thio--D-galactopyranoside as a substrate and an inducer, respectively. Positive clones were further identified by restriction digestion patterns. Finally, E. coli JM109 cells harboring the desired pHC1 or pHC2 plasmid were first grown at 37 °C overnight in 20 ml of LB medium containing 100 mg liter of ampicillin. Four-ml aliquots were each inoculated into 1 liter of fresh medium for cell growth at 23 °C for 48 h. Isopropyl-1-thio--D-galactopyranoside (0.2 mM) was added 4 h prior to harvest.

Purification of Individual Subunits

All procedures were carried out at 2-4 °C. The method of Waddle and Baldwin (11) was followed for the purification of individual and subunits. For the former, the procedure was modified to apply 0.4 M P, pH 7.0, instead of a phosphate gradient for the DEAE-Sephadex A-50 column chromatography. Purities of the isolated and subunits were determined to be both 90% on the basis of SDS-polyacrylamide gel electrophoresis.

Bioluminescence Assays

Unless stated otherwise, 0.05 M P, pH 7.0, was used as a standard buffer. Bioluminescence was measured using either a calibrated home-made photometer or a Turner TD-20e luminometer. Several non-turnover assay methods were used to examine various kinetic parameters and the effects of ligand binding. These assays are designated by the method of flavin reduction and the condition of luciferase-substrate (or inhibitor) equilibration prior to the initiation of bioluminescence. In one series, FMN was reduced by Cu(I) (16, 17) for the Cu(I)-EAO and Cu(I)-EO assays(17) . The former involves the injection of an FMNH solution into an aerobic buffer containing the enzyme sample and aldehyde to initiate the bioluminescence. The latter assay differs only by including the aldehyde in the FMNH solution rather than the aerobic enzyme solution. A standard dithionite assay(18, 19) , now designated D-EF, was also used in which an aerobic aldehyde solution was injected into an anaerobic buffer containing enzyme and FMNH. Last, for an alternative dithionite assay (designated D-EAO), a dithionite-reduced FMNH solution was injected into an aerobic buffer containing the enzyme and the aldehyde.

Stabilities of Flavin 4a-Hydroperoxide Intermediates

Individual subunits and the native luciferase in 1 ml of 0.1 M P, pH 7.0, were each mixed with 50 µM FMN and a few milligrams of dithionite powder to reduce the flavin at 1 °C. The solution was stirred gently until the free reduced flavin was reoxidized as indicated by the yellowing of the solution. At this point, the bound FMNH had also been converted to the FMN 4a-hydroperoxide intermediate. The sample was kept at 1 °C and 50-µl aliquots were withdrawn after different times for the determination of the remaining quantities of the flavin 4a-hydroperoxide intermediate on the basis of the bioluminescence activities upon mixing with 1 ml of 0.05 M P, pH 7.0, containing saturating decanal at 23 °C. The first-order decay rates of various flavin 4a-hydroperoxide intermediate species can be determined from semilogarithmic plots of remaining bioluminescence activities versus times of incubation at 1 °C.

Kinetics of Inactivations of , , and Native Luciferase

Trypsin (0.5 mg) was added to 10 ml of 0.1 M P, pH 7.0, containing a designated level of , , or luciferase. Aliquots were withdrawn after different times of incubation at 23 °C and the remaining bioluminescence activities were determined using the Cu(I)-EO assay. The apparent first-order rate constants for inactivation were determined by semilogarithmic plots of remaining activities versus times of proteolysis. Native luciferase and the individual and were each also treated with 0.2 mMN-ethylmaleimide at 23 °C for inactivation by sulfhydryl group modification. Aliquots were withdrawn after different times and diluted into 1 ml of standard P buffer containing 1 mM dithiothreitol. Remaining bioluminescence activities and the apparent first-order rate constants of inactivation were determined as described for trypsin digestion.

Molecular Weights of the Catalytically Active and

Sephadex G-150 columns were equilibrated with 0.1 M P, pH 7.0, and calibrated with dextran blue, bovine serum albumin, ovalbumin, cytochrome c (monomer), and FMN for the construction of a molecular weight standard curve. The isolated and were each mixed with dextran blue and FMN in 1 ml of the standard buffer and applied to a calibrated column. Elutions were maintained at 15 ml h, and fractions were collected for the measurements of A and bioluminescence activities using the Cu(I)-EO assay. Partition coefficients were calculated for the A and activity peak fractions and the corresponding molecular weights were subsequently determined.

Aldehyde and Flavin Binding Stoichiometries as Determined by Activity Measurements

The principle of Job analysis (20) was applied to determine the number of aldehyde binding site by activity measurements. A series of samples were set up all containing a constant total concentration, at 1 µM, of a designated luciferase subunit species plus decanal in 1 ml of standard buffer but with the mole fraction of decanal varying from 0 to 1. Bioluminescence activities were measured by the D-EAO assay using a saturating level of FMNH as a cosubstrate, and were plotted against the mole fractions of decanal. The observed data were compared with theoretical plots for designated molar ratios of aldehyde binding by luciferase subunit. The stoichiometries of FMNH binding by luciferase subunits were determined under the same conditions except that the mole fractions of FMNH were varied and the activities were determined by the D-EF assay in the presence of a saturating level of decanal.

Aldehyde Binding Stoichiometry as Determined by Gel Filtration

The method of Hummel and Dreyer (21) was followed. A 0.2-ml sample containing designated levels of an individual subunit and decanal in 0.02 M P, pH 7.0, was applied to a Sephadex G-25 column (1.5 3.5 cm) pre-equilibrated and eluted with the same decanal-containing buffer. Fractions (50 µl) were collected in tubes each containing 0.95 ml of ethanol. The amount of decanal in all fractions were determined by a method described previously(17) .

Aldehyde Binding as Determined by Relaxation Kinetics

Designated levels of an individual subunit and decanal were mixed in the standard buffer and incubated for >3 min to allow the aldehyde binding to reach equilibrium. This solution was then diluted 5-fold with buffer. After a designated period of incubation, bioluminescence was initiated by the injection of 1 ml of buffer containing 0.5 mM decanal and 50 µM FMNH (reduced by the Cu(I) method). The time period of post-dilution incubation was lengthened gradually until no further change in bioluminescence activity was detected.

Aldehyde Consumption Assay

To test the abilities of individual subunits to consume aldehyde, a limiting level of decanal (1 µM) was mixed with an excess amount of an individual subunit (8.3-9 µM) in 1 ml of standard buffer. An equal volume of buffer containing 50 µM FMNH (reduced by Cu(I)) was injected to initiate the light emission. After the cessation of bioluminescence, aliquots (20 µl) were taken for the determination of levels of the remaining aldehyde following a method detailed previously (22).


RESULTS

General Catalytic Properties of Individual Subunits

The and subunits expressed from recombinant plasmids pHC1 and pHC2, respectively, and isolated as described above were both found to be active in catalyzing bioluminescence. Substrate titrations were carried out at 23 °C using either decanal (in the Cu(I)-EO assay) or FMNH (in the D-EF assay) as the varying substrate, and values of K and V were determined by double reciprocal plots. The was found to have a specific activity of 8.4 10 q s mg and a K for FMNH of 0.15 µM, whereas the was found to have a specific activity of 1.1 10 q s mg and a K for FMNH of 0.58 µM. These values are similar to those reported earlier by Sinclair et al.(12) . In addition, values of 0.9 and 2.0 µM were also determined as the K for decanal for and , respectively, under conditions without any aldehyde inhibition (discussed in more detail later). The light decay rate (k) of the non-turnover reaction catalyzed by the native luciferase is quite sensitive to the type of aldehyde utilized. For , k values of 0.052, 0.32, and 0.053 s were detected for octanal, decanal, and dodecanal, respectively. In comparison, k values of 0.040, 0.29, and 0.043 s were observed for octanal, decanal, and dodecanal, respectively, for . Again, these values are quite close to those found by Sinclair et al.(12) . The general catalytic properties of and described thus far along with other kinetic properties to be detailed later are summarized in .

Activity Perturbations of Individual Subunits and Native Luciferase

The responses of the native luciferase and the individual subunits to activity perturbations by proteolysis and chemical modification were compared. Upon treatment by 50 µg ml of trypsin in 0.1 M P, pH 7.0, apparent first-order inactivations of , , and the native luciferase were observed with the respective rate constants of 0.29, 0.15, and 0.62 min (Fig. 1). The activities of both individual subunits were more resistant to trypsin digest than that of the native luciferase. The native luciferase is known to be quite sensitive to sulfhydryl modification as a means of inactivation. As a reference, an apparent first-order rate constant of 0.19 min was observed for the inactivation of the native luciferase upon treatment with 0.2 mMN-ethylmaleimide (Fig. 2). Under identical treatment, the showed an apparent first-order rate constant of 0.12 min for inactivation and was slightly more stable than the native luciferase, whereas no significant activity loss was observed for (Fig. 2). For the two subunits individually ( Fig. 1and Fig. 2) and within the native luciferase(23, 24) , the appears to be more resistant to tryptic digest and sulfhydryl modification than the .


Figure 1: Inactivation of , , and native luciferase by tryptic digestion. Trypsin (0.5 mg) was added to 10 ml of 0.1 M P, pH 7.0, containing 1.4 mg of (), 1.8 mg of (), or 0.9 mg of native luciferase (). Bioluminescence activities were measured using 1-ml aliquots after different times as indicated.




Figure 2: Inactivation of , , and native luciferase by sulfhydryl group modification. N-Ethylmaleimide was added at 0.2 mM to 1 ml of 0.1 M P, pH 7.0, containing 0.23 mg of (), 0.16 mg of (), or 0.23 mg of native luciferase (). After different times, 0.1-ml aliquots were withdrawn for the measurement of bioluminescence activities.



Stabilities of Flavin 4a-Hydroperoxide Intermediates

Flavin 4a-hydroperoxide, designated intermediate II, is a key luciferase intermediate which has been characterized extensively. Using the native luciferase as a reference, the flavin peroxide intermediate II was found to have a decay rate (k) of 0.089 min at 1 °C (Fig. 3). In comparison, the flavin peroxide intermediates formed with the isolated and exhibited decay rates of 0.036 and 0.031 min, respectively, under identical conditions and were, hence, significantly more stable than that of the native luciferase (Fig. 3; ).


Figure 3: Stabilities of flavin 4a-hydroperoxide intermediates. Flavin 4a-hydroperoxide intermediates were formed at 1 °C in 1 ml of 0.1 M P, pH 7.0, containing 50 µM FMNH and 0.3 mg of (), 0.8 mg of (), or 0.3 mg of native luciferase (). Aliquots (50 µl) were withdrawn after different times of incubation at 1 °C and bioluminescence activities were measured at 23 °C by injection into 1 ml of 0.05 M P, pH 7.0, containing saturating decanal.



Molecular Weights of the Bioluminescence-active Forms of and

The isolated and subunits were each subject to Sephadex G-150 molecular sieve chromatography. For each subunit, only a single peak was observed when the fractions were tested for either A or bioluminescence activity. Moreover, the protein peak and the activity peak were comigrating during elution, indicating that for each individual subunit the predominant protein species was the catalytically active form. When compared with protein molecular weight standards, the catalytically active forms of and showed molecular weights of 42,000 and 67,000, respectively, indicating a monomeric form for and a dimeric form for .

Effects of Ligand Preincubation on Activities

The native V. harveyi luciferase is sensitive to inhibition by high concentrations of aldehyde when luciferase is equilibrated with aldehyde prior to the binding of reduced flavin(25, 26) . Aldehyde inhibitions of individual and under the same condition of aldehyde pre-equilibration have also been reported recently(12) . This claim has now been confirmed by our observations of effective inhibitions of and at 10M decanal in the Cu(I)-EAO assay (Fig. 4). In addition, we have used two other assay methods to examine the effects of ligand preincubation on the activities of individual subunits. At decanal concentrations up to 1.7 mM for and 2 mM for , no aldehyde inhibition was detected when either subunit was pre-equilibrated with FMNH prior to aldehyde addition as in the D-EF assay. Moreover, only slight inhibitions were observed for the two individual subunits when 1.7 to 2.0 mM aldehyde was added together with FMNH as in the Cu(I)-EO assay (Fig. 4). These properties of the individual subunits are qualitatively the same as that of the native luciferase(17) . The latter has been shown to have an aldehyde substrate site and an independent aldehyde inhibitor site which competes against the FMNH binding. Furthermore, the binding of the aldehyde inhibitor is substantially slower than that of the FMNH and aldehyde substrate(17) .


Figure 4: Effects of ligand pre-equilibration on activity. Activities of 0.35 µM (panel A) and 0.6 µM (panel B) were measured at 25 µM FMNH and different decanal concentrations by three methods. In the Cu(I)-EAO assay (), enzymes were preincubated with aldehyde for >5 min. For the Cu(I)-EO assay (), decanal and FMNH were added to an aerobic solution containing the enzyme sample for the initiation of bioluminescence. For the dithionite assay (), the enzyme sample was first equilibrated with FMNH. Each titration curve was normalized with the highest activity assumes a value of 1.0.



Number of Aldehyde and Flavin Binding Sites in Absence of Aldehyde Inhibition

The abilities of and subunits to bind FMNH and decanal (at micromolar levels to avoid inhibition) were examined by using the Job plot(20) . For aldehyde binding by , a series of samples were set up to maintain a total concentration of 1 µM for monomer plus decanal but with the mole fraction of decanal varying from zero to unity. When assayed in the presence of saturating FMNH, the degrees of aldehyde binding are reflected by the observed bioluminescence activities. In a plot of activity versus mole fraction of decanal, the linear portions of the two sides of the curve can be extrapolated to obtain an interception point. A single decanal site per monomer would correspond to an interception point at 0.5 mol fraction of decanal, whereas a mole fraction of 0.33 would support a single decanal site per dimer. The observed data show a close fit to the theoretical plot for a binding stoichiometry of one decanal per monomer (Fig. 5A, ). However, when was tested under identical conditions, the data indicate a binding stoichiometry of one decanal per (Fig. 5A, ). Similar experiments were carried out to determine the binding of FMNH by and . Again, a single flavin site was detected for monomer (Fig. 5B, ) and for (Fig. 5B, ).


Figure 5: Determination of number of substrate binding sites in the absence of aldehyde inhibition. For panel A, samples all contained the same 1 µM total concentration of decanal plus monomeric () or () but the mole fraction of decanal was varied from 0 to 1. For panel B, samples all contained the same 1 µM total concentration of FMNH plus monomeric () or () but the mole fraction of flavin was varied from 0 to 1. The activity of each sample was measured in the presence of a saturating level of co-substrate. Activity measurements were made in triplet for each mole fraction. The solid lines are theoretical plots for points of interception at 0.5 and 0.33 mol fractions.



Number of Aldehyde Site at High Aldehyde Concentrations

The equilibrium bindings of decanal, at levels effective for inhibition, by individual subunits were determined using the gel filtration method of Hummel and Dryer(21) . At 60 µM decanal, 2.5 nmol of was found to bind 4.6 and 4.0 nmol of aldehyde on the basis of the areas of the leading peak and tailing through, respectively (Fig. 6A), giving an average of 1.7 decanal molecules bound per monomer. For 2.3 nmol of in the presence of 53 µM decanal, 3.8 and 4.0 nmol of decanal were bound according to the leading peak and tailing through areas, respectively, yielding an average of 1.7 decanal molecules bound per dimer (Fig. 6B). These results indicate the existence of a second aldehyde site on and at aldehyde levels effective for inhibition.


Figure 6: Decanal binding by individual subunits as determined by gel filtration. A 0.2-ml 0.02 M P, pH 7.0, sample solution containing 12.5 µM and 60 µM decanal (panel A) or 11.5 µM and 53 µM decanal (panel B) was applied to a Sephadex G-25 column. The column was pre-equilibrated and, subsequent to the sample application, eluted with the same buffer containing decanal at the same concentration as in the original sample. The amount of decanal in each 50-µl fraction was determined as described under ``Experimental Procedures.''



Equilibrium and Kinetic Constants for the Binding of Aldehyde Inhibitor

The kinetic and equilibrium constants of aldehyde binding by individual subunits were determined in chemical relaxation experiments similar to those detailed recently for the native luciferase(17) . Each individual subunit was first equilibrated with decanal and then diluted 5-fold with buffer. After the dilution, the capacity of the sample to catalyze bioluminescence was measured at different times to determine the amount of aldehyde inhibitor-free subunit which increased over >20 s until a new equilibrium was reached. The experiment was repeated with 4 different initial decanal concentrations. The relaxation times () of such processes can be determined from semilogarithmic plots of v/vversus time of post-dilution incubation (Fig. 7A), where v is the difference of the final activity minus the initial activity prior to dilution and v is the difference of the final activity minus the activity at a given time after the dilution. Furthermore, the relationship 1/ = k([E] + [A]) + k exists where k and k are rate constants for binding and dissociation of aldehyde inhibitor (A) by individual subunit, respectively, and [E] and [A] are the concentrations of A-free subunit and free aldehyde at the new equilibrium state after dilution, respectively. By plotting 1/ against [E] + [A], values of k and k can be obtained and, subsequently, the dissociation constant for A can be determined as K = k/k (Fig. 7B). Analyses of experimental data as described above yield k values of 0.9 and 2.7 mM s, k values of 0.025 and 0.054 s, and K values of 27 and 20 µM for and , respectively (). The half-lives of the binding and dissociation of decanal inhibitor at 20 µM, for example, would be in the order of 10-40 s for both and . In non-turnover assays, the bioluminescence intensity reaches a maximum in only 2 s when the reaction is initiated either by the substrate decanal (as in the D-EF assay) or FMNH (as in the Cu(I)-EAO assay). In comparison, the on and off rates for the decanal inhibitor binding are substantially slower than the binding of the decanal and FMNH substrates.


Figure 7: Relaxation kinetics of decanal binding by individual subunits. A solution containing either 35 µg of and 79.5 µM decanal, or 4 µg of and 60 µM decanal in 0.2 ml of standard buffer was incubated to reach equilibrium, and then diluted 5-fold by the standard buffer at time 0. At different times after the dilution, a 1-ml solution containing 50 µM FMNH and saturating decanal was injected for the measurement of bioluminescence activity. Panel A, semilogarithmic plots of v/vversus post-dilution time were constructed for () and (), and the relaxation times () were determined from the slopes. Panel B, the experiment described for panel A was repeated three more times with the total decanal concentrations before the dilution set at 106, 159, and 185.5 µM for , and 80, 120, and 160 µM for . Plots of 1/ against [E]+[A] are shown for () and ().



In another series of experiments, a limiting level of an individual subunit was first equilibrated with varying levels of aldehyde, denoted as [A]. Subsequently, a solution containing saturating levels of aldehyde and FMNH was injected to initiate the bioluminescence. Assuming independent bindings of the aldehyde substrate (A) and inhibitor (A), the equilibrium binding of A during the first incubation period follows the relationship K = [E][A]/[EA]. Since the binding and dissociation of the decanal inhibitor are much slower than the binding of the aldehyde and flavin substrates, the bioluminescence activity observed upon the secondary injection provides a measure of the amount of individual subunit free from the aldehyde inhibitor during the initial incubation. When the final aldehyde concentration is much greater than the K for the aldehyde substrate, the observe activity would follow the relationship (17) shown in Equation 1. By plotting 1/v against [A] (Fig. 8), values of K were obtained as 19 and 23 µM for and , respectively ().

On-line formulae not verified for accuracy


Figure 8: Linear graphic analyses for the determination of K for decanal inhibitor binding by individual subunits. Varying concentrations (0-50 µM) of decanal were first incubated with either 15 µg of () or 24.5 µg of () in 1 ml of standard buffer to reach equilibrium. Subsequently, 1 ml of standard buffer containing 50 µM FMNH (reduced by the Cu(I) method) and 0.2 mM decanal was injected to initiate the bioluminescence. Reciprocals of the observed activities are plotted against the decanal concentrations present during the initial incubation.



Aldehyde Consumptions

A limiting level of decanal was first added to an excess amount of a subunit and, subsequently, a saturating level of FMNH was introduced to initiate the bioluminescence. Upon completion of the bioluminescence, aliquots were taken and tested for the remaining levels of decanal. The same type of measurements were also carried out using the native luciferase as a positive control. Under our experimental conditions, 85% of the aldehyde was consumed by the native luciferase. whereas only 15% of aldehyde consumptions were detected for either of the two individual subunits.


DISCUSSION

Two earlier studies (11, 12) claim that the individual and subunits of V. harveyi luciferase were each active in bioluminescence. However, one recent study states that the individual was inactive in vitro(13) and another reports that no activity was detected for or in vitro and in vivo(14) . In this work, we found that the isolated individual subunits were indeed active in bioluminescence. A number of general catalytic parameters such as specific activities, Kfor FMNH, K for decanal, and light decay rates in the non-turnover assays using octanal, decanal, and dodecanal were determined for and (). Values for these parameters, with the exception of the K for decanal, were also reported by Sinclair et al.(12) and are in good accord with those shown in .

The individual subunits used in the studies by Waddle and Baldwin (11) and Sinclair et al.(12) as well as in this work were each expressed from a recombinant plasmid harboring either the luxA gene encoding only the or the luxB gene encoding only the . It is unlikely that the individual subunits so obtained were contaminated by their respective counter subunits. However, the specific activities of both individual subunits are about 5 orders of magnitude lower than that of the native luciferase (Ref. 12; ). Moreover, the general catalytic properties mentioned above for the individual subunits either are the same as or differ only slightly from those of the native luciferase. The possibility exists that the very low levels of bioluminescence activities observed for the individual subunits could be due to trace contaminations of the respective counter subunits. These considerations together with the apparently contradictory findings by different laboratories regarding the activities of individual subunits prompted us to more rigorously examine whether individual subunits are truly active.

If the low activities observed with the isolated individual subunits were due to trace contaminations of the respective counter subunits, then the subunit samples should respond the same way as the native luciferase does to activity perturbation under identical conditions. Instead, we found that and, especially, were significantly more resistant than the native luciferase to inactivations by trypsin (Fig. 1) and N-ethylmaleimide (Fig. 2). In another series of experiments, the flavin 4a-hydroperoxide intermediate species formed with and subunits were found to have decay rate constants of 0.036 and 0.031 min, respectively, at 1 °C. Both were close to three times more stable than the flavin peroxide intermediate formed with the native luciferase (Fig. 3). When each of the isolated subunits was chromatographed on a calibrated molecular sieve column, a single protein peak was observed which comigrated with the activity peak corresponding to 42,000 M for monomer and 67,000 M for dimer. If the bioluminescence activities in the isolated subunit samples were due to the presence of trace amounts of , the activity peaks should be eluted at a position corresponding to 76,500 M and should be nondetectable by A reading. This is certainly not the case. Taking the results of activity perturbations, flavin peroxide intermediate stabilities, and molecular weights of the active subunit species together, it is clear that the observed bioluminescence activities are truly associated with individual subunits.

Sensitivities to aldehyde inhibition have been reported long ago for the native V. harveyi luciferase (25) and recently for the individual subunits(12) . We have now carried out several investigations to characterize the aldehyde and flavin binding by the individual subunits. Regarding the effects of ligand preincubation on the bioluminescence activity, both individual subunits behave qualitatively the same as the native luciferase. Substantial inhibitions by decanal were observed when either subunit was preincubated with decanal before the flavin addition, whereas no inhibitions were detected when decanal was added after the flavin binding and very little inhibitions were observed when decanal and FMNH were added together (Fig. 4). These findings indicate that the bindings of decanal inhibitor by individual subunits were significantly slower than the binding of reduced flavin.

At decanal concentrations low enough to obviate aldehyde inhibition, a single flavin site and a single aldehyde site were detected for both the monomeric and the dimeric (Fig. 5). The existence of a single active center per monomeric and dimeric is in good accord with the findings described earlier regarding and being the predominant and catalytically active species in the isolated individual subunit samples. At decanal concentrations effective for inhibition, the existence of a second aldehyde inhibitor site per and was observed (Fig. 6). The binding and dissociation rate constants for the decanal inhibitor were determined for both subunits using the approach of chemical relaxation (Fig. 7; ). These processes were much slower than the bindings of the flavin and aldehyde substrates.

The properties of the individual subunits with respect to aldehyde and flavin bindings are qualitatively quite similar to that of the native luciferase. Therefore, using the native luciferase as a model, the same kinetic scheme (17) can be proposed for the individual subunits regarding the mechanism of aldehyde inhibition. Briefly, and each has a tighter aldehyde substrate site and a weaker aldehyde inhibitor site. The binding of the aldehyde inhibitor blocks the FMNH binding thus resulting in inhibition. Moreover, the binding and dissociation of the aldehyde inhibitor are significantly slower than the bindings of aldehyde substrate and FMNH. Hence, pronounced aldehyde inhibition is only detected when each subunit is equilibrated with aldehyde before the reduced flavin addition.

On the basis of such a kinetic scheme, a linear relationship is expected between the reciprocal of the bioluminescence activity and the concentration of aldehyde during the initial incubation as described for Fig. 8. The expected linear relationships were indeed observed for and . From such a graphic analysis, values of K for the decanal inhibitor binding were determined to be 19 and 23 µM for and , respectively. These values are in good agreements with those determined by chemical relaxation ().

Interestingly, for subunits derived from the V. harveyi luciferase, the predominant and active form of the isolated is monomeric, whereas that of is dimeric. It should be pointed out that the individual subunit of the Vibrio fischeri (originally designated as Photobacterium fischeri) luciferase is long known to assume a dimeric and/or higher polymeric form in aqueous solution(1) . In the case of V. harveyi (formerly designated as MAV) luciferase, the apparent existence of dimer has also been noted(25) . These earlier studies, however, did not establish whether the V. fischeri or the V. harveyi has any low level activity.

At the present, at least two possibilities should be considered regarding the relationship between the subunit active sites and the single active site of the dimeric luciferase. First, and both contribute to the makeup of a single active site at the subunit interface of the native luciferase. In individual subunit forms, each retains sufficient characteristics of the native enzyme active site to be catalytically active but with a much reduced specific activity. Second, the single active site of luciferase is not a combination of two partial sites at the subunit interface. Upon subunit association, either the initial active sites of the two individual subunits are both inactivated and a new active site is generated in the dimeric luciferase or the active site of one subunit is inactivated while the other is greatly activated.

The much reduced specific activities of and could be due to a number of reasons. It is known that the bioluminescence emission is coupled with the conversion of aldehyde to the acid product (4). The substantially diminished abilities to oxidize the aldehyde substrate by and were shown to, at least in part, contribute to their low light-emitting activities. The yields of FMN 4a-hydroperoxide intermediate by the individual subunits may also be compromised. Work is in progress to examine this possibility. The excited state of flavin 4a-hydroxide species has generally been regarded as the emitter in bacterial bioluminescence in the absence of secondary photo-proteins. Free flavin 4a-hydroxide model compounds are only very weakly fluorescent in solution (quantum yield 10 or lower), whereas much enhanced fluorescence was observed upon freezing(27) . It is thus likely that the luciferase flavin emitter (quantum yield 0.1) is tightly bound by the luciferase and well shielded from the medium. While individual subunits retain the ability to catalyze bioluminescence, their active sites may well be more accessible to medium, hence, resulting in reduced emission efficiencies of the bound emitter.

  
Table: Kinetic and equilibrium parameters of luciferase individual subunits

The decay rates of flavin peroxide intermediate II (k) were determined at 1 °C, whereas all other parameters were measured at 23 °C.



FOOTNOTES

*
This work was supported by Grant GM25953 from the National Institutes of Health and Grant E-1030 from The Robert A. Welch Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Dept. of Chemistry, National Tsing Hua University, Hsinchu, Taiwan 30043, Republic of China. Fax: 011-886-35-711082.

The abbreviations used are: FMNH, reduced riboflavin 5`-phosphate; E, luciferase; A, aldehyde; A, aldehyde substrate; A, aldehyde inhibitor; O, molecular oxygen; q, quantum.


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