Mini-Series: Modern Metabolic Concepts. Substrate Channeling MOLECULAR BASES*

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1 2003 by The International Union of Biochemistry and Molecular Biology BIOCHEMISTRY AND MOLECULAR BIOLOGY EDUCATION Printed in U.S.A. Vol. 31, No. 4, pp , 2003 Mini-Series: Modern Metabolic Concepts Substrate Channeling MOLECULAR BASES* Received for publication, April 3, 2002, and in revised form, October 14, 2002 Mario Milani, Alessandra Pesce, Martino Bolognesi, Alessio Bocedi, and Paolo Ascenzi ** From the Giannina Gaslini Institute, Largo G. Gaslini 5, I Genoa, Italy, the Department of Physics, National Institute for the Physics of Matter and Center of Excellence for Biomedical Research, University of Genoa, Via Dodecaneso 33, I Genoa, Italy, and the Department of Biology, University Roma Tre, Viale G. Marconi 446, I Rome, Italy Substrate channeling (or tunneling) is the process of non-covalent direct transfer of a reaction intermediate from the active site of one enzyme to the catalytic center of a second enzyme without prior dissociation into the bulk solvent. Substrate channeling can occur within protein matrix tunnels or along electrostatic highways crossing the surface of multifunctional enzymes, of tightly associated multienzyme complexes, or of transient multienzyme complexes. Substrate channeling has been proposed (i) to decrease the transit time of reaction intermediates, (ii) to prevent the loss of reaction intermediates by diffusion, (iii) to protect labile reaction intermediates from solvent, (iv) to sequester reaction intermediates that are toxic to the cell, (v) to circumvent unfavorable equilibria, (vi) to forestall the entrance of reaction intermediates into competing metabolic pathways, (vii) to prevent the build-up of excess reaction intermediates, and (viii) to closely regulate a block of consecutive reactions within a metabolic pathway or in a multistep catalytic cycle. The three-dimensional structures of Escherichia coli carbamoyl-phosphate synthetase and Leishmania major dihydrofolate reductase-thymidylate synthase beautifully exemplify the concept of substrate channeling. Keywords: Substrate channeling, protein matrix tunnels, electrostatic highways, Escherichia coli carbamoylphosphate synthetase, Leishmania major dihydrofolate reductase-thymidylate synthase. * This study was supported in part by grants from the Italian Space Agency (ASI; IR/294/02 (to M. B.)), from Università Roma Tre (Fondi per lo Sviluppo 2001 (to P. A.)), from the National Research Council of Italy (CNR; Target-oriented Project Biotecnologie and Agenzia 2000 (to M. B. and P. A.)), and from the Giannina Gaslini Institute (to M. B.). This paper is dedicated to Professor Maurizio Brunori who pioneered the function of protein cavities. ** To whom correspondence should be addressed. Tel.: ; Fax: ; ascenzi@bio.uniroma3. it. 228 Substrate channeling (or tunneling) is the process by which the products of an enzymatic reaction are converged directly to the next enzyme in a biosynthetic pathway rather than being transferred by diffusion through the bulk solvent. Substrate channeling can occur within protein matrix tunnels or along electrostatic highways crossing the surface of multifunctional enzymes, of tightly associated multienzyme complexes, or of transient multienzyme complexes. Substrate channeling has many advantages over the free diffusion of reaction intermediates through the bulk solvent. The transit time for transferring the reaction intermediates from one active site to the next is reduced. Chemically labile reaction intermediates can be protected from decomposition due to the aqueous external environment. Reaction intermediates toxic to the cell can be sequestered. Unfavorable equilibria can be circumvented, and reaction intermediates can be segregated from competing enzymatic transformations. The build-up of excess reaction intermediates may be prevented by allosteric communications between the active sites that synchronize enzymatic actions. A block of consecutive reactions, within a metabolic pathway or in a multistep catalytic cycle, may be regulated tightly [1 14]. Examples of substrate channeling have been reported for numerous biochemical pathways, including purine and pyrimidine biosynthesis, amino acid metabolism, lipid metabolism, glycolysis, the tricarboxylic acid cycle, DNA replication, RNA synthesis, and protein biosynthesis. However, direct and compelling experimental evidence for substrate channeling is lacking in many cases claimed for transient multienzyme complexes and a large number of the proposed examples of metabolic channeling. In fact, protein matrix tunnels and electrostatic highways crossing the protein surface may be not apparent from the available three-dimensional structures due to crystallization conditions and/or to their dynamic nature. Moreover the build-up of intramolecular tunnels and electrostatic highways crossing the protein surface is often induced by substrates, cofactors, and allosteric effectors [1 14]. Here the concept of substrate channeling is illustrated through inspection of the three-dimensional structures of Esche- This paper is available on line at

2 229 richia coli carbamoyl-phosphate synthetase (CPS) 1 [14 22] and Leishmania major dihydrofolate reductase-thymidylate synthase (DHFR-TS) [6, 8, 23]. E. coli CPS: TUNNELING OF REACTION INTERMEDIATES WITHIN THE PROTEIN MATRIX The synthesis of carbamoyl phosphate serves as the gateway for two distinct important metabolic pathways: the biosynthesis of arginine and urea and the de novo production of pyrimidine nucleotides. In the urea cycle and in arginine biosynthesis, the carbamoyl moiety of carbamoyl phosphate is transferred to ornithine, whereas in the pyrimidine pathway the same group is condensed with aspartate [12, 14 22, 24, 25]. E. coli CPS catalyzes carbamoyl phosphate production from bicarbonate, glutamine, and two molecules of MgATP via four distinct chemical steps and three reaction intermediates (i.e. ammonia, carboxyl phosphate, and carbamate; see Scheme 1): SCHEME 1 When one or more of the substrates are absent from the reaction mixture, E. coli CPS also catalyzes the three partial Reactions 1 3. Gln H 2 O 3 Glu NH 3 REACTION 1 MgATP H 2 O 3 MgADP P i REACTION 2 MgADP carbamoyl phosphate 3 MgATP NH 2 CO 2 REACTION 3 Moreover ammonia can substitute for glutamine (see Scheme 1) as an alternative source of nitrogen [12, 14 22, 26, 27]. E. coli CPS is a non-covalent (, )-heterodimer composed of the small monofunctional glutamine amidotransferase subunit and the large bifunctional synthetase 1 The abbreviations used are: CPS, carbamoyl-phosphate synthetase; DHFR, dihydrofolate reductase; TS, thymidylate synthase; CH 2 H 4 folate, (6R)-L-5,10-methylenetetrahydrofolate; dtmp, 2 -deoxythymidylate; dump, 2 -deoxyuridylate; FdUMP, 5-fluoro- 2 -deoxyuridylate; H 2 folate, dihydrofolate; H 4 folate, tetrahydrofolate; MTX, methotrexate; PDDF, 10-propargyl-5,8-dideazafolate. All other abbreviations are those recommended by IUPAC. subunit (Fig. 1). The small subunit, a member of the Triad class of amidotransferases, is distinctly bilobal in appearance (Fig. 1); the active site is located at the interface between the N- and C-terminal domains. The small subunit delivers ammonia to the large synthetase subunit, which catalyzes the formation of carbamoyl phosphate. The N-terminal half of the large subunit, the carboxyl phosphate domain (Met 1 Glu 403 ), catalyzes the phosphorylation of bicarbonate and the addition of ammonia to carboxyl phosphate, leading to carbamate (Fig. 1). The C-terminal region of the large subunit, the carbamoyl phosphate domain (Asn 554 Asn 936 ), catalyzes the phosphorylation of carbamate to the final product carbamoyl phosphate (Fig. 1). The carboxyl phosphate and the carbamoyl phosphate domains share 40% amino acid sequence identity and structurally belong to the ATP-grasp superfamily. E. coli CPS is allosterically regulated by binding of the metabolites ornithine, IMP, and UMP to the allosteric domain (Ser 937 Lys 1073 ) of the large subunit. Ornithine and IMP function as activators and promote the formation of the (, ) 4 -heterotetramer, whereas UMP is an inhibitor favoring the formation of the (, ) 2 -heterodimer. However, the oligomerization state and the catalytic activity of E. coli CPS are unlinked, indicating that a complex allosteric mechanism is operative. Direct molecular contacts occur between identical residues within the allosteric domains of two adjacent (, )-heterodimers. The fourth region of the large subunit, the oligomerization domain (Val 404 Ala 553 ), bridges the two homologous synthetase domains, contacts the small subunit in the (, )-heterodimer, and participates in the formation of the (, ) 4 - structure (Fig. 1) [12, 14 22, 28]. By far the most unexpected result from the first structural analysis of E. coli CPS [15] was the extent of intramolecular distances separating the three active sites of the (, )-heterodimer. Indeed the active site in the amidotransferase domain of the small subunit is located at 45-Å distance from the active site in the carboxyl phosphate domain of the large subunit, which in turn is 35 Å away from the active site in the carbamoyl phosphate motif. Notably these three active sites are connected by a 100-Å-long preformed intramolecular tunnel, which leads from the base of the glutamine binding site within the small subunit to the two phosphorylation sites of the large subunit (Fig. 1) [12, 14 21, 26, 27]. The intramolecular tunnel allows channeling of ammonia and carbamate (see Scheme 1) within the catalytic centers of E. coli CPS. Therefore, ammonia and carbamate are not lost to solution during the enzymatic transformations and are not chemically modified by the aqueous external environment. Direct support for the tunneling of ammonia and carbamate within the interior of E. coli CPS has been provided through the tunnel blockage strategy. The degree of constriction within the ammonia tunnel of E. coli CPS mutants correlates with the extent of uncoupling of the partial reactions occurring at the small subunit and at the phosphorylation sites in the large subunit, with the decrease of carbamoyl phosphate formation, and with the percentage of the internally produced ammonia that is channeled to the carboxyl phosphate domain. Moreover the Gly 359 to Phe substitution in the small subunit results

3 230 BAMBED, Vol. 31, No. 4, pp , 2003 FIG. 1. -Carbon trace of (, )-heterodimeric E. coli CPS (Protein Data Bank entry 1JDB) [16]. Blue spheres trace the course of the molecular tunnel that leads from the amidotransferase active site in the small subunit to the carboxyl phosphate catalytic center and finally to the carbamoyl phosphate active site in the large subunit. The tunnel is 100 Å long. The picture has been drawn with the program BOBSCRIPT [59] and has been subsequently rendered with the program RASTER3D [60]. in a complete change in the conformation of the Glu 355 Ala 364 loop, thereby providing an escape route for the ammonia reaction intermediate directly to the bulk solvent and affecting the geometry of key catalytic residues in the amidotransferase domain active site. Channeling of carbamate is impaired by mutation of residues that line the interior walls of the tunnel within the large subunit. However, the blockage of the carbamate tunnel does not affect appreciably partial reactions occurring at the active sites of E. coli CPS and the allosteric communication between and subunits [12, 14 21, 26, 27]. The portion of the tunnel channeling ammonia from the amidotransferase domain in the small subunit to the carboxyl phosphate domain in the large subunit is lined primarily with backbone atoms and unreactive side chains with the exception of Glu 217 and Cys 232. Unreactive residues that define the interior wall of the intramolecular tunnel channeling NH 3 may prevent the protonation of ammonia as NH 4 is incapable of reacting with the carboxyl phosphate intermediate. Moreover the NH 3 formed from glutamine must be channeled because the K m value for free NH 3 is 3 orders of magnitude greater than that of glutamine. The portion of the tunnel channeling carbamate from the carboxyl phosphate domain to the carbamoyl phosphate domain in the large subunit is more polar and includes groups contributed by Glu 577, Glu 604, Arg 848, Lys 891, and Glu 916. The presence of few charged side chains in this portion of the tunnel is consistent with the need to avoid the hydrolysis of the labile carbamate reaction intermediate during intramolecular channeling. The average radius of the intramolecular tunnel connecting the active sites present in the amidotransferase domain, in the carboxyl phosphate domain, and in the carbamoyl phosphate domain is 3.3 Å with constrictions of 2.1 and 2.5 Å that occur at the side chains of Glu 217 and Ile 20, respectively, in the large subunit. Considering the dimensions of ammonia and carbamate, readjustment of residues lining the tunnel wall and/or ligand-induced conformational changes are needed to allow substrate channeling. However, the detailed mechanism for reaction intermediate channeling in E. coli CPS has yet to be determined [12, 14 21, 26 28]. The reaction stoichiometry dictates the precise coupling of the individual parallel and sequential chemical events during the assembly of carbamoyl phosphate. Synchronization of the chemical transformations occurring in E. coli CPS is controlled by coupling active sites within the (, )- heterodimer. In particular, the phosphorylation of bicarbonate within the carboxyl phosphate domain acts as a gate keeper for the intramolecular tunnel in E. coli CPS. Thus, only after bicarbonate is phosphorylated is the hydrolysis of glutamine fast enough to inject a molecule of ammonia into the tunnel. Interestingly the rate of glutamine hydrolysis increases by 3 orders of magnitude in the presence of bicarbonate and ATP. On the other hand, no communication(s) seems to occur between the two ATP binding sites in the large subunit and between the carbamoyl phosphate domain and the glutamine binding site [12, 14 21, 28]. L. major DHFR-TS: ELECTROSTATIC CHANNELING OF REACTION INTERMEDIATES ACROSS THE PROTEIN SURFACE Thymidylate synthase (TS) and dihydrofolate reductase (DHFR) catalyze sequential reactions in the thymidylate cycle, which supplies cells with their sole de novo source of 2 -deoxythymidylate (dtmp) for DNA synthesis. TS catalyzes a reductive methylation of 2 -deoxyuridylate (dump) to form dtmp in which the cofactor for the reaction, (6R)-L-5,10-methylenetetrahydrofolate (CH 2 H 4 folate), is converted to dihydrofolate (H 2 folate). DHFR then reduces H 2 folate to tetrahydrofolate (H 4 folate) in a reaction requiring NADPH. H 2 folate represents the reaction intermediate (see Scheme 2): SCHEME 2 In sources as diverse as bacteriophages, prokaryotes, fungi, mammalian viruses, and vertebrates, TS and DHFR are distinct monofunctional enzymes. Protozoa and at least some plants are unusual in having the DHFR and TS enzymes coded in a single polypeptide [6, 8, 9, 23, 29 36]. L. major DHFR-TS is a homodimeric enzyme ( 2 ) with the N-terminal DHFR domain connected to the C-terminal TS region by a short linker sequence that is absent in monofunctional enzymes. Extensive intersubunit contacts occur between the respective -sheets of the individual TS domains. The DHFR domains, however, are not in contact,

4 231 each one being tethered to its TS domain by the short linker peptide that directly connects the TS N-terminal helix. The TS domain is further stabilized by the DHFR N terminus, which encircles the opposite side of the attached TS domain [23]. The DHFR domain of L. major DHFR-TS consists of an eight-stranded mixed -sheet flanked by -helices, thus displaying an overall fold similar to that characteristic of monofunctional DHFRs. However, the L. major DHFR domain fold is more similar to that of vertebrate monofunctional DHFRs than to that of monofunctional bacterial enzymes. Methotrexate (MTX), a DHFR inhibitor structurally similar to folic acid, as well as the nicotinamide portion of NADPH bind in a deep crevice formed by the three central strands A, E, and F of -sheet, by -helix B, and by loops connecting -strand-a to -helix-b and -helix-c to -strand-c. Residues that directly contact MTX or nicotinamide are conserved in all monofunctional and bifunctional DHFRs [23, 37, 38]. The TS amino acid sequence is among the most highly conserved of known proteins, and indeed the overall secondary and tertiary structures of the TS domain of L. major DHFR-TS are very similar to those found in monofunctional enzymes. TSs consist of a large five- or six-stranded mixed -sheet flanked by predominantly parallel -helices and covered by helical segments and extended surface loops. The binding modes of the potent antitumor agent 5-fluoro-2 -deoxyuridylate (FdUMP) and of the structural analogue of folic acid 10-propargyl-5,8-dideazafolate (PDDF) with the TS domain of L. major DHFR-TS are nearly identical to those observed in the corresponding E. coli TS FdUMP PDDF ternary complex. There are 23 amino acid residues of L. major DHFR-TS that interact directly with FdUMP or PDDF, 19 of which are identical in all known TS sequences and 2 of which are conservatively substituted [6, 23, 39 47]. Unlike E. coli CPS [12, 14 21, 26, 27], the juxtaposition of domains in L. major DHFR-TS does not form bulk solvent-shielded transport paths capable of shuttling H 2 folate between TS and DHFR active sites. The TS active site of one subunit is 70 Å away and on the opposite side of the enzyme molecule relative to the DHFR active site of the second subunit. The two intrasubunit DHFR and TS active sites are located on the same side of the L. major bifunctional enzyme and are separated by a distance of 40 Å (Fig. 2). While dynamic, transient association of the DHFR and TS active sites on one subunit cannot be formally excluded, the DHFR domain does not appear capable of flexing to bring the DHFR and TS H 2 folate binding sites closer together without disrupting the DHFR N-terminal tethering or without severe deformation. Thus, an intersubunit or intrasubunit transfer mechanism of H 2 folate based simply on transient proximity of the DHFR and TS active sites appears unlikely [23]. The analysis of the three-dimensional structure of L. major DHFR-TS suggested a novel mechanism of substrate channeling of H 2 folate across the surface of the bifunctional enzyme. The negatively charged H 2 folate reaction intermediate was proposed to move along a positively charged electrostatic highway that links the TS active site to the DHFR catalytic region of L. major DHFR- FIG. 2. Representation of the electrostatic potential of the solvent-accessible surface of L. major DHFR-TS [23]. MTX and PDDF are bound at the DHFR and TS active sites, respectively. The DHFR and TS catalytic centers are connected by a strong positive potential pathway crossing the bifunctional enzyme surface. This electrostatic highway is 40 Å long. Blue areas represent electrostatically positive regions, and red areas indicate electrostatically negative regions. This picture has been kindly provided by Dr. D. R. Knighton. TS. The L. major DHFR-TS charge distribution results in a positive electrostatic potential surface around and between both H 2 folate binding sites set against a generally negative surrounding protein surface (Fig. 2). Interestingly the negatively charged glutamate moieties of H 2 folate analogues MTX and PDDF present at the DHFR and TS binding regions, respectively, lie in a groove along the electropositive highway between the two sites and point approximately to one another. The possibility that the DHFR-TS bifunctional enzyme has evolved to enhance substrate channeling is supported by the finding that the DHFR domain of L. major DHFR-TS has 6 extra positively charged residues located between the two DHFR and TS H 2 folate binding sites within a monomer, which may function in binding the (poly)glutamate tail of the reaction intermediate. However, H 2 folate electrostatic channeling may occur also between the active sites of monofunctional TSs and DHFRs as suggested by the conservation of some positively charged patches across the enzyme surface [6, 23]. Substrate channeling along the electrostatic highway crossing the surface of L. major DHFR-TS is supported by Brownian dynamics simulation studies and kinetic investigations. In the most active conformer under condition of substrate channeling, H 2 folate is formed at the TS active site (2.6 s 1 ) and does not accumulate but is transferred to the DHFR catalytic center at a rate of 1000 s 1.Atthe DHFR active site, H 2 folate is rapidly converted to H 4 folate (120 s 1 ). Remarkably almost all ( 95%) H 2 folate mole-

5 232 BAMBED, Vol. 31, No. 4, pp , 2003 cules leaving the TS active site reach the DHFR catalytic center. The substrate-dependent activation and interplay between the TS and DHFR active sites are most likely modulated via changes in the protein conformation and indicate that there is a tight coupling of TS-DHFR catalytic activity (Scheme 2) and that domain-domain communication is a prerequisite for efficient channeling of H 2 folate [8, 9, 34, 35]. Some possible mechanisms by which electrostatic channeling might deliver H 2 folate from the TS to the DHFR active sites can be envisioned. If specific charge-mediated hydrogen bonds between H 2 folate and L. major DHFR-TS Lys and Arg side chains are important in binding, then there may be a preferred channeling pathway involving sequential formation and disruption of specific salt bridges as H 2 folate is steered from the TS to the DHFR active site. It appears that basic side chains of the DHFR domain approach the H 2 folate binding site of TS in such a way that this handing off mechanism is an attractive possibility. Alternatively it is simply the overall positive electrostatic potential between the two active sites surrounded by a generally repulsive negative potential that promotes H 2 folate channeling from one site to the other. A third possibility is that elements of both mechanisms may be operative. The electrostatics-based mechanism for channeling H 2 folate from TS to DHFR active sites would be even more efficient for highly negative charged polyglutamylated forms of H 2 folate [6, 23]. Finally the electrostatic highway connecting the TS and DHFR active sites of protozoan bifunctional enzymes may represent a binding region for species-specific drugs to treat some of the most important diseases in the world (e.g. malaria, Chagas disease, African trypanosomiasis, and leishmaniasis) [6]. CONCLUSIONS A possible rationale might be at the basis for the two different substrate channeling mechanisms here illustrated: the substrate channeling within the protein matrix tunnel in E. coli CPS and the substrate channeling along the electrostatic highway crossing the enzyme surface in L. major DHFR-TS. The NH 3 channeling within the protein matrix tunnel prevents the protonation of ammonia, and the intramolecular tunneling of carbamate avoids the decomposition of this labile reaction intermediate due to the aqueous external environment. Electrostatic surface adhesion may have less need for physical confinement. The electrostatic channeling of H 2 folate may not need physical confinement because H 2 folate is a stable intermediate [6, 12, 14]. Substrate channeling within protein matrix tunnels or along electrostatic highways crossing the surface of multifunctional enzymes, of tightly associated multienzyme complexes, or of transient enzyme complexes appears to be a rather widespread means of sustaining biochemical processes. In addition to E. coli CPS, other remarkable examples of ammonia channeling within intramolecular tunnels in allosteric enzymes are Azospirillum brasilense glutamate synthase, Bacillus subtilis and E. coli glutamine phosphoribosylpyrophosphate amidotransferase, E. coli asparagine synthetase B, E. coli glucosamine-6-phosphate synthase, E. coli GMP synthetase, and Saccharomyces cerevisiae imidazole-glycerol-phosphate synthase. Interestingly the intramolecular tunnel for channeling of reaction intermediates in E. coli CPS is preformed. On the other hand, the protein matrix tunnels in E. coli glutamine phosphoribosylpyrophosphate amidotransferase and in E. coli glucosamine-6-phosphate synthase are both allosterically induced, only being observed when substrate analogs, and presumably substrates as well, are bound at both active sites [12, 14 22, 28, 48 58]. A large number of enzymes have been suggested to form stable or transient multienzymatic complexes and to exhibit channeling or direct transfer of reaction intermediates. Thus, S. cerevisiae CPS is part of a single polypeptide that also encodes aspartate transcarbamoylase. This binary enzyme complex catalyzes the first two steps in the pyrimidine biosynthetic pathway. In mammals, CPS is part of an even larger protein that encodes not only aspartate transcarbamoylase but also dihydroorotase. This ternary enzyme complex, named CAD (carbamoyl-phosphate synthetase-aspartate carbamoyltransferase-dihydroorotase), catalyzes the first three steps of the pyrimidine pathway. The interaction of CPS and aspartate transcarbamoylase has been proposed to promote effective coordination of these two enzymatic activities and channeling of the labile carbamoyl phosphate reaction intermediate [12, 14, 22]. As a whole, substrate channeling is a result of the following essential elements: (i) the presence of preformed or allosterically induced protein matrix tunnels or electrostatic highways crossing the enzyme surface to connect different catalytic centers, (ii) the rapid rate of the tunneling event, (iii) the rapid rate of intermediate reaction(s), and (iv) the allosteric communications between active sites that result in full coupling of enzymatic reactions [6, 8 12, 14, 23]. Acknowledgments We thank Dr. Daniel R. Knighton for providing the DHFR-TS picture shown in Fig. 2. 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