Determinants of Substrate Specificity in ω-Aminotransferases

Abstract

Ornithine aminotransferase and 4-aminobutyrate aminotransferase are related pyridoxal phosphate-dependent enzymes having different substrate specificities. The atomic structures of these enzymes have shown (i) that active site differences are limited to the steric positions occupied by two tyrosine residues in ornithine aminotransferase and (ii) that, uniquely among related, structurally characterized aminotransferases, the conserved arginine that binds the α-carboxylate of α-amino acids interacts tightly with a glutamate residue. To determine the contribution of these residues to the specificities of the enzymes, we analyzed site-directed mutants of ornithine aminotransferase by rapid reaction kinetics, x-ray crystallography, and 13C NMR spectroscopy. Mutation of one tyrosine (Tyr-85) to isoleucine, as found in aminobutyrate aminotransferase, decreased the rate of the reaction of the enzyme with ornithine 1000-fold and increased that with 4-aminobutyrate 16-fold, indicating that Tyr-85 is a major determinant of specificity toward ornithine. Unexpectedly, the limiting rate of the second half of the reaction, conversion of ketoglutarate to glutamate, was greatly increased, although the kinetics of the reverse reaction were unaffected. A mutant in which the glutamate (Glu-235) that interacts with the conserved arginine was replaced by alanine retained its regiospecificity for the δ-amino group of ornithine, but the glutamate reaction was enhanced 650-fold, whereas only a 5-fold enhancement of the ketoglutarate reaction rate resulted. A model is proposed in which conversion of the enzyme to its pyridoxamine phosphate form disrupts the internal glutamate-arginine interaction, thus enabling ketoglutarate but not glutamate to be a good substrate. Ornithine aminotransferase and 4-aminobutyrate aminotransferase are related pyridoxal phosphate-dependent enzymes having different substrate specificities. The atomic structures of these enzymes have shown (i) that active site differences are limited to the steric positions occupied by two tyrosine residues in ornithine aminotransferase and (ii) that, uniquely among related, structurally characterized aminotransferases, the conserved arginine that binds the α-carboxylate of α-amino acids interacts tightly with a glutamate residue. To determine the contribution of these residues to the specificities of the enzymes, we analyzed site-directed mutants of ornithine aminotransferase by rapid reaction kinetics, x-ray crystallography, and 13C NMR spectroscopy. Mutation of one tyrosine (Tyr-85) to isoleucine, as found in aminobutyrate aminotransferase, decreased the rate of the reaction of the enzyme with ornithine 1000-fold and increased that with 4-aminobutyrate 16-fold, indicating that Tyr-85 is a major determinant of specificity toward ornithine. Unexpectedly, the limiting rate of the second half of the reaction, conversion of ketoglutarate to glutamate, was greatly increased, although the kinetics of the reverse reaction were unaffected. A mutant in which the glutamate (Glu-235) that interacts with the conserved arginine was replaced by alanine retained its regiospecificity for the δ-amino group of ornithine, but the glutamate reaction was enhanced 650-fold, whereas only a 5-fold enhancement of the ketoglutarate reaction rate resulted. A model is proposed in which conversion of the enzyme to its pyridoxamine phosphate form disrupts the internal glutamate-arginine interaction, thus enabling ketoglutarate but not glutamate to be a good substrate. Ornithine aminotransferase (Orn-AT) 3The abbreviations used are:Orn-ATornithine aminotransferaseGABA4-aminobutyrateGABA-ATGABA aminotransferasePLPpyridoxal 5′-phosphateELenzyme with cofactor bound as an aldimine of PLP with Lys-292EMenzyme with cofactor bound as pyridoxamine 5′-phosphateEPPS4-(2-hydroxyethyl)-1-piperazinepropanesulfonic acidTricineN-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycineWTwild type and 4-aminobutyrate aminotransferase (GABA-AT) are members of a large family of pyridoxal 5′-phosphate (PLP)-dependent enzymes that catalyze a wide range of reactions on amino acids (1Mehta P. Christen P. Adv. Enzymol. Relat. Areas Mol. Biol. 2000; 74: 129-184PubMed Google Scholar). Each enzyme operates by a mechanism, common to all aminotransferases (Fig. 1), in which the cofactor shuttles between pyridoxaldimine and pyridoxamine forms by means of two coupled half-reactions (2John R.A. Fowler L.J. Biochem. J. 1976; 155: 645-651Crossref PubMed Scopus (64) Google Scholar, 3Williams J.A. Bridge G. Fowler L.J. John R.A. Biochem. J. 1982; 201: 221-225Crossref PubMed Scopus (24) Google Scholar). The half-reaction converting ketoglutarate to glutamate is the same for both enzymes as well as for the majority of other aminotransferases. In this half-reaction, the chemical changes occur at the α-carbon. The substrate specificity of the enzymes thus arises from the other half-reaction that transfers an amino group distant from the α-carbon. For this reason, the enzymes are known as “ω-aminotransferases” (4John R.A. Fowler L.J. Christen P. Metzler D. Transaminases. John Wiley & Sons, Inc., New York1985: 413-421Google Scholar). In the case of GABA-AT, the amino group transferred is the only amino group in the substrate molecule, whereas Orn-AT specifically selects the ω-amino group of ornithine despite the presence of a second amino group on Cα with the same configuration as that in glutamate (3Williams J.A. Bridge G. Fowler L.J. John R.A. Biochem. J. 1982; 201: 221-225Crossref PubMed Scopus (24) Google Scholar). ornithine aminotransferase 4-aminobutyrate GABA aminotransferase pyridoxal 5′-phosphate enzyme with cofactor bound as an aldimine of PLP with Lys-292 enzyme with cofactor bound as pyridoxamine 5′-phosphate 4-(2-hydroxyethyl)-1-piperazinepropanesulfonic acid N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine wild type The three-dimensional structures of both enzymes have been solved in the unliganded form as well as in complex with various substrate analogues (5Storici P. Capitani G. Müller R. Schirmer T. Jansonius J.N. J. Mol. Biol. 1999; 285: 297-309Crossref PubMed Scopus (54) Google Scholar, 6Storici P. Capitani G De Biase D. Moser M. John R.A. Jansonius J.N. Schirmer T. Biochemistry. 1999; 38: 8628-8634Crossref PubMed Scopus (81) Google Scholar, 7Liu W. Peterson P.E. Carter R.J. Zhou X. Langston J.A. Fisher A.J. Toney M.J. Biochemistry. 1994; 43: 10896-10905Crossref Scopus (59) Google Scholar). The enzymes have the same basic fold as the best studied member of the largest family of PLP-dependent enzymes, namely aspartate aminotransferase (8Ford G.C. Eichele G. Jansonius J.N. Proc. Natl. Acad. Sci. U. S. A. 1980; 77: 2559-2563Crossref PubMed Scopus (249) Google Scholar). The root mean square deviation of Cα positions in Orn-AT and GABA-AT is relatively large at 2.1 Å, and the enzymes have only 17% sequence identity (6Storici P. Capitani G De Biase D. Moser M. John R.A. Jansonius J.N. Schirmer T. Biochemistry. 1999; 38: 8628-8634Crossref PubMed Scopus (81) Google Scholar). Nevertheless, the arrangements of residues at the active sites of the two enzymes are strikingly similar (Fig. 2). All of the direct contacts between cofactor and enzyme protein are conserved. Sterically, there are only two significant differences in the active site regions. One is a straightforward substitution in which Tyr-85 in Orn-AT is replaced by isoleucine in GABA-AT. 4In this study, residues occupying homologous sequence positions will be numbered as in Orn-AT. The other is an exchange of Tyr-55 in Orn-AT for Phe-351* in GABA-AT 5The asterisk indicates that the residue is from the neighboring subunit. . Despite arising from completely different parts of the molecule, the Cϵ atoms of the aromatic rings of these last named residues occupy the same steric position, but the planes of their aromatic rings are perpendicular to each other. The necessary space for the aromatic ring of Tyr-55 in Orn-AT exists because residue 351 is glycine. Conversely, space for the Phe-351* aromatic ring is available because the homologue of residue 55 in GABA-AT is alanine. One of the aims of this study was to determine the extent to which Orn-AT can be converted to GABA-AT by mutating the relevant residues together as well as individually. A further aim of the study was to test a hypothesis advanced to explain the ability of these enzymes to act at ω- and α-amino groups in alternate half-reactions (5Storici P. Capitani G. Müller R. Schirmer T. Jansonius J.N. J. Mol. Biol. 1999; 285: 297-309Crossref PubMed Scopus (54) Google Scholar). We addressed this particular aspect of specificity in the case of Orn-AT because ornithine has both an α- and an ω-amino group. The “Glu-235 switch” hypothesis was inspired by the observation that, uniquely in Orn-AT and GABA-AT, a conserved arginine (Arg-413) is engaged in tight H-bond/salt bridge interactions with the side chain carboxylate of Glu-235 (5Storici P. Capitani G. Müller R. Schirmer T. Jansonius J.N. J. Mol. Biol. 1999; 285: 297-309Crossref PubMed Scopus (54) Google Scholar). This arginine is one of only four residues that are conserved in all the aminotransferases from the same family (9Mehta P.K. Hale T.I. Christen P. Eur. J. Biochem. 1993; 214: 549-561Crossref PubMed Scopus (359) Google Scholar). The crystal structure of Orn-AT bound to an ornithine analogue shows that Arg-413 remains masked by Glu-235 and that the α-carboxylate of ornithine is bound by Arg-180 with the non-reacting α-amino group held between the OH groups of Tyr-55 and Tyr-85 (5Storici P. Capitani G. Müller R. Schirmer T. Jansonius J.N. J. Mol. Biol. 1999; 285: 297-309Crossref PubMed Scopus (54) Google Scholar). These observations led to the proposal that the interaction between Glu-235 and Arg-413 is important in preventing binding of ornithine in an orientation that would lead to transamination of the α-amino group of this substrate. It was further proposed that in the second half-reaction, Glu-235 switches its position and uncovers Arg-413 so that ketoglutarate and glutamate can bind productively through their respective α-carboxylate groups (5Storici P. Capitani G. Müller R. Schirmer T. Jansonius J.N. J. Mol. Biol. 1999; 285: 297-309Crossref PubMed Scopus (54) Google Scholar). To assess the contribution of the proposed Glu-235 switch in determining Orn-AT substrate specificity, we produced Orn-AT E235S and E235A mutants and analyzed their kinetic properties. Mutagenesis, Expression, Preparation, and Assay of Enzymes—A DNA fragment encoding a fusion protein consisting of residues 26-439 of wild-type Orn-AT precursor protein coupled to maltose-binding protein in the expression vector pMal-c2 (New England Biolabs) was transformed into XL1 Escherichia coli cells and used for expression of the wild-type enzyme (5Storici P. Capitani G. Müller R. Schirmer T. Jansonius J.N. J. Mol. Biol. 1999; 285: 297-309Crossref PubMed Scopus (54) Google Scholar). The same vector was used for mutagenesis with the QuikChange™ site-directed mutagenesis system (Stratagene). Expressed proteins were purified according to the standard protocol described in the manual provided with the pMal™ expression and purification system except that ketoglutarate (2 mm) and pyridoxal phosphate (20 μm) were included in the solution used to lyse the bacteria. In this solution and in solutions used for affinity and DE-Sepharose columns, 20 mm Tris/HCl, pH 7.6, was replaced by 20 mm EPPS, pH 7.6. This purification resulted in enzyme that was completely in the EL form. For experiments requiring the EM form of the enzyme, the EL form (0.5 ml, 100 μm) was treated with 5 μl of 100 mml-ornithine and separated on Sephadex G-25 equilibrated in 20 mm EPPS, pH 7.6. Orn-AT and GABA-AT were assayed by the methods of Peraino et al. (10Peraino C. Bunville L.G. Tahmisian T.N. J. Biol. Chem. 1969; 255: 10284-10289Google Scholar) and Silverman and Biochemistry. PubMed Scopus (59) Google and of the half-reactions of EL and EM with were on a All the reactions were at in 20 mm to pH with was at and was the with the All of the of reactions well to experiments were at each substrate and rate were the provided with the was used to the of on substrate for the reactions of ornithine and glutamate with EL forms of the enzyme and of ketoglutarate with EM was used to the of on in its reaction with EL forms (0.5 ml, mm) in was to wild-type mutant enzyme in and at for a of protein a the NMR of the ornithine were at 20 in on a at for and for The 13C were with of the Orn-AT mutants were in by the similar to described for the wild-type protein M. Jansonius J.N. Schirmer T. J. Mol. Biol. PubMed Scopus Google mm mm pH and mm the wild-type Orn-AT to space group in the and have similar Å, for the a Å, for the For the were transferred to a solution and at 100 in of the Orn-AT mutant at were at the A Orn-AT mutant crystal was analyzed and a was on an a with All were with and with from the Biol. 1994; PubMed Scopus Google Scholar). The structures of the two mutants were from a the wild-type Orn-AT structure M. Jansonius J.N. Schirmer T. J. Mol. Biol. PubMed Scopus Google Scholar) as a was with Biol. 1994; PubMed Scopus Google Scholar) and included for the Biol. 1993; PubMed Google was included as of the the were the of the Orn-AT mutant but were in the case of the and were with M. A. PubMed Scopus Google Scholar). The of the was with the R.A. J. 1993; Google Scholar). The have been in the and for and and 5 were with A. a system for of Scholar). of determine the kinetic the half-reactions with different we experiments in which we changes in as the enzyme between its pyridoxaldimine form and its pyridoxamine form of the kinetics of the half-reactions of wild-type Orn-AT with ornithine, glutamate, and ketoglutarate has been (3Williams J.A. Bridge G. Fowler L.J. John R.A. Biochem. J. 1982; 201: 221-225Crossref PubMed Scopus (24) Google Scholar). In the similar were on wild-type Orn-AT to a for determining the of the A study of the half-reaction of wild-type Orn-AT with GABA was in from all the half-reactions well to and the rate on substrate 1), The of all these kinetic are in rate and for the half reactions of wild-type and mutant forms of Orn-AT with in a rate and for the half reactions of wild-type and mutant forms of Orn-AT with increased range (0.5 increased range (0.5 increased range (0.5 in a rate and for the half reactions of wild-type and mutant forms of Orn-AT with in a rate and for the half reactions of wild-type and mutant forms of Orn-AT with in a of the found for the wild-type enzyme that is at the reaction of EL with ornithine that with The between the kinetic toward the two was in the an observation that well with the hypothesis that Glu-235 from its interaction with Arg-413 glutamate can bind productively to We that this would be necessary for ketoglutarate to bind the EM form of the we found that the reaction of EM with ketoglutarate is the of the half-reactions of the wild-type enzyme in this study and has a that for ornithine in its reaction with This observation that, in the case of Arg-413 is available for binding because the interaction between Arg-413 and Glu-235 is which will be in this study, this of of structure of Orn-AT bound to ornithine analogues shows that the α-amino group of ornithine is held between the OH groups of Tyr-55 and Tyr-85 (5Storici P. Capitani G. Müller R. Schirmer T. Jansonius J.N. J. Mol. Biol. 1999; 285: 297-309Crossref PubMed Scopus (54) Google Scholar). Mutation of residue resulted in a similar of for Tyr-55 and 1000-fold for the contribution of each of these interactions to the for the reaction is relatively that both of these are major to the binding of ornithine. The that and led to a relatively of for the reactions with glutamate and ketoglutarate that the by of the Tyr-55 side chain has not the structure of the active the half-reaction with GABA was the only mutant to a in toward GABA was In of the EL form of this mutant with GABA the wild-type enzyme (Fig. shows that the in for the reaction increased with GABA indicating that the from EM the of reaction with ornithine and of the relevant that this converted the wild-type enzyme, which has a for ornithine, to an enzyme with a 5-fold for the resulted in a of specificity in the The of for the half-reaction of with GABA mm is to that for wild-type GABA-AT R.A. Fowler L.J. Biochem. J. 1976; 155: 645-651Crossref PubMed Scopus (64) Google Scholar) which has and that are both the for the presence of tyrosine isoleucine as residue was a major determinant of the ability of the enzyme to between GABA and ornithine. Despite the major of the on the reaction with ornithine, the reaction with glutamate was we a and on the reaction of the mutant with ketoglutarate (Fig. for the reaction of wild-type EM with this substrate was the for the reaction of was to be because increased with ketoglutarate the reaction to be was the of the The of was at ketoglutarate this increased the of for the reaction by at and the of by at We were that the so greatly the ketoglutarate reaction but on the glutamate This observation would be the reaction of EL with glutamate an in which Glu-235 is from Arg-413 by a that is to a major contribution to for the reaction in this of have solved the structure of to a of from the there were significant differences between wild-type and mutant the and and atoms of Tyr-85 in wild-type Orn-AT and in mutant Orn-AT are in positions (Fig. differences between the structures of the mutant and wild-type enzymes be in the the group of into a between the aldimine and the of This was in the wild-type in the the substrate binding was because of the of the atoms of the tyrosine side chain (Fig. These together with the of contacts between Tyr-85 side chain and the substrate (5Storici P. Capitani G. Müller R. Schirmer T. Jansonius J.N. J. Mol. Biol. 1999; 285: 297-309Crossref PubMed Scopus (54) Google explain the significant in toward ornithine for the in toward GABA to the kinetic that not have a major on substrate binding as shown by the relatively in the in was to a in of the structures of GABA-AT and (Fig. shows that is by to the homologous of GABA-AT. with this the two different because GABA-AT with which is not in the Orn-AT that GABA binds to the active site as in GABA-AT with its carboxylate bound to Arg-180 (6Storici P. Capitani G De Biase D. Moser M. John R.A. Jansonius J.N. Schirmer T. Biochemistry. 1999; 38: 8628-8634Crossref PubMed Scopus (81) Google that the side chain of Tyr-85 would although the increased indicates that binding to form the aldimine is by the We that the in arises because the orientation of GABA substrate so that its is held perpendicular to the cofactor the that is to Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). aminotransferase has in a position similar to that of in (8Ford G.C. Eichele G. Jansonius J.N. Proc. Natl. Acad. Sci. U. S. A. 1980; 77: 2559-2563Crossref PubMed Scopus (249) Google Scholar). One of the atoms is to with of the substrate analogue aspartate in the we for and GABA in the major in the kinetic of and the mutant the large of both and for the reaction of EM with These were to the of of into a position to which the and to the atoms of the cofactor and of between which the is the of the cofactor was to be to the cofactor and of were to reaction between the EL form of this mutant μm) and ornithine at to mm and to The the reactions between the EL form of this mutant were with both glutamate and In both decreased and increased with substrate to the this of glutamate, of the protein was of and not be for these the of the with substrate is to The of this was for both for the wild-type enzyme and all other mutants The reaction of the EM form of with ketoglutarate was 100 the reaction of the EL form with glutamate and the on ketoglutarate was the of was for the wild-type enzyme with both and of the structures of the active sites of GABA-AT and the mutant (Fig. shows that the side chain into Orn-AT not occupy the same steric position as in GABA-AT, although the atoms of the residues are in The of with ornithine was not of the tyrosine residues that bind the α-amino group of ornithine and the reaction with ornithine and only were both these but the ring of occupied the space by the α- and of ornithine was bound to the wild-type It that the major of toward glutamate, and ketoglutarate is by of the carboxylate substrate binding site by A for the observation that the side chain not occupy the space by the Tyr-55 side chain and so not a similar orientation to that of Phe-351* in GABA-AT is that in GABA-AT, Phe-351* with the ring of a residue arising from a at a conserved residue in GABA-AT, in The Orn-AT residue to in GABA-AT was glycine. It that, in the of the and interactions with the homologue of a position in which these interactions with of Glu-235 kinetics of the half-reactions of the EL forms of and with ornithine were and The structure of with that the and of Glu-235 with the Cα and of the ornithine It that these contacts both to binding and to orientation of this substrate. E235S and E235A led to a of and an in although the relatively of the that the group of this residue remains to the atoms of its side chain to with ornithine. The E235S produced the same on for the reaction of EL with glutamate as for the reaction with ornithine. whereas for the ornithine reaction was increased for glutamate was decreased The E235A produced similar but the were For glutamate, the of and were and with the that was increased of the relevant that of Glu-235 to produced a switch in specificity from ornithine and toward glutamate of whereas of this residue to alanine produced a switch in the same by a of These observations that a major of the interaction between the side of and Glu-235 is to reaction with glutamate to a to reaction with ornithine. The further observations that the Glu-235 on the reaction of EM with ketoglutarate to the in this that Glu-235 interacts not at with in the EM form of the of E235A Mutation on the of the with experiments to test the proposal that of by Glu-235 from binding the α-carboxylate of ornithine, thus that transamination at the ω-amino group of this substrate. The transamination from the the transferred amino group a half-reaction is in the relevant is A.J. and Wiley New Scholar). This exchange because the half-reaction between amino substrate and to in both is although there is in substrate and It has been shown by NMR that Orn-AT the on of ornithine (3Williams J.A. Bridge G. Fowler L.J. John R.A. Biochem. J. 1982; 201: 221-225Crossref PubMed Scopus (24) Google Scholar). In this we this observation by that the the to the two on of ornithine mm) was to half in the presence of Orn-AT We used 13C NMR of the same ornithine to that the exchange at was 13C NMR was a for and of by at atoms because all the 13C as was replaced by shows the 13C NMR of ornithine and with Orn-AT in The to was a and a This that only one of the atoms on has been replaced of both atoms would have resulted in a The to the Cα was a in both We in the same with the mutant we used the same enzyme as in the with the wild-type enzyme the solution ornithine a of protein by and protein by 13C NMR a of at not A similar at a enzyme mm) for resulted in and (Fig. The to was of a and a This that of one of the on been replaced by to the The was to which two The to Cα as a that exchange at of the of the and the the of to be as by mutating Orn-AT retained its ability to the ω-amino group of ornithine in to the α-amino that the interaction is not for the regiospecificity of the wild-type enzyme in its reaction with ornithine. of kinetic the important Orn-AT EL enzyme with ornithine with glutamate because of a affinity for ornithine. This was Glu-235 was to to alanine. both resulted in for this was not we the second half-reaction by the enzyme, that between EM and Mutation of Glu-235 on that E235S and E235A increased but 100 that of the To this different toward the two we in that in the EL form that with glutamate, the cofactor is bound as a aldimine with whereas in the EM form that with the is pyridoxamine phosphate and is not bound to the The structure of the enzyme, solved the EL form (6Storici P. Capitani G De Biase D. Moser M. John R.A. Jansonius J.N. Schirmer T. Biochemistry. 1999; 38: 8628-8634Crossref PubMed Scopus (81) Google Scholar, 7Liu W. Peterson P.E. Carter R.J. Zhou X. Langston J.A. Fisher A.J. Toney M.J. Biochemistry. 1994; 43: 10896-10905Crossref Scopus (59) Google shows a of Glu-235 and Arg-413 to of the cofactor the side chain of It is that the cofactor is pyridoxamine this is so as to the interaction between Arg-413 and We thus a system in which the Glu-235 switch is in the EL binding of ornithine but binding of glutamate and in the EM Arg-413 available to bind This model well with the that, in Orn-AT operates in the in which ornithine and ketoglutarate are the (4John R.A. Fowler L.J. Christen P. Metzler D. Transaminases. John Wiley & Sons, Inc., New York1985: 413-421Google Scholar). the hypothesis is by the structure Peterson P.E. Langston J.A. Zhou Fisher A.J. Toney Biochemistry. PubMed Scopus Google Scholar) of a mutant form of GABA-AT that is in the EM form and in which the homologue of Glu-235 is to be from the homologous conserved the structure of the homologue of in which the switch be shows the enzyme to be in the EM form Peterson P.E. Langston J.A. Zhou Fisher A.J. Toney Biochemistry. PubMed Scopus Google the proposal that the of the cofactor and the Glu-235 switch are