A Substrate-induced Biotin Binding Pocket in the Carboxyltransferase Domain of Pyruvate Carboxylase

Biotin-dependent enzymes catalyze carboxyl transfer reactions by efficiently coordinating multiple reactions between spatially distinct active sites. Pyruvate carboxylase (PC), a multifunctional biotin-dependent enzyme, catalyzes the bicarbonate- and MgATP-dependent carboxylation of pyruvate to oxaloacetate, an important anaplerotic reaction in mammalian tissues. To complete the overall reaction, the tethered biotin prosthetic group must first gain access to the biotin carboxylase domain and become carboxylated and then translocate to the carboxyltransferase domain, where the carboxyl group is transferred from biotin to pyruvate. Here, we report structural and kinetic evidence for the formation of a substrate-induced biotin binding pocket in the carboxyltransferase domain of PC from Rhizobium etli. Structures of the carboxyltransferase domain reveal that R. etli PC occupies a symmetrical conformation in the absence of the biotin carboxylase domain and that the carboxyltransferase domain active site is conformationally rearranged upon pyruvate binding. This conformational change is stabilized by the interaction of the conserved residues Asp590 and Tyr628 and results in the formation of the biotin binding pocket. Site-directed mutations at these residues reduce the rate of biotin-dependent reactions but have no effect on the rate of biotin-independent oxaloacetate decarboxylation. Given the conservation with carboxyltransferase domains in oxaloacetate decarboxylase and transcarboxylase, the structure-based mechanism described for PC may be applicable to the larger family of biotin-dependent enzymes

Insights Into the Carboxyltransferase Reaction of Pyruvate Carboxylase From the Structures of Bound Product and Intermediate Analogs

Pyruvate carboxylase (PC) is a biotin-dependent enzyme that catalyzes the MgATP- and bicarbonate-dependent carboxylation of pyruvate to oxaloacetate, an important anaplerotic reaction in central metabolism. The carboxyltransferase (CT) domain of PC catalyzes the transfer of a carboxyl group from carboxybiotin to the accepting substrate, pyruvate. It has been hypothesized that the reactive enolpyruvate intermediate is stabilized through a bidentate interaction with the metal ion in the CT domain active site. Whereas bidentate ligands are commonly observed in enzymes catalyzing reactions proceeding through an enolpyruvate intermediate, no bidentate interaction has yet been observed in the CT domain of PC. Here, we report three X-ray crystal structures of the Rhizobium etli PC CT domain with the bound inhibitors oxalate, 3-hydroxypyruvate, and 3-bromopyruvate. Oxalate, a stereoelectronic mimic of the enolpyruvate intermediate, does not interact directly with the metal ion. Instead, oxalate is buried in a pocket formed by several positively charged amino acid residues and the metal ion. Furthermore, both 3-hydroxypyruvate and 3-bromopyruvate, analogs of the reaction product oxaloacetate, bind in an identical manner to oxalate suggesting that the substrate maintains its orientation in the active site throughout catalysis. Together, these structures indicate that the substrates, products and intermediates in the PC-catalyzed reaction are not oriented in the active site as previously assumed. The absence of a bidentate interaction with the active site metal appears to be a unique mechanistic feature among the small group of biotin-dependent enzymes that act on α-keto acid substrates

Restoration of woodpasture on former agricultural land: The importance of safe sites and time gaps before grazing for tree seedlings

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Space station attached payload program support

The USRA is providing management and technical support for the peer review of the Space Station Freedom Attached Payload proposals. USRA is arranging for consultants to evaluate proposals, arranging meeting facilities for the reviewers to meet in Huntsville, Alabama and management of the actual review meetings. Assistance in developing an Experiment Requirements Data Base and Engineering/Technical Assessment support for the MSFC Technical Evaluation Team is also being provided. The results of the project will be coordinated into a consistent set of reviews and reports by USRA. The strengths and weaknesses analysis provided by the peer panel reviewers will by used NASA personnel in the selection of experiments for implementation on the Space Station Freedom

Gauge dependence and self-force from Galilean to Einsteinian free fall, compact stars falling into black holes, Hawking radiation and the Pisa tower at the general relativity centennial

(Short abstract). In Galilean physics, the universality of free fall implies an inertial frame, which in turns implies that the mass m of the falling body is omitted. Otherwise, an additional acceleration proportional to m/M would rise either for an observer at the centre of mass of the system, or for an observer at a fixed distance from the centre of mass of M. These elementary, but overlooked, considerations fully respect the equivalence principle and the identity of an inertial or a gravitational pull for an observer in the Einstein cabin. They value as fore-runners of the self-force and gauge dependency in general relativity. The approximate nature of Galilei's law of free fall is explored herein. When stepping into general relativity, we report how the geodesic free fall into a black hole was the subject of an intense debate again centred on coordinate choice. Later, we describe how the infalling mass and the emitted gravitational radiation affect the free fall motion of a body. The general relativistic self-force might be dealt with to perfectly fit into a geodesic conception of motion. Then, embracing quantum mechanics, real black holes are not classical static objects any longer. Free fall has to handle the Hawking radiation, and leads us to new perspectives on the varying mass of the evaporating black hole and on the varying energy of the falling mass. Along the paper, we also estimate our findings for ordinary masses being dropped from a Galilean or Einsteinian Pisa-like tower with respect to the current state of the art drawn from precise measurements in ground and space laboratories, and to the constraints posed by quantum measurements. The appendix describes how education physics and high impact factor journals discuss the free fall. Finally, case studies conducted on undergraduate students and teachers are reviewed