The Role of Arginine 310 in Catalysis and Substrate Specificity in Xanthine Dehydrogenase from Rhodobacter capsulatus

The rapid reaction kinetics of wild-type xanthine dehydrogenase from Rhodobacter capsulatus and variants at Arg-310 in the active site have been characterized for a variety of purine substrates. With xanthine as substrate, kred (the limiting rate of enzyme reduction by substrate at high [S]) decreased ∼20-fold in an R310K variant and 2 × 104-fold in an R310M variant. Although Arg-310 lies on the opposite end of the substrate from the C-8 position that becomes hydroxylated, its interaction with substrate still contributed ∼4.5 kcal/mol toward transition state stabilization. The other purines examined fell into two distinct groups: members of the first were effectively hydroxylated by the wild-type enzyme but were strongly affected by the exchange of Arg-310 to methionine (with a reduction in kred greater than 103), whereas members of the second were much less effectively hydroxylated by wild-type enzyme but also much less significantly affected by the amino acid exchanges (with a reduction in kred less than 50-fold). The effect was such that the 4000-fold range in kred seen with wild-type enzyme was reduced to a mere 4-fold in the R310M variant. The data are consistent with a model in which “good” substrates are bound “correctly” in the active site in an orientation that allows Arg-310 to stabilize the transition state for the first step of the overall reaction via an electrostatic interaction at the C-6 position, thereby accelerating the reaction rate. On the other hand, “poor” substrates bound upside down relative to this “correct” orientation. In so doing, they are unable to avail themselves of the additional catalytic power provided by Arg-310 in wild-type enzyme but, for this reason, are significantly less affected by mutations at this position. The kinetic data thus provide a picture of the specific manner in which the physiological substrate xanthine is oriented in the active site relative to Arg-310 and how this residue is used catalytically to accelerate the reaction rate (rather than simply bind substrate) despite being remote from the position that is hydroxylated. The rapid reaction kinetics of wild-type xanthine dehydrogenase from Rhodobacter capsulatus and variants at Arg-310 in the active site have been characterized for a variety of purine substrates. With xanthine as substrate, kred (the limiting rate of enzyme reduction by substrate at high [S]) decreased ∼20-fold in an R310K variant and 2 × 104-fold in an R310M variant. Although Arg-310 lies on the opposite end of the substrate from the C-8 position that becomes hydroxylated, its interaction with substrate still contributed ∼4.5 kcal/mol toward transition state stabilization. The other purines examined fell into two distinct groups: members of the first were effectively hydroxylated by the wild-type enzyme but were strongly affected by the exchange of Arg-310 to methionine (with a reduction in kred greater than 103), whereas members of the second were much less effectively hydroxylated by wild-type enzyme but also much less significantly affected by the amino acid exchanges (with a reduction in kred less than 50-fold). The effect was such that the 4000-fold range in kred seen with wild-type enzyme was reduced to a mere 4-fold in the R310M variant. The data are consistent with a model in which “good” substrates are bound “correctly” in the active site in an orientation that allows Arg-310 to stabilize the transition state for the first step of the overall reaction via an electrostatic interaction at the C-6 position, thereby accelerating the reaction rate. On the other hand, “poor” substrates bound upside down relative to this “correct” orientation. In so doing, they are unable to avail themselves of the additional catalytic power provided by Arg-310 in wild-type enzyme but, for this reason, are significantly less affected by mutations at this position. The kinetic data thus provide a picture of the specific manner in which the physiological substrate xanthine is oriented in the active site relative to Arg-310 and how this residue is used catalytically to accelerate the reaction rate (rather than simply bind substrate) despite being remote from the position that is hydroxylated. The molybdenum-containing hydroxylases represent a unique solution to the hydroxylation of carbon centers. Other monooxygenases introduce an oxygen atom derived from O2 and consume two reducing equivalents (along with the two removed from the substrate to be hydroxylated) in reducing O2 to water. The molybdenum enzymes, on the other hand, utilize water itself as the ultimate source of the oxygen atom incorporated into product. In the case of enzymes such as xanthine dehydrogenase, not only are molybdenum enzyme reducing equivalents not consumed in carrying out the catalyzed reaction, but in fact physiologically useful reducing equivalents in the form of NADH are generated. As such, these enzymes represent a unique solution to the chemistry of hydroxylation, and the requisite cleavage of a carbon-hydrogen bond that accompanies it (1Hille R. Chem. Rev. 1996; 96: 2757-2816Crossref PubMed Scopus (1477) Google Scholar). The xanthine dehydrogenase from Rhodobacter capsulatus is an (αβ)2 heterotetramer comprising two copies each of the XdhA and XdhB gene products (2Leimkühler S. Kern M. Solomon P.S. McEwan A.G. Schwarz G. Mendel R.R. Klipp W. Mol. Microbiol. 1998; 27: 853-869Crossref PubMed Scopus (88) Google Scholar). XdhA possesses two different [2Fe-2S] iron-sulfur clusters of the spinach ferredoxin variety as well as FAD, with each redox-active center located in a separately folded and contiguous domain of the polypeptide. XdhB possesses a molybdenum center with a square pyramidal LMoVIOS(OH) coordination sphere, with L representing a unique pyranopterin cofactor coordinated to the metal via an enedithiolate sidechain. This organic cofactor is common to all molybdenum- and tungsten-containing enzymes, with the sole exception of the multinuclear molybdenum- and iron-containing active site of nitrogenase (1Hille R. Chem. Rev. 1996; 96: 2757-2816Crossref PubMed Scopus (1477) Google Scholar). The structure of the R. capsulatus enzyme is known (3Truglio J.J. Theis K. Leimkühler S. Rappa R. Rajagopalan K.V. Kisker C. Structure (Camb.). 2002; 10: 115-125Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar), and it bears strong structural as well as sequence homology to other members of the molybdenum hydroxylase family of enzymes (including bovine xanthine oxidoreductase (4Enroth C. Eger B.T. Okamoto K. Nishino T. Pai E.F. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 10723-10728Crossref PubMed Scopus (586) Google Scholar, 5Okamoto K. Matsumoto K. Hille R. Eger B.T. Pai E.F. Nishino T. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 7931-7936Crossref PubMed Scopus (248) Google Scholar) and quinoline 2-oxidoreductase from Pseudomonas putida (6Bonin I. Martins B.M. Purvanov V. Fetzner S. Huber R. Dobbek H. Structure (Camb.). 2004; 12: 1425-1435Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar)). The structure of the active site of the R. capsulatus xanthine dehydrogenase is shown in Fig. 1. The molybdenum hydroxylases, which also include aldehyde oxidases from vertebrate, plant, and bacterial sources, are thought to share a common reaction mechanism, which has been best worked out in the case of the bovine enzyme (7Huber R. Hof P. Duarte R.O. Moura J.J.G. Moura I. LeGall J. Hille R. Archer M. Romão M. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 8846-8851Crossref PubMed Scopus (254) Google Scholar, 8Hille R. Trends Biochem. Sci. 2002; 27: 360-367Abstract Full Text Full Text PDF PubMed Scopus (357) Google Scholar, 9Hille R. Arch. Biochem. Biophys. 2005; 433: 107-116Crossref PubMed Scopus (257) Google Scholar). Catalysis is initiated by abstraction of a proton from the Mo-OH group by a universally conserved active site glutamate residue (7Huber R. Hof P. Duarte R.O. Moura J.J.G. Moura I. LeGall J. Hille R. Archer M. Romão M. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 8846-8851Crossref PubMed Scopus (254) Google Scholar) followed by nucleophilic attack on the carbon center to be hydroxylated and concomitant hydride transfer to the Mo=S of the molybdenum center. This reaction yields an LMoIV(SH)(OR) intermediate, with OR representing the now hydroxylated product coordinated to the molybdenum via the newly introduced hydroxyl group. The catalytic sequence is completed by displacement of the bound product from the molybdenum coordination sphere by hydroxide from solvent, electron transfer out of the molybdenum center to the FAD (via the [2Fe-2S] centers), and deprotonation of the Mo-SH to give the original, oxidized LMoOS(OH) form of the center. The specific sequence of these latter events varies with the substrate used and the reaction conditions, but the LMoIV(SH)(OR) species is considered an obligatory intermediate. In addition to the glutamate residue thought to act as a general base, another highly conserved residue in the active site of the xanthine-utilizing enzymes (but not the aldehyde-utilizing ones) is Arg-310 (Arg-880 in the bovine enzyme) (Fig. 1). This residue lies some 10 Å from the molybdenum center, too far to participate directly in catalysis. In the structure of the enzyme with the mechanism-based inhibitor alloxanthine, however, Arg-310 is hydrogen-bonded to the back side of the heterocycle via one of the carbonyl groups of the latter (3Truglio J.J. Theis K. Leimkühler S. Rappa R. Rajagopalan K.V. Kisker C. Structure (Camb.). 2002; 10: 115-125Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar). Also, in a model of urate binding to reduced enzyme based on the crystal structure of the aldehyde oxidoreductase from Desulfovibrio gigas (7Huber R. Hof P. Duarte R.O. Moura J.J.G. Moura I. LeGall J. Hille R. Archer M. Romão M. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 8846-8851Crossref PubMed Scopus (254) Google Scholar), the equivalent Arg is suggested to interact similarly with bound product. In the present work, we examined the catalytic role of Arg-310 and found, surprisingly, that its exchange to methionine results in a 20000-fold decrease in kred (the limiting rate of enzyme reduction upon mixing with substrate). Thus, ∼4.5 kcal/mol of the free energy available to the enzyme from its interaction with substrate is used to accelerate reaction rate rather than to stabilize the E·S complex. An analysis of the reaction of wild-type and R310M or R310K enzymes with a series of other purine substrates provides information on how substrate orients itself in the active site for effective catalysis. Protein Purification—Recombinant R. capsulatus xanthine dehydrogenase was purified using the procedure described by Leimkühler et al. (11Leimkühler S. Hodson R. George G.N. Rajagopalan K.V. J. Biol. Chem. 2003; 278: 20802-20811Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar), with affinity chromatography on a Sepharose 4B/folate gel as the final step. By using PCR mutagenesis, amino acid exchanges R310M and R310K were introduced into R. capsulatus xanthine dehydrogenase. The generated variants were expressed under the same conditions as the wild-type enzyme and purified by nickel-nitrilotriacetate chromatography, Q-Sepharose, and size exclusion chromatography. The purified enzymes were concentrated by ultrafiltration, gel-filtered using a PD-10 gel filtration column (GE Healthcare), equilibrated with 50 mm Tris, 1 mm EDTA, 2.5 mm DTT, pH 7.5, and stored at -70 °C until used. The iron content of wild-type xanthine dehydrogenase and the R310M and R310K variants was determined to be in a range of 93 to 95%, whereas the molybdenum content varied for the proteins: 99% for wild-type xanthine dehydrogenase, 67% for the R310M variant, and 50% for the R310K variant, as analyzed by ICP-OES (Perkin Elmer Optima, DV2100). The levels of molybdenum saturation correspond well with the amount of the pterin cofactor present in the enzymes as determined by conversion to Form A described previously (11Leimkühler S. Hodson R. George G.N. Rajagopalan K.V. J. Biol. Chem. 2003; 278: 20802-20811Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). The wild-type enzyme was determined to be approximately 80% active. Enzyme Assays and Rapid Reaction Kinetics—Routine enzyme assays were carried out as described previously (10Leimkühler S. Stockert A.L. Igarishi K. Nishino T. Hille R. J. Biol. Chem. 2004; 279: 4037-4044Abstract Full Text Full Text PDF Scopus (61) Google Scholar) at 25 °C in 20 mm Tris, 0.2 mm EDTA, pH 7.8, monitoring the absorbance change at 340 nm due to reduction of NAD+ to NADH. The enzyme concentration was determined from the absorbance at 465 nm using an extinction coefficient of 31.6 mm-1 cm-1 (11Leimkühler S. Hodson R. George G.N. Rajagopalan K.V. J. Biol. Chem. 2003; 278: 20802-20811Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). Reductive half-reaction experiments were performed under anaerobic conditions using an Applied Photophysics SX-18MV kinetic spectrophotometer with a 1-cm observation path length. Standard reaction conditions were 20 mm Tris, 0.2 mm EDTA, pH 7.8, at 4 °C. In a typical experiment, enzyme at a concentration of 12–14 was with an of substrate the latter at from 20 to The reaction was at and nm an and the kinetic to to substrates for which varied with substrate was substrate concentration to the limiting rate of reduction at high and the substrates as seen previously with the bovine enzyme Hille R. J. Biol. Chem. Full Text PDF PubMed Google for which not the of the was as was from and were from from and from the of Other were of the available and were used of Arg-310 of R. capsulatus active site and in the R. capsulatus enzyme have been shown to be for catalysis. is thought to act as an active site in the step of the reaction, and its to an the limiting rate of enzyme reduction by substrate by a of at is in substrate binding and transition state as its to results in a decrease in kred and a in (10Leimkühler S. Stockert A.L. Igarishi K. Nishino T. Hille R. J. Biol. Chem. 2004; 279: 4037-4044Abstract Full Text Full Text PDF Scopus (61) Google Scholar). the role of Arg-310 for R. capsulatus xanthine dehydrogenase, the variants R310K and R310M were generated as described under The of the purified variants were to the wild-type and the seen in the nm is due to a rather Mo=S content of the (Fig. The amino acid exchanges were on the of amino acid of and aldehyde-utilizing enzymes, which that these share an of are some in the cofactor binding domain and aldehyde-utilizing enzymes, and the highly conserved and Arg-310 capsulatus in substrate binding in the R. capsulatus enzyme are to and in aldehyde oxidases (Fig. The structure of R. capsulatus xanthine dehydrogenase with at the active site that Arg-310 a bond with the oxygen atom of the of to the of we to the of an exchange of to and methionine on substrate binding and the catalytic The Reaction of R. capsulatus and with half-reaction of wild-type R. capsulatus xanthine dehydrogenase a of the rate on substrate at 4 °C for kred and of and 25 with a for of × in with (10Leimkühler S. Stockert A.L. Igarishi K. Nishino T. Hille R. J. Biol. Chem. 2004; 279: 4037-4044Abstract Full Text Full Text PDF Scopus (61) Google Scholar). of Arg-310 with the are and × that this significantly but not the catalytic power of the to on the other hand, kred to a reduction of than from the seen with the wild-type In the case of the R310M variant, substrate concentration of the rate was seen the range of 20 to as has been previously with the substrate with bovine xanthine Hille R. J. Biol. Chem. Full Text PDF PubMed Google Scholar). This of the wild-type enzyme and R310M variant it is from a of kred that Arg-310 ∼4.5 kcal/mol toward transition state in the wild-type As in the R310K variant was active than the R310M variant for a variety of purine that the at position to less on the kinetic of the is thus that a residue at position to transition state in the reaction kinetic for wild-type R. capsulatus xanthine dehydrogenase and R310K and R310M not of data using two kinetic of data using one kinetic S. Stockert A.L. Igarishi K. Nishino T. Hille R. J. Biol. Chem. 2004; 279: 4037-4044Abstract Full Text Full Text PDF Scopus (61) Google not of data using two kinetic of data using one kinetic step in a The effect on kred upon Arg-310 to methionine is that Arg-310 lies Å from the active site molybdenum and on the opposite end of the substrate from the site that is hydroxylated that a amount of the free energy available to the from the interaction substrate and Arg-310 is used to stabilize the transition state rather than simply bind to substrate). As seen in Fig. however, a in the of the C-6 position is to the on the as a of nucleophilic attack at As the fact that this at C-6 rather than has the orientation of substrate in the active site for catalysis. The Reaction of with addition to we also examined the reaction of R. capsulatus xanthine dehydrogenase with and for which the are shown in Fig. purines were into that xanthine dehydrogenase is also to at and C-6 as well as these are each of the present is in such a that it only be hydroxylated at the C-8 position is and as such they a series in which to substrate The results of this kinetic are in 1. and were to be effective substrates for the R. capsulatus xanthine dehydrogenase, with kred and with seen with two have also previously been to be effective substrates for bovine xanthine A.L. S. Hille R. J. Chem. 2002; PubMed Scopus Google Scholar). By a substrate for the bovine a reduced kred and with the R. capsulatus Although it is the best substrates for the bovine enzyme A.L. S. Hille R. J. Chem. 2002; PubMed Scopus Google Scholar), it is rather less effective than xanthine as a substrate for the R. capsulatus On the other hand, and are substrates than xanthine of the for the wild-type R. capsulatus with kred of and The Reaction of with the R310K and R310M of R. capsulatus The seen with xanthine as substrate was for the other purines used in this with reduced in the R310K variant and significantly reduced in the R310M variant. The exception was seen with the R310K variant was active than the wild-type by a of was a range of for kred for the R310M variant with the substrates a of only 4 as with for wild-type enzyme for the wild-type bovine enzyme A.L. S. Hille R. J. Chem. 2002; PubMed Scopus Google Scholar)). Arg-310 a role in substrate the of the substrates a typical of with wild-type whereas substrate concentration of the rate the range of 20 to mm with the R310M variant. On the of the experiments described it is that the substrates into two groups: members of the first are effective substrates of the wild-type enzyme and are hydroxylated significantly less effectively by the R310M variant, whereas members of the second are substrates of the wild-type enzyme but are much less affected by of Arg-310 to in the first group is the physiological substrate in which hydroxylation than 104-fold in the variant than in the wild-type that Arg-310 is removed from the site hydroxylation it is that kred is affected to the that it is upon of Arg-310 to is that a amount of the free energy available to the enzyme from the interaction of this residue with substrate is used not to stabilize the E·S but to stabilize the transition thereby accelerating the this is in Fig. it be seen that interaction of Arg-310 with the C-6 carbonyl group of substrate on the heterocycle that accompanies nucleophilic attack at of on substrate in the of the reaction is best substrate in the active site with the orientation shown in Fig. with the carbonyl in a position to interact with In the crystal structure of the reduced R. capsulatus enzyme with the inhibitor alloxanthine, however, it is the carbonyl equivalent to of xanthine the of is such that the equivalent carbonyl of is V. H. G. J. Biol. Chem. Full Text PDF PubMed Google that with Arg-310 (3Truglio J.J. Theis K. Leimkühler S. Rappa R. Rajagopalan K.V. Kisker C. Structure (Camb.). 2002; 10: 115-125Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar). is to however, that in the with reduced Å to the molybdenum than product in the of the reaction, directly to the molybdenum via of its whereas the C-8 position of product is to the metal via the hydroxyl oxygen that is introduced catalytically (Fig. removed from the it is that the catalytically orientation of substrate is in fact relative to the orientation seen in the complex. a model for urate bound to xanthine oxidoreductase based on the structure of the gigas aldehyde oxidoreductase the aldehyde-utilizing enzymes, possesses Arg at position equivalent to Arg-310 in the R. capsulatus enzyme) has with the rather than carbonyl group of the product (7Huber R. Hof P. Duarte R.O. Moura J.J.G. Moura I. LeGall J. Hille R. Archer M. Romão M. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 8846-8851Crossref PubMed Scopus (254) Google Scholar). this it was to exchange in the structure of the gigas enzyme to a and to that the hydroxyl groups of and which are in the xanthine-utilizing enzymes, not the of substrate is to be that of the gigas which was not shown in the model (7Huber R. Hof P. Duarte R.O. Moura J.J.G. Moura I. LeGall J. Hille R. Archer M. Romão M. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 8846-8851Crossref PubMed Scopus (254) Google Scholar), a significantly different orientation relative to the molybdenum center than the in in the determined R. capsulatus and bovine these the orientation with with Arg-310 that of substrate at the of catalysis. now the two substrates for the wild-type and an of that be to interact effectively with Arg-310 via by an amino group and a is thus that the of these substrates is due to binding in an orientation relative to this the the catalytic power from Arg-310 be but as is in fact be effect upon of the upon to methionine the was not to in the first The data thus that the in of the two groups of substrate is of different in the active An additional observation the half-reaction of the R310M variant is a With each of the substrates shown in Fig. the reaction with the R310M variant as followed at nm an absorbance decrease followed by a the of that the of the absorbance The effect was at high substrate the specific concentration at which this varied from one substrate to the The absorbance decrease was only a of the absorbance change in the of a than was distinct to the of in the of this form of xanthine dehydrogenase. This is under In in the present we have that the active site Arg-310 of R. capsulatus xanthine dehydrogenase an role in ∼4.5 kcal/mol toward transition state and for a 2 × 104-fold in rate Arg-310 is in the active site well from the molybdenum center and the C-8 position of substrate that is hydroxylated, but it the energy for the reaction by on the heterocycle an electrostatic interaction with the carbonyl oxygen of This in that substrate in the opposite orientation to that seen in the structure of the enzyme in with the mechanism-based inhibitor (but that seen in the model of urate bound to the gigas aldehyde oxidoreductase (7Huber R. Hof P. Duarte R.O. Moura J.J.G. Moura I. LeGall J. Hille R. Archer M. Romão M. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 8846-8851Crossref PubMed Scopus (254) Google Scholar)). This is by the results of a kinetic a series of that are effective substrates of the wild-type binding in an orientation to xanthine and of the catalytic of are strongly affected by the exchange of Arg-310 to whereas that with enzyme and have groups at position and that interaction with that these substrates bind in an orientation to that seen with for these purines are not to of transition state by Arg-310 in wild-type they are to be less to its to as was in fact The in reaction rate the purine substrates used with the R310M variant is less than with the wild-type that Arg-310 a role in the substrate of xanthine dehydrogenase. it is that the catalytic role for Arg-310 also provides an as to the is not conserved in the aldehyde with these enzymes is to be to the carbonyl oxygen of substrate, much the molybdenum center. was Nishino and T. K. Okamoto K. Nishino T. J. Biochem. Scholar) have the of of xanthine an to the R310M considered with the R. capsulatus xanthine dehydrogenase. By kinetic these in the consistent with observation that kred is reduced by a of and in with the bacterial these that toward as substrate is only affected by the in the for of the xanthine dehydrogenase variant