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

Abstract

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, k red (the limiting rate of enzyme reduction by substrate at high [S]) decreased ϳ20-fold in an R310K variant and 2 ؋ 10 4 -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 ϳ4.5 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 k red greater than 10 3 ), 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 k red less than 50-fold). The effect was such that the 4000-fold range in k red 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 O 2 and consume two reducing equivalents (along with the two removed from the substrate to be hydroxylated) in reducing O 2 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 (1).The xanthine dehydrogenase from Rhodobacter capsulatus is an (␣␤) 2 heterotetramer comprising two cop...

Substrate Orientation and Catalytic Specificity in the Action of Xanthine Oxidase

“…With each substrate, one orientation has C-2 positioned appropriately for hydroxylation by the molybdenum center, and the second has C-8 so positioned instead. Both of the orientations seen here have the exocyclic functional group at the C-6 position adjacent to a conserved arginine residue (Arg 880 in the bovine enzyme), consistent with our previous conclusions regarding the catalytic role of Arg 880 in stabilizing negative charge accumulation on the heterocycle at this position in the course of the reaction (12)(13)(14). In light of these results, we have undertaken further kinetic studies and confirm that xanthine is indeed the sole product of enzyme action on hypoxanthine; to within our detection limits 6,8-dihydroxypurine is not formed, despite the fact that hypoxanthine is able to bind in an orientation that permits such hydroxylation.…”

“…We have previously suggested (12) that Arg 880/881 (Arg 310 in the R. capsulatus enzyme) is involved in transition state stabilization by compensation of negative charge accumulation on the C-6ϭO oxygen of xanthine in the course of hydroxylation at C-8, and the orientation we see here indicates that this is also likely to be the case for hydroxylation of hypoxanthine at C-2, because the C-6 carbonyl is again oriented toward the arginine. Similarly, we have suggested (14,34) that Glu 802/803 (Glu 232 in the R. capsulatus enzyme) is involved in proton tautomerization from N-3 to N-9 of xanthine in the course of hydroxylation at C-8, as shown in Fig.…”

“…Although the active site of the enzyme is shaped such that in-plane rotation of newly formed xanthine between the two phenylalanines so as to position C-8 appropriately for hydroxylation is probably sterically allowed, doing so positions xanthine such that its C-6ϭO is oriented away from Arg 880 rather than toward. We have previously argued that this is a catalytically less effective orientation, with neither Arg 880 nor Glu 802 positioned appropriately relative to the purine ring for optimal catalysis (12). The slower rate of reaction thus would allow dissociation of xanthine to a greater extent than would otherwise be the case, allowing it to accumulate in solution as is observed experimentally.…”

The Reductive Half-reaction of Xanthine Dehydrogenase from Rhodobacter capsulatus

“…Mutation of Glu 730 to Ala abolishes all activity toward xanthine in rapid reaction kinetic studies at neutral pH, with a minimum reduction in k red (and, correspondingly, the steady-state k cat ) of 10 7 , consistent with the role of this residue as a general base, abstracting the Mo-OH proton to initiate catalysis (6). Mutation of Arg 310 to Met also results in a large reduction in k red , by a factor of 10 4 , an effect attributed to Arg 310/880 stabilizing negative charge on the heterocycle in the course of nucleophilic attack (11). The E232A variant exhibited a more modest 12-fold decrease in k red and a 12-fold increase in K d in the reductive half-reaction, relative to wild-type enzyme, comparable with the effect seen on the steady-state kinetic behavior of an E803V variant of the human enzyme (13).…”

The Mo-Se active site of nicotinate dehydrogenase

“…S3). R880 of bXOR was suggested to stabilize the developing negative charge on the substrate during the hydroxylation step, and its mutation results in an increase of the dissociation constant, K D , for substrate binding and a decrease in the rate constant of enzyme reduction, k red (36). The conservation of amino acids essential for catalysis indicates common ways of substrate binding and transition state stabilization in NDH and XORs.…”