From the
The lumazine synthase/riboflavin synthase complex of Bacillus subtilis consists of an icosahedral capsid of 60
Sigmoidal
kinetics would be expected for the formation of riboflavin from PYR and
DHB and are indeed observed with mixtures of artifactual
The lumazine synthase/riboflavin synthase complex of Bacillus subtilis consists of 3
The riboflavin synthase reaction
(
The structure of
the
More recently, it was found that the overexpression of
X-ray structures for the
icosahedral
The
Unfortunately, the
crystals of the
Preliminary studies by
Neuberger and Bacher(1986) had indicated that the
In experiments with the
The lumazine synthase/riboflavin synthase complex catalyzes
two consecutive reactions as shown in Fig. 1. Steady state
kinetic studies of the second reaction step (i.e. the
formation of riboflavin from the intermediate LUM) have been reported
earlier (Bacher et al., 1980). More recently, kinetic studies
of the first reaction step, i.e. the formation of the lumazine
LUM from PYR and DHB have been performed (Kis et al., 1995).
Steady state kinetic parameters of both partial reactions are
summarized in . Kinetic parameters for
The
formation of riboflavin from the substrates PYR (initial concentration,
800 µM) and DHB (initial concentration, 105
µM) by the
As
an approximation, the formation of lumazine can be therefore treated as
a one substrate reaction depending on the concentration of DHB. The
velocity of lumazine formation is given by Equation
1,
On-line formulae not verified for accuracy
where [DHB] is the concentration of the substrate DHB
at time t, K
The reaction
catalyzed by the
On-line formulae not verified for accuracy
where [LUM] is the concentration of LUM,
V
Using the kinetic parameters for the two reaction
steps of the
It appeared likely at this stage of the analysis that the kinetic
anomalies are a direct consequence of the tight physical association of
Fig. 4shows a reconstruction experiment using
A
simplified kinetic network for the partial reactions catalyzed by the
On-line formulae not verified for accuracy
where [RIB] is the concentration of riboflavin and
[LUM
On-line formulae not verified for accuracy
The formation of transient lumazine can be given by Equation
5.
On-line formulae not verified for accuracy
The formation of riboflavin via free diffusion can be described
by Equation 6.
On-line formulae not verified for accuracy
The total riboflavin formation rate results from the
contributions of the reactions via the channel and via free diffusion,
respectively. Thus, we obtain Equation
7.
On-line formulae not verified for accuracy
The total formation of riboflavin is described by the
differential Equations 4 and 6, which can be integrated numerically.
The integral was fit to the experimental data in Fig. 2by
numerical iteration yielding the refined parameters shown in I. The lines in Fig. 2A represent the
calculated values using these refined parameters. The agreement between
the numerical simulation and the experimental data is excellent. The
rate of riboflavin formation as calculated by this numerical approach
is shown in Fig. 2B.
In the experiment with the
The partitioning factor
Substrate channeling in biological systems has been the focus
of considerable controversy. The arguments have been summarized in a
series of recent discussion papers (Ovádi, 1991).
Convincing
and detailed evidence in favor of channeling has been obtained for
tryptophan synthase of Escherichia coli (Creighton, 1970; Dunn et al., 1990; Anderson et al., 1991). The enzyme
complex consists of an
In other systems, results
formerly attributed to channeling have been reinterpreted in terms of
free diffusion kinetics on the basis of more detailed kinetic studies
(Kvassman and Pettersson, 1989a, 1989b). Whereas a universally accepted
definition of the criteria for channeling is not available, the absence
of a lag phase for the second step in a sequential reaction is crucial.
The formation of riboflavin by the
An important
consequence of channeling is the high velocity for the conversion of
``nascent'' LUM. The velocity of riboflavin formation has its
maximum value at t = 0 and remains high, while the
lumazine intermediate LUM is being generated from the precursors PYR an
DHB by the
The slow rate of riboflavin formation from transient
LUM (after consumption of PYR or DHB) is a direct consequence of the
high apparent K
The structure of the icosahedral
On the other hand, the
presumptive temporary entrapment of the intermediate LUM inside the
capsid where it is generated could provide a straightforward
explanation of the unusual kinetic properties of the enzyme complex.
The molecule temporarily trapped inside the capsid would have time to
find the active site of the
The pyrimidine PYR is both a product
of the
In summary, the
The chemical stability of the intermediates PYR and
DHB is limited. Thus, DHB can decompose spontaneously under formation
of diacetyl, and the reaction is accelerated by the presence of acids
and bases. The diaminopyrimidine PYR is sensitive to molecular oxygen
and can decompose under formation of a tricyclic pyrimidopteridine
(Cresswell et al., 1960). Moreover, the isolation of
6-methyl-7-dihydroxyethyl-8-ribityllumazine from the culture fluid of
riboflavin-deficient B. subtilis mutant strains (Bresler et al., 1976) suggests that PYR can react with carbohydrates
in bacterial cells.
The riboflavin requirement of B. subtilis under laboratory culture conditions is low. Riboflavin mutants
reach their maximum growth rate at a concentration of about 50 µg
of riboflavin/liter. Nevertheless, B. subtilis has developed a
complex regulatory system for the riboflavin pathway, which allows
up-regulation of expression of biosynthetic enzymes by a factor of at
least 30 from the basic level (Bacher and Mailänder, 1978). Under
laboratory conditions, B. subtilis wild strains are
continually in a state of maximum repression of the riboflavin pathway.
The potential for up-regulation becomes manifest only in
riboflavin-starved riboflavin mutants. Under conditions of maximum
derepression, the lumazine synthase/riboflavin synthase complex
accounts for as much as about 0.5% of cellular protein. This regulatory
potential should enable the microorganism to produce riboflavin at a
level by far exceeding its basic metabolic requirement.
The
evolution of this regulatory system could reflect a need for massive
riboflavin overproduction in natural habitats of B. subtilis.
The selective pressure that was responsible for the evolution of the
regulatory system could also have worked to streamline the metabolic
process by the device of substrate channeling.
Our understanding of
metabolic and regulatory aspects of riboflavin biosynthesis in B.
subtilis is still incomplete. A more detailed understanding of
channeling in the lumazine synthase/riboflavin synthase will require
the determination of the individual rate constants in the kinetic
network shown in Fig. 5.
V
The refined values were
obtained by numerical iteration and yield the line curves in Fig. 4. V
The refined values were obtained by numerical
iteration and yield the line curves in Fig. 2. V
This paper is dedicated to Professor Ivar Ugi on the occasion of
his 65th birthday.
We thank Angelika Kohnle and Cornelia Krieger for
skillful technical assistance and for help with the preparation of the
manuscript. We are grateful to J. Rudolph and J. Stubbe for sharing of
unpublished data.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
subunits surrounding a core of three
subunits. The
subunits catalyze the condensation of
5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione (PYR) with
3,4-dihydroxy-2-butanone 4-phosphate (DHB) yielding
6,7-dimethyl-8-ribityllumazine. This intermediate is converted to
riboflavin by the
subunits via an unusual dismutation. The second
product of this reaction is PYR, which is also a substrate of the
subunits and can be recycled in the catalytic process.
capsids and
subunit trimers. In contrast, the formation of
riboflavin from PYR and DHB by the native
is characterized by a finite initial rate, which is similar to
the rate of lumazine formation. Most notably, the rate of riboflavin
formation has its maximum value at t = 0 and decreases
dramatically after the consumption of PYR and DHB despite the presence
of transiently formed lumazine. These data suggest that a significant
fraction of DHB is converted to riboflavin by substrate channeling,
which is conducive to an improved overall catalytic rate of riboflavin
formation at low substrate concentrations. The channel is leaky, and
the intermediate lumazine is therefore transiently accumulated in the
bulk solution. The partitioning factor relating the direct formation of
riboflavin via substrate channeling and the formation of transient
6,7-dimethyl-8-ribityllumazine increases at low concentrations of the
substrates PYR and DHB and has a maximum value at pH 7.5. Channeling
appears to result from the compartmentalization of the
subunits
inside the icosahedral
subunit capsid whose catalytic sites are
located close to the inner capsid surface.
subunits and 60
subunits (Bacher et al., 1980). The 1-MDa protein complex
catalyzes the terminal two reaction steps in the biosynthesis of
riboflavin (Neuberger and Bacher, 1986; Volk and Bacher, 1990; Kis et al., 1995). More specifically, the
subunits catalyze
the condensation of 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione
(PYR)
(
)(Fig. 1) with
3,4-dihydroxy-2-butanone 4-phosphate (DHB) under formation of
6,7-dimethyl-8-ribityllumazine (LUM) and orthophosphate. The
subunits catalyze the dismutation of the lumazine substrate yielding
riboflavin and the pyrimidine PYR, which serves both as a product of
the
subunits and also as a substrate of the
subunits. Thus
the pyrimidine produced by the catalytic action of the
subunits
can be recycled by the
subunits. The overall stoichiometry
implicates the formation of one molecule of riboflavin and two
molecules of inorganic phosphate from one molecule of PYR and two
molecules of DHB. Every second molecule of PYR must be processed twice
by the
subunits of the enzyme complex in order to allow for
quantitative conversion of PYR to the product, riboflavin.
Figure 1:
Biosynthesis of
riboflavin. , 6,7-dimethyl-8-ribityllumazine synthase;
,
riboflavin synthase.
The
reaction mechanism of the lumazine synthase ( subunit) has been
studied in some detail (Kis et al., 1995). It appears likely
that the initial reaction step consists in the formation of a
Schiff's base by reaction of the 5-amino group of PYR with the
carbonyl group of DHB. Formation of the imine motif may facilitate the
abstraction of a proton from position 3 of DHB, and the resulting
carbanion could eliminate phosphate. The resulting intermediate could
cyclize under formation of LUM.
subunit) has been studied in considerable detail by Plaut and
co-workers using the enzyme from bakers' yeast. This elegant work
has been reviewed repeatedly (Plaut et al., 1970; Plaut and
Harvey, 1971; Plaut and Beach, 1975; Bacher, 1990).
complex of B. subtilis has been studied in considerable detail (Bacher et al.,
1986; Ladenstein et al., 1986, 1994; Bacher and Ladenstein,
1990; Ritsert et al., 1995). The 60
subunits form an
icosahedral capsid with triangulation number T = 1, in
which all protomers occupy equivalent positions. The central core of
the icosahedral capsid is occupied by a trimer of
subunits.
Dissociation of the enzyme complex under mild conditions (e.g. Tris hydrochloride, pH 8) is conducive to the formation of
subunit trimers and of large, heterogeneous aggregates consisting of
more than 100
subunits, which may represent incomplete
icosahedral structures with triangulation number T = 3
or 4 (Bacher et al., 1986). In the presence of substrate
analogs such as 5-nitro-6-ribitylamino-2,4(1H,3H)-pyrimidinedione,
these aggregates can be converted to hollow, icosahedral capsids of 60
subunits, which differ from the native enzyme by the absence of
subunits from the central core space (Schott et al.,
1990a).
subunits in a recombinant Escherichia coli strain is conducive
to the formation in vivo of hollow
capsids.(
)
The artifactual
capsids catalyze the formation of LUM from PYR and DHB at
virtually the same rate as the native
enzyme complex (Kis et al., 1995). Moreover, it was
found that even the large, heterogeneous
subunit aggregates with
molecular masses in the range of 3-5 MDa catalyze the formation
of LUM. However, it is possible that the catalytic activity of these
aggregates depends on ligand-induced reaggregation conducive to the
formation of
capsids.
subunit capsid have been obtained with crystals of
the native
capsids and with
artifactual
capsids obtained by ligand-driven
renaturation of
subunits (Ladenstein et al., 1988, 1994;
Ritsert et al., 1995). The two respective structures are
virtually identical, but the artifactual
capsids
diffract x-rays to significantly higher resolution than the native
species.
subunit
capsid is best described as an assembly of 12 densely packed pentamers.
Channels formed by five skew
helices surround the 5-fold axes.
The 60 equivalent active sites are located at the interface between
adjacent pentamer subunits in close proximity of the inner capsid
surface. Since the capsid is rather densely packed, it is as yet an
open question how the substrates and products can exchange with the
bulk solvent. The channels along the five-fold axes could allow the
passage of substrates but appear too narrow for the passage of LUM and
of riboflavin (Ladenstein et al., 1994).
complex yield no
structural information for the
subunits. It appears likely that
the
subunits can be located on any of the 10 three-fold axes of
the icosahedral capsid. This would allow for 20 different positions
with respect to the crystal lattice. Sequence analysis has suggested
that each
subunit forms two homologous domains (Schott et
al., 1990a). Moreover, NMR studies suggest that the
subunit
trimer may not obey strict C
symmetry.(
)
However, the trimers might be characterized by pseudo-C
or pseudo-D
symmetry.
complex has unexpected kinetic
properties. This paper describes a more detailed analysis of steady
state kinetics indicative of substrate channeling between the
and
subunit sites.
Materials
5-Nitro-6-ribitylamino-2,4(1H,3H)-pyrimidinedione
(Cresswell et al., 1960) and 6,7-dimethyl-8-ribityllumazine
(Bacher, 1986) were prepared by published procedures. PYR was freshly
prepared by hydrogenation of
5-nitro-6-ribitylamino-2,4(1H,3H)-pyrimidinedione over palladium on
charcoal in aqueous solution (Sedlmaier et al., 1987). Both
enantiomers of DHB were prepared as described elsewhere (Kis et
al., 1995).
Proteins
The lumazine synthase/riboflavin synthase
complex (``heavy riboflavin synthase'') was purified from the
derepressed B. subtilis mutant H94 (Schott et al.,
1990b). Recombinant capsids were kindly provided by
Markus Fischer and Karl Kugelbrey.
Estimation of Protein Concentration
Protein
concentration was monitored photometrically using absorbance
coefficients of = 7.0
for the lumazine synthase/riboflavin synthase complex (Bacher et
al., 1980) and of
= 8.0 for isolated
subunits (Bacher et al.,
1986).
Enzyme Assay
Reaction mixtures contained DHB (S or R) and freshly prepared PYR in the
concentrations indicated. The mixtures also contained 100 mM
potassium phosphate, pH 7.0, 2 mM EDTA, and 2 mM dithiothreitol unless otherwise noted. They were incubated at 37
°C. Aliquots were taken at intervals, and the reaction was quenched
by the addition of trichloroacetic acid to a final concentration of 0.4 M. Concentrations of LUM and riboflavin were determined by
high performance liquid chromatography analysis. Columns of Nucleosil
RP18 (4 250 mm) were used throughout. The effluent was
monitored fluorometrically. For the analysis of LUM, an eluent
containing 7% methanol and 30 mM formic acid was used
(excitation, 408 nm; emission, 487 nm). The retention volume was 8.4
ml. For the analysis of riboflavin, the eluent contained 40% methanol
and 100 mM ammonium formate (excitation, 445 nm; emission, 516
nm). The retention volume was 6.0 ml.
complex, the total amount of
lumazine formed by the catalytic activity of the
subunits was
calculated as the sum of the molar amount of LUM plus 2 times the molar
amount of riboflavin to account for the stoichiometry of the
dismutation reaction, which generates one molecule of riboflavin from
two molecules of LUM.
Calculations
Numerical simulations were performed
using the program Microsoft Excel Solver on a PC with a 386DX
processor from Intel
and a 387DX coprocessor from Cyrix
.
subunit
trimers (``light riboflavin synthase'') and for recombinant
capsids devoid of
subunits are also shown. It
should be noted that the K
value for LUM of the
complex is about 10 times larger as
compared to light riboflavin synthase (
).
complex is
shown in Fig. 2. The initial reaction phase is characterized by
the formation of riboflavin at a rate of 2,600 nmol mg
h
, whereas the formation of the intermediate,
LUM, proceeds with an apparent rate of 6,100 nmol mg
h
. After the consumption of the substrate DHB,
the concentration of the transient intermediate LUM shows a slow
decrease. Concomitantly, the velocity of riboflavin formation decreases
by a factor of about 30 as compared to the initial reaction phase. This
kinetic behavior is unexpected. Most notably, the initial rate of the
second reaction step in a sequential reaction would be expected to have
a value of zero, and the time course of riboflavin formation would be
expected to have a sigmoidal character. On the contrary, the rate of
riboflavin formation by the
complex
has its maximum value at t = 0 and shows first a slow
decrease and later, when the substrate DHB has been consumed, a very
marked decrease. Numerically, the initial rate of riboflavin formation
in this experiment corresponds to 1.3 times the value of V
for the conversion of exogenous LUM into
riboflavin by the
complex.
Figure 2:
Kinetics of product formation by the
complex. A, formation of
LUM (
) and riboflavin (
) from S-DHB and PYR. B, velocity of riboflavin formation. The reaction mixture
contained 800 µM PYR, 105 µMS-DHB,
and 74 µg of the
complex in 1
ml of potassium phosphate buffer (100 mM, pH 7.0) at 37
°C. The reaction was started by the addition of protein. The lines
were calculated as described under ``Results'' using the
refined parameters in Table III.
The
reaction catalyzed by the subunit is a bisubstrate reaction.
Thus, this reaction step is inherently first order for each of the
substrates PYR and DHB. However, the experiment described above can be
treated as zero order for the pyrimidine for the following reasons. (i)
The pyrimidine was present in an 8-fold excess over the carbohydrate.
(ii) The K
for the pyrimidine (5 µM) is
small as compared to the actual pyrimidine concentration (800
µM at t = 0). (iii) The second reaction
step, i.e. the formation of riboflavin by the
subunit,
regenerates a certain amount of PYR as the second product of the
dismutation reaction, thus slowing down the consumption of PYR.
is the Michaelis
constant of the
complex for the
substrate DHB (130 µM), and V
is the maximum velocity of formation of LUM.
subunits, i.e. the dismutation of the
lumazine under formation of riboflavin and the pyrimidine PYR, is also
a bisubstrate reaction. Surprisingly, this reaction has been shown to
follow Michaelis-Menten kinetics over a wide range of concentrations in
experiments by Harvey and Plaut(1966) using the enzyme from
bakers' yeast. Later, the
subunit trimer (light riboflavin
synthase) of B. subtilis has also been shown to obey
Michaelis-Menten kinetics over a wide range of substrate concentrations
(Bacher et al., 1980). Thus, the velocity of riboflavin
formation from LUM can be given as
is the maximum velocity of riboflavin
formation, and K
is the Michaelis constant of
the
complex for LUM (130
µM).
complex given in and the starting conditions of the experiment in Fig. 2, the formation of riboflavin by the sequential reaction
DHB + PYR
LUM
RIB can be predicted by numerical
integration of Equations 1 and 2. A comparison of the numerical
prediction with the experimental data is shown in Fig. 3. It is
obvious from this comparison that the model of independent, consecutive
reactions as specified by Equations 1 and 2 is not an appropriate
description of the experimental data. Most notably, the absence of a
lag phase for the experimentally observed formation of riboflavin
indicates that the formation of riboflavin does not depend on the
presence of the intermediate LUM in finite concentration in the bulk
solvent. This could be explained by the hypothesis that a significant
fraction of the
subunit product is directed to the
subunit
active site by substrate channeling rather than by equilibration with
the bulk solution (for a more detailed definition of substrate
channeling, see ``Discussion'').
Figure 3:
Formation of riboflavin by the
complex by substrate channeling (solid line, data from Fig. 2) and prediction of riboflavin
formation by the free diffusion model (dashed line, calculated
with Equations 1 and 2 and kinetic constants from Table
I).
The transient formation
of LUM in substantial concentration also indicates that only a fraction
of the intermediate LUM is handled by substrate channeling, whereas the
remaining part reacts via the standard consecutive model after
equilibration with the bulk solvent. In other words, we propose
partitioning between a channeled pathway and a pathway involving
equilibration with the solvent, (i.e. a ``leaky
channel'' model following the terminology of Ovádi (1991)).
and
subunits in the
complex. This hypothesis can be tested directly, because the
subunits as well as the
subunits can assemble separately to
yield catalytically active oligomers. Thus,
subunits can form
stable trimers (light riboflavin synthase), which have approximately
1.7 times the turnover number as the
complex and a K
value of 13
µM as compared to 130 µM for the
complex. Moreover, the recombinant
expression of
subunits in E. coli in the absence of
subunits yields icosahedral particles consisting of 60
subunits whose V
and K
values are similar to the
complex () (Kis et al., 1995).
molecules and recombinant
molecules at a molar
ratio of 1:1 (i.e. 3
subunits/60
subunits as in
the native complex). The concentrations of LUM and of riboflavin are
shown. Most notably, the experiment shows the expected lag phase with a
value of zero for the initial rate of riboflavin formation.
Figure 4:
Kinetics
of product formation by a 1:1 mixture of and
molecules. A, formation of LUM (
) and
riboflavin (
) from PYR and DHB. B, velocity of riboflavin
formation. The reaction mixture contained 600 µM PYR and
230 µMS-DHB in 100 µl of 100 mM potassium phosphate, pH 7.0. The reaction was started by the
addition of 34.2 µg of recombinant lumazine synthase
(
) and of 2.5 µg of light riboflavin synthase
(
). The lines were calculated as described under
``Results'' using the refined parameters from Table
II.
Using
Equations 1 and 2, the formation of riboflavin by the
/
mixture (Fig. 4) was
simulated using the values of V
and K
for
and
molecules as shown in . Subsequently, the values for V
and K
were
refined by numerical iteration. A close fit was obtained with the
refined parameters given in . The lines in Fig. 4A show the numerical fit. Also shown is the
calculated velocity of riboflavin formation (Fig. 4B).
The fundamental difference between the experiments in Fig. 2and Fig. 4is best explained by the involvement of substrate
channeling in the enzyme complex but not in the mixture.
complex is shown in Fig. 5.
We assume that the transfer of the lumazine LUM from the
subunits
to the
subunits can either occur by equilibration of the
intermediate with the bulk solvent by free diffusion, or by substrate
channeling without equilibration with the bulk solution. The simplified
scheme does not take into account that the two lumazine molecules
required for the dismutation reaction must be supplied to the
subunits one by one. Moreover, the dissociation of products from the
subunits as well as the binding of substrate to the
subunits may be more complex than expressed in the scheme.
Figure 5:
Kinetic
model of the reactions catalyzed by the complex.
,
subunits;
,
subunits.
Enzyme-bound ligands are shown in parentheses; RIB,
riboflavin.
Even for
the simplified scheme, the rate constants for most of the partial
reactions shown are unknown. It is therefore not possible at this time
to perform a comprehensive numerical simulation of the proposed leaky
channel behavior of the complex.
However, a successful attempt was made to simulate the data in Fig. 2by a heuristic kinetic model. It is obvious that the
contributions of the channeled and the free diffusion pathway must be
additive. We assume that the free diffusion pathway can be described,
in principle, by Equations 1 and 2. Moreover, we assume that, under a
given set of starting conditions, each lumazine molecule formed after
the initiation of the reaction has a finite probability to enter the
channeled or the unchanneled pathway. This probability of a molecule to
react via the channel is given by the partitioning factor
defined
as shown by Equation
3,
] is the apparent concentration of LUM, i.e. the concentration of the transient intermediate in Fig. 2. The factor 2 must be introduced because two lumazine
molecules must enter the channeled pathway in order to obtain one
molecule of riboflavin. The experimental value of
at t = 0 is easily determined from the initial velocities in Fig. 2. The formation of riboflavin via channeling is rigidly
coupled to the velocity of the first reaction step and can be given by
Equation 4.
complex, rate acceleration by
substrate channeling can of course only occur while the substrates PYR
and DHB are available. After their exhaustion, the consumption of the
transient lumazine LUM occurs by the unchanneled reaction. Since the K
of the
complex for LUM is relatively large as compared to free
trimers, the consumption of the transient LUM is slow in the experiment
with the
complex (Fig. 2) as
compared to the
/
mixture (Fig. 4).
depends on the
substrate concentration. For a systematic study, the initial
concentration of PYR or of DHB was varied, while the other substrate
was proffered at a high concentration well above the respective K
value (Fig. 6). In both
experiments, the partitioning factor increased at lower substrate
concentrations. Values close to 0.9 were observed at low concentration
of the pyrimidine PYR, even in the presence of DHB at high
concentration. Values of
close to 1 can be expected when both
substrates are present at low concentrations.
Figure 6:
Partitioning coefficient derived
from initial rates according to Equation 3 at different substrate
concentration. The reaction mixtures contained 1.5 mM DHB (A) or 2 mM PYR (B) and 1.5 µg of
complex in 100 µl of 100 mM potassium phosphate buffer, pH 7.0.
The data indicate that
substrate channeling is favored by a low velocity of lumazine
formation. This suggested that a substrate analog for lumazine synthase
with an inherently low conversion rate should be conducive to efficient
substrate channeling. We have found earlier that the R-enantiomer of DHB is converted to LUM with a velocity of
about 1/6 as compared to the natural S-enantiomer. Fig. 7shows an experiment where the R- and S-enantiomers were converted to riboflavin by the
complex under identical conditions.
Numerical modeling was performed as described above. The partitioning
factor is 0.4 in the case of the S-enantiomer and 0.6 in the
case of the R-enantiomer. The initial rate of riboflavin
formation is of course lower with the R-enantiomer due to its
slow conversion into the intermediate LUM.
Figure 7:
Kinetics of product formation by
complex. Formation of riboflavin
(
) and LUM (
) from PYR and S-DHB (A) and
from R-DHB (B) is shown. C, velocities of
riboflavin formation from S-DHB (solidline)
and R-DHB (dashedline). The reaction
mixtures contained 180 µMS-DHB or R-DHB, 800 µM PYR, and 90 µg of
complex in 1 ml potassium phosphate
buffer, 100 mM, pH 7.0, at 37 °C. The lines were
calculated as described under
``Results.''
As shown in Fig. 8,
the partitioning factor has a maximum value at a pH around 7.5. Both
lumazine synthase and riboflavin synthase have their respective pH
optimum in the same range.
Figure 8:
pH dependence of the partitioning
coefficient . The reaction mixture contained 200 µM
PYR, 50 µMS-DHB and 22 µg of
complex in 120 µl of potassium
phosphate/Tris buffer (100 mM) at 37
°C.
The partitioning factor characterizing
the flow of intermediate through the channeled and unchanneled pathway
is independent of protein concentration (data not shown).
subunit catalyzing the formation of indole
from indolyl glycerol phosphate and a
subunit converting indole
to tryptophan. The indole moiety is supposed to pass rapidly through a
channel inside the dimeric protein from the active site of the
subunit to the active site of the
subunit. The activity of the
subunit is low unless the
subunit is charged with the
second substrate, serine, which is required for the conversion of
indole to tryptophan. As a consequence, virtually no free indole is
formed, and channeling is perfect.
complex is best described by a leaky channel model. This is
especially true when both substrates PYR and DHB are present in high
concentrations. At low concentrations of the pyrimidine substrate, the
partitioning factor reaches values greater than 0.8.
subunits but decreases by a factor of more than 30
after the consumption of one of the
subunit substrates, despite
the high concentration of the transient lumazine intermediate in the
bulk solvent.
of the
subunits in
the
complex for the lumazine
substrate. Thus, at a maximum concentration of 45 µM LUM
in Fig. 2, the rate of riboflavin formation can be determined to
be less than 5% of V
. It also follows that the
formation of riboflavin by channeling in the initial part of the
experiment in Fig. 2proceeds at a velocity equivalent to V
even at t = 0, i.e. under conditions where the bulk concentration of LUM is virtually
zero. The efficiency of the
subunits is thus dramatically
enhanced by channeling.
subunit capsid has been determined at high resolution. Each of the 60
active sites is located at the interface of two adjacent
subunits
within the pentamer substructures of the capsid, and in close proximity
of the inner capsid surface. Thus, substrates and products could easily
find access to the active site from the central cavity. On the other
hand, it is not clear how the exchange of substrates and products
between the bulk solvent and the active site proceeds. The channels
running parallel to the five-fold symmetry axes of the capsid would
allow the passage of substrates PYR and DHB, but are too narrow for
riboflavin and even for LUM (Ladenstein et al., 1994). Major
dynamic motions of the capsid may be necessary to enable the exit of
LUM or riboflavin. Under saturation conditions for the substrates, PYR
and DHB, the
molecule can produce
about 0.7 molecules of riboflavin per second. In addition, the enzyme
molecule will produce about 1.5 molecules of LUM. Local molecular
motions proceed on the time scale of picoseconds. The catalytic cycle
of the enzyme proceeds about 11 orders of magnitude more slowly, and
this would leave ample time for major dynamic motions of the capsid as
a whole, which could enable molecules as large as LUM and riboflavin to
slip in and out of the protein molecule.
subunits where it is converted to
riboflavin. The increase of K
by a factor
of 10, which accompanies the enclosure of the
subunit trimer in
the
subunit capsid, could be a consequence of hampered diffusion
of LUM through the capsid wall.
subunit and a substrate of the
subunit. Thus, the
intermediate PYR could enhance the overall velocity by channeling from
the
subunits to the
subunits, i.e. in the opposite
direction as LUM. However, no experimental evidence for this plausible
mechanism has been obtained up to now.
complex is a very efficient device
for the rapid formation of riboflavin at low concentration of PYR and
DHB. This raises the question how an enzyme complex with these
remarkable properties could have developed. Factors providing the
necessary selective pressure for the evolution of the complex could
have been (i) a requirement to produce riboflavin at a high rate with a
minimum amount of catalyst or (ii) a requirement to avoid the
accumulation of unstable intermediates which might be lost by wasteful
side reactions.
Table: Steady
state kinetic parameters of the proteins under study (data from Bacher
et al., 1980, and from Kis et al., 1985)
, V
, maximum velocities of
lumazine synthase (
) and riboflavin synthase
(
), respectively. K
, K
, K
, Michaelis constants for
the substrates indicated.
Table: Kinetic
simulation of riboflavin formation by the
/
mixture
and V
, maximum velocities of
lumazine synthase (
) and riboflavin synthase
(
), respectively. K
, K
, Michaelis constants for
the substrates indicated.
Table: Kinetic simulation
of riboflavin formation by the complex
, V
, maximum velocities of
lumazine synthase (
) and riboflavin synthase
(
), respectively. K
, K
, Michaelis constants for
the substrates indicated.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.