From the
Contradictory findings have recently been reported regarding the
(in)abilities of individual subunits of the Vibrio harveyi
Bacterial luciferase, an
On-line formulae not verified for accuracy
On-line formulae not verified for accuracy
The noncovalent interaction between the
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
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
We
believe that the question as to whether the individual
On-line formulae not verified for accuracy
Two earlier studies (11, 12) claim that the
individual
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
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
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
At
decanal concentrations low enough to obviate aldehyde inhibition, a
single flavin site and a single aldehyde site were detected for both
the monomeric
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,
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
Interestingly, for subunits derived from
the V. harveyi luciferase, the predominant and active form of
the isolated
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,
The
much reduced specific activities of
The decay rates of flavin peroxide intermediate
II (k
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.
heterodimeric
flavin-dependent monooxygenase, catalyzes the light-emitting oxidation
of reduced riboflavin 5`-phosphate (FMNH
)
(
)and a long-chain aliphatic aldehyde by molecular
oxygen.
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.
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.
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.
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.
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
Trypsin (0.5 mg) was added to 10 ml of 0.1 M P,
, and Native
Luciferase
, 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
Sephadex G-150 columns were equilibrated with 0.1 M P and
, 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).
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
The isolated and
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
10
M 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/
v
versus 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 ().
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.
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, K
for
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 .
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.
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.
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.
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.
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.
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 ().
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.
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.
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
) were determined at 1 °C, whereas all
other parameters were measured at 23 °C.
,
reduced riboflavin 5`-phosphate; E, luciferase; A, aldehyde;
A
, aldehyde substrate; A
, aldehyde inhibitor;
O, molecular oxygen; q, quantum.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
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