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J Biol Chem, Vol. 274, Issue 18, 12193-12196, April 30, 1999
From the
Substrate channeling is the process of direct
transfer of an intermediate between the active sites of two enzymes
that catalyze sequential reactions in a biosynthetic pathway (for
reviews see Refs. 1 and 2). The active sites can be located either on separate domains in a multifunctional enzyme or on separate subunits in
a multienzyme complex.
Substrate channeling has been proposed to decrease transit time of
intermediates, prevent loss of intermediates by diffusion, protect
labile intermediates from solvent, and forestall entrance of
intermediates into competing metabolic pathways (2). Loss of an
intermediate by diffusion may be especially important in the case of a
neutral species, such as indole, which could escape from the cell by
passive diffusion across cell membranes (2-5). Nevertheless, there has
been considerable debate over whether channeling actually occurs and
whether it is advantageous (5, 6).
X-ray crystallographic studies on several enzyme complexes have
revealed two molecular mechanisms for channeling. The discovery of an
intramolecular tunnel in tryptophan synthase (7) provided the first
molecular mechanism for channeling. For a long time this was a unique
example, but recent structure determinations have revealed plausible
evidence for tunneling of ammonia and carbamate in carbamoyl-phosphate
synthase (CPS)1 (8) and of
ammonia in phosphoribosylpyrophosphate amidotransferase (GPATase)
(9).
The structure of dihydrofolate reductase-thymidylate synthase, however,
showed no evidence for a tunnel between the two active sites (10).
Neither are the two active sites adjacent to one another. Instead,
there are positively charged residues along the surface between the
active sites that form an electrostatic highway sufficient to channel
the negatively charged dihydrofolate with high efficiency (10, 11).
The focus of this minireview will be on the structural basis of
channeling in the four enzyme structures cited above and on solution
studies of these systems.2
Scheme I summarizes the reactions
catalyzed by the four enzymes. A comparison of the structural and
kinetic results reveals that these enzymes frequently exhibit
allosteric interactions that synchronize the reactions to prevent the
build-up of excess intermediate (12-16).
Tryptophan Synthase--
Tryptophan synthase catalyzes the last
two reactions in the biosynthesis of L-tryptophan (Scheme
I, A) (for reviews see Refs. 14 and 17-19). In bacteria,
the two reactions are catalyzed by separate
High resolution studies of the native enzyme showed that the tunnel was
blocked by the side chain of
Kinetic studies have established that the tunnel actually functions to
channel indole between the two sites (12, 29, 30). Transient kinetic
analysis shows that the rate of indole transfer through the tunnel is
very rapid (>1000 s
The kinetic results also indicate that intersubunit communication keeps
the reactions catalyzed by the wild-type
The crystallographic data suggest mechanisms for the allosteric
interactions between the two active sites. The ligand-induced closure
and ordering of the
The combined structural and kinetic results provide a model for the
allosteric properties of tryptophan synthase (14, 32). Allosteric
signals derived from covalent transformations at the CPS--
A remarkable example of channeling occurs in the case of
CPS, which catalyzes the synthesis of carbamoyl phosphate from
bicarbonate, glutamine, and two molecules of ATP (Scheme I,
B) (for recent reviews see Refs. 34-36). Earlier solution
studies (37) provided evidence that the synthesis involves a series of
four separate reactions that generate three reactive and unstable
intermediates: NH3, carboxyphosphate, and carbamate (Scheme
I, B). These intermediates must be efficiently channeled and
the reactions effectively coupled because the reaction stoichiometry is
precisely 2 mol of ADP and 1 mol of glutamate for every mole of
carbamoyl phosphate (37). The NH3 formed from glutamine
must be channeled because the Km for free
NH3 is 3 orders of magnitude greater than that of
glutamine. In addition, recent results using 13C NMR to
measure isotopic oxygen exchange are consistent with a mechanism
that requires channeling of the carbamate intermediate (38).
CPS from Escherichia coli is composed of two subunits: a
small monofunctional glutamine amidotransferase (
The 2.8-Å resolution crystal
structure4 (Fig.
2) of the enzyme in the presence of ADP,
Mn2+, phosphate, and ornithine (an allosteric effector) (8,
35) revealed that the three active sites are very far apart; the
glutamine binding site of the small subunit (
The coordination of the three active sites of CPS to work synchronously
in the synthesis of carbamoyl phosphate must result from allosteric
communication between these distant sites (16). However, the mechanism
of communication has yet to be determined. CPS catalyzes a very slow
hydrolysis of glutamine in the absence of bicarbonate and ATP. Addition
of these substrates increases the rate of glutamine hydrolysis by 3 orders of magnitude.
GPATase--
GPATase catalyzes the transfer of the amide nitrogen
from glutamine to PRPP, producing phosphoribosylamine, pyrophosphate, and glutamate (Scheme I, C) (for a recent review, see Ref.
34). GPATase from E. coli is a bifunctional enzyme that has
two separate catalytic domains on a single polypeptide chain: an
N-terminal glutaminase domain (
GPATase can utilize either glutamine or free NH3 for the
biosynthesis of phosphoribosylamine (Scheme I, C). Early
studies provided evidence that glutamine and free NH3 bind
at distinct sites (39). First, free NH3 inhibits glutamine
hydrolysis and competes with glutamine as an ammonia source. Second,
alkylation of an essential catalytic residue by a glutamine analog or
by a sulfhydryl reagent inhibited the glutamine-dependent
activity but not the NH3-dependent activity.
These results support a mechanism for glutamine utilization that
involves amide transfer (i.e. channeling) to the
NH3 site.
Crystal structures have been reported for GPATase from E. coli in an open, inactive form (13, 40) and in a closed, active form containing analogs of glutamine and PRPP at the two active sites
(9) (Fig. 3). Comparison of these
structures reveals that activation of GPATase results in the formation
of a 20-Å tunnel that connects the two active sites (9). This tunnel is created by the ordering of a flexible loop in the C-terminal domain,
which closes over the space between the active sites, effectively
sequestering both sites from bulk solvent. This change is accompanied
by the kinking of the adjacent C-terminal TS and DHFR catalyze sequential reactions in the thymidylate
pathway, which supplies 2-deoxythymidylate for DNA synthesis (Scheme I,
D). Although TS and DHFR are distinct monofunctional enzymes
in most species, the two activities are found on a single polypeptide
chain in protozoa and some plants. DHFR-TS from Leishmania major is a homodimer, ( Electrostatic channeling in DHFR-TS is also supported by Brownian
dynamics simulation and experimental kinetic studies (41-44). A recent
transient kinetic analysis of DHFR-TS from L. major (15) demonstrates that the dihydrofolate intermediate is channeled efficiently as the result of two features of the reaction kinetics: the
rate of dihydrofolate transfer between the two sites is very rapid
(>1000 s It is predicted that the highly negatively charged polyglutamylated
molecules of dihydrofolate, which occur in cells (45), would be even
more efficiently channeled than the monoglutamylated folates (10). The
crystal structure of DHFR-TS reveals that the negatively charged
glutamate moieties of the folate analogs at the two sites lie in a
groove along the electropositive highway between the two sites. The
possibility that the bifunctional enzyme has evolved to enhance
channeling is supported by the finding that the DHFR domain in the
bifunctional enzyme has six extra positively charged residues located
between the two sites, which may function in binding the polyglutamate tail.
A comparison of the structural and kinetic features of the
channeling enzymes described above indicates some common features. These enzymes are either multifunctional proteins or stable multienzyme complexes. Because allosteric signals coordinate the activities of each
of these enzymes (12-16), allosteric communication may be a general
and essential feature of channeling enzymes. These allosteric signals
coordinate activities at two or more sites and promote efficient
channeling that prevents the buildup of intermediates and their loss
into solution.
The four examples described above suggest that channeling may be a more
general feature of biochemical processes involving enzyme complexes.
The once isolated example of indole channeling to prevent the escape of
the neutral indole from the cell is now supported by examples of
channeling of reactive intermediates. A large number of enzymes have
been suggested to form stable or transient complexes and to exhibit
channeling or direct transfer of intermediates (2). For example, the
finding of NH3 tunnels in two families of amidotransferases
(GPATase and CPS) suggests that other amidotransferases in these
families may have similar mechanisms of NH3 transfer
(34).
The products of these reactions may themselves be channeled to other
enzymes. For example, there is kinetic and biochemical evidence for
channeling between GPATase and glycinamide ribonucleotide synthetase
(46). This evidence is supported by docking experiments between the
crystal structures of GPATase and glycinamide ribonucleotide synthetase
(47). Another example involves CPS, which catalyzes the first step in
the pyrimidine biosynthetic pathway. In Saccharomyces cerevisiae, CPS is part of a larger polypeptide that catalyzes the
first two steps in this pathway (CPS and aspartyl transcarbamoylase). In mammals, CPS is part of an even larger polypeptide, termed CAD,
which catalyzes the first three steps in this pathway. The interaction
of CPS and aspartyl transcarbamoylase has been proposed to promote
effective coordination of the two activities and channeling of the
labile carbamoyl phosphate intermediate in CAD (48) and in the enzyme
from yeast (49).
*
This minireview will be reprinted
in the 1999 Minireview Compendium, which
will be available in December, 1999.
§
To whom reprint requests should be addressed: Laboratory of
Biochemistry and Genetics, NIDDK, Bldg. 8, Rm. 225, 8 Center Dr. MSC
0830, Bethesda, MD 20892-0830. Tel.: 301-496-2763; Fax: 301-402-0240; E-mail: EdithM{at}intra.niddk.nih.gov.
2
We will not consider enzymes in which the
intermediate is covalently bound (e.g. pyruvate
dehydrogenase and
3
We use the hyphen to indicate that two different
catalytic domains (e.g.
4
Currently at 2.1-Å resolution (50).
The abbreviations used are:
CPS, carbamoyl-phosphate synthase;
GPATase, glutamine
phosphoribosylpyrophosphate amidotransferase;
DHFR, dihydrofolate
reductase;
TS, thymidylate synthase;
PRPP, phosphoribopyrophosphate.
MINIREVIEW
The Molecular Basis of Substrate Channeling*
§, Enzyme Structure and Function Section,
INTRODUCTION
TOP
INTRODUCTION
Tunneling
Electrostatic Channeling
Concluding Remarks
REFERENCES
View larger version (30K):
[in a new window]
Scheme I.
Enzymes that exhibit substrate channeling
in their reactions. Channeled intermediates in reactions are
boxed. Domains in multifunctional enzymes are separated by
hyphens. Additional abbreviations used are: IGP,
indole-3-glycerol phosphate; PLP, pyridoxal phosphate;
G3P, D-glyceraldehyde 3-phosphate;
H2folate, dihydrofolate;
H4folate, tetrahydrofolate;
C2H4folate, methylene tetrahydrofolate;
Glu-NH2, glutamine; PRA,
phosphoribosylamine.
Tunneling
TOP
INTRODUCTION
Tunneling
Electrostatic Channeling
Concluding Remarks
REFERENCES
and 
subunits, which
combine to form a stable multienzyme complex, ()2. In
yeast and molds, the two reactions are catalyzed by separate and
sites in a single bifunctional polypeptide chain,
(-)2.3
Evidence that indole is not liberated as a free intermediate in the
overall conversion of indole-3-glycerol phosphate and
L-serine to L-tryptophan suggested that indole
passes directly from the site to the site without release to
the surrounding solvent (20-23). The 2.8-Å resolution crystal
structure of tryptophan synthase from Salmonella typhimurium
showed that the active sites of the and subunits are about 25 Å apart (7). The structure also revealed that the and sites
are connected by a largely hydrophobic intramolecular tunnel with
dimensions sufficient to accommodate up to four molecules of indole
(Fig. 1). Further refinement of the
native structure and determination of several structures in the
presence of different ligands have revealed a number of ligand-induced conformational changes that may be important for channeling indole and
also for allosteric communication between the and subunits (24-28). In the absence of ligands, the active sites of both the and subunits are accessible to solvent. Dramatic conformational changes occur upon addition of ligands that bind to both and subunits (25, 28). These changes include ordering of disordered loop
structures in the vicinity of the active site of the subunit together with movement of a subunit subdomain (residues 93-189). These conformational changes restrict the access of solvent to the two
active sites and to the tunnel and may also prevent escape of the
indole intermediate into bulk solvent (14, 25, 28).
View larger version (55K):
[in a new window]
Fig. 1.
Tryptophan synthase
22
complex. A substrate analog, indole-3-propanol phosphate
(IPP), locates the subunit active site, and pyridoxal
phosphate (PLP) locates the subunit active site. The
indole tunnel between the active sites of the subunit
(blue) and subunit is shown in the left part
of the complex (7). This tunnel passes between the N-domain
(yellow) and the C-domain (red) of the subunit.
Phe-280. However, crystal structures of
the enzyme in the presence of different cations showed that exchange of
K+ or Cs+ for Na+ results in
movement of the side chain of Phe-280 out of the tunnel (24),
suggesting that Phe-280 can act as a gate to control passage through
the tunnel. This change in structure is accompanied by movements of
many tunnel-associated residues that link the and active sites.
1), that the reaction of indole at
the site is fast and irreversible, and that the reaction of
L-serine at the site to form aminoacrylate increases
the site cleavage of indole-3-glycerol phosphate by approximately
30-fold (12). These three features of the reaction kinetics promote
efficient channeling of indole and prevent accumulation of the indole
intermediate. However, a mutation (E109D) that reduces the rate of
reaction of indole at the site (12) and another mutation (C170F
or C170W) that partially blocks the indole tunnel (31) result in the
accumulation of indole.
and subunits in phase
so that the indole intermediate does not accumulate (12). These kinetic
results and others (32, 33) argue that the reaction of
L-serine at the site to form a pyridoxal
phosphate-aminoacrylate intermediate produces a conformational change
that is transmitted to the site and enhances the rate of indole
formation from indole-3-glycerol phosphate. Thus, indole is produced at
the site with a significant rate only when the site is ready to
receive it.
subunit loop 6 is accompanied by enhanced
interactions with loop 2. Specifically, interactions between the
loop 6 residue, Thr-183, and the loop 2 residue, Asp-60, as well
as main chain interactions decrease the flexibility of loop 2 and are
accompanied by increased interactions of loop 2 with residues in the
subunit mobile subdomain (residues 93-189) (25, 28). These
interactions could provide a route of communication from the site
to the site and vice versa.
and sites
are proposed to switch the enzyme from an open, low activity state to
which ligands bind to a closed high activity state, which prevents the
escape of indole.
), which belongs to
the Triad class, one of the two classes of amidotransferases (34), and
a large, bifunctional subunit () in which the N- and C-terminal
halves are homologous, each having an ATP binding site. The N-terminal
half (N) catalyzes the production of carboxyphosphate, which reacts with NH3 to form carbamate, whereas the
C-terminal half (C) catalyzes the phosphorylation of carbamate.
) is 45 Å from the ADP
binding site of the carboxyphosphate domain (N), which
is 35 Å from the ADP site in the carbamoyl phosphate domain
(C). Examination of the structure reveals a tunnel with
a length of at least 96 Å by which intermediates might pass from the
active site of the small subunit to the carbamoyl phosphate site (Fig.
2). Although the average minimum radius (3.2-3.5 Å) of the proposed
tunnel is sufficient for passage of the NH3 and carbamate
intermediates, constrictions (2.1-2.5 Å) are observed in at least two
places. Movements of residues in the tunnel wall or ligand-induced
conformational changes may enlarge the dimensions of the tunnel.
Interestingly, the proposed NH3 tunnel between the
glutamine and carboxyphosphate sites is lined with hydrophilic residues
and is thus quite different from the hydrophobic NH3 tunnel
in GPATase. In contrast, in the proposed tunnel between the
carboxyphosphate and carbamoyl phosphate sites, there are few charged
side chains that might hydrolyze the labile carbamate intermediate.
View larger version (56K):
[in a new window]
Fig. 2.
CPS. The upper small subunit
(red) catalyzes the release of ammonia from glutamine. The
large subunit contains the carboxyphosphate and the carbamoyl phosphate
active sites. A tunnel, 96 Å long, connects these three sites
(8).
) and a C-terminal acceptor domain
(). The glutaminase domain belongs to a second class of
amidotransferases (Ntn) that have a catalytic N-terminal nucleophile.
The enzyme is a homotetramer, (-)4, and is regulated
by nucleotides and by allosteric control.
-helix. In contrast to
CPS, the walls of the tunnel are lined with conserved hydrophobic
residues. The hydrophobic tunnel may function not only as a passageway
for NH3 but also to exclude water and ensure that
NH3 is the only nucleophile able to enter the tunnel and to
react with PRPP. The conformational differences between the active and
inactive forms provide a structural basis for understanding how PRPP
binding in the acceptor domain stimulates glutaminase activity in the
glutamine domain 200-fold (13).
View larger version (55K):
[in a new window]
Fig. 3.
Structure of GPATase showing the
conformational changes that occur when substrate analogs are bound to
the two active sites. The changes result in closing off the active
sites from solvent and in formation of a 20-Å hydrophobic tunnel for
ammonia transfer (9).
Electrostatic Channeling
TOP
INTRODUCTION
Tunneling
Electrostatic Channeling
Concluding Remarks
REFERENCES
-)2, with an N-terminal
DHFR domain connected to the C-terminal TS domain. The 2.8-Å
resolution x-ray structure of this bifunctional enzyme (10) suggests a
novel mechanism of "electrostatic channeling" across the surface of
the protein. The negatively charged dihydrofolate intermediate is
proposed to move along a positively charged electrostatic "highway"
that links the TS active site to the DHFR site 40 Å away (10, 11) (Fig. 4). The proposed channeling
mechanism is supported by the conservation of positively charged
patches across the surface of monofunctional TS and DHFR from other
sources, suggesting that the monofunctional enzymes may also channel
substrates in vivo (11).
View larger version (88K):
[in a new window]
Fig. 4.
The surface of the DHFR-TS showing the
electrostatic potential distribution. Active site ligands are
shown in yellow. The two sites are connected by a strong
positive pathway along the surface (10). MTX, methotrexate;
PDDF, 10-propargyl-5,8-dideazafolate.
1) and each site is activated by ligand binding
to the other site. Thus, reciprocal communication between the two sites
leads to tight coupling of the reactions at the two sites.
Concluding Remarks
TOP
INTRODUCTION
Tunneling
Electrostatic Channeling
Concluding Remarks
REFERENCES
FOOTNOTES
-ketoglutarate dehydrogenase).
and ) are located on a single
polypeptide chain in a bifunctional or multifunctional enzyme.
ABBREVIATIONS
REFERENCES
TOP
INTRODUCTION
Tunneling
Electrostatic Channeling
Concluding Remarks
REFERENCES
22 multienzyme complex from Salmonella typhimurium. J. Biol. Chem.
263,
17857-1787122 complex.
Biochemistry
35,
4211-4221[CrossRef][Medline]
[Order article via Infotrieve]K87T) tryptophan synthase 22 complex with ligands bound to the active sites of the and subunits reveal ligand-induced conformational changes.
Biochemistry
36,
7664-7680[CrossRef][Medline]
[Order article via Infotrieve]D60N) tryptophan synthase 22 complex reveals the correct orientation of active site Glu49.
J. Biol. Chem.
273,
8553-8555D60N) tryptophan synthase 22 complex reveals allosteric roles of Asp-60.
Biochemistry
37,
10653-10659[CrossRef][Medline]
[Order article via Infotrieve]- and -sites via a 25-30 Å long tunnel.
Biochemistry
29,
8598-8607[CrossRef][Medline]
[Order article via Infotrieve]22 complex: kinetic studies with a mutant enzyme (K87T) provide evidence for allosteric activation by an aminoacrylate intermediate.
Biochemistry
34,
12704-12711[CrossRef][Medline]
[Order article via Infotrieve]
Copyright © 1999 by The American Society for Biochemistry and Molecular Biology, Inc.
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