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

Paper Promises for Drug Innovation

Innovation does not stop when new medicines are launched. Companies with approved drugs and biologics continue to study their products for years after the initial approval-for instance, exploring new ways to formulate their drugs or modifying the active ingredients to introduce versions with different, and sometimes better, safety and effectiveness profiles. They also routinely study their products for usefulness in treating new conditions. This continued research requires time and money, and companies will not invest that time and money without adequate reason to do so. This Article examines the incentives federal law provides for new-use research, concluding that current incentives are little more than paper promises

Outpatient Civil Commitment in North Carolina: Constitutional and Policy Concerns

This article examines preventive outpatient commitment, which targets those not ill or dangerous enough to be committed to inpatient facilities under state commitment laws. After discussing the history and design of the NC scheme, it explores constitutional and practical difficulties. Ultimately, it argues that individualized case management through local mental health clinics is the more effective and humane way of serving the interests of both the individual and the state

Early Access to Unapproved Medicines in the United States and France

In 2018, President Trump signed a federal right to try law, claiming that it would give desperately ill patients earlier access to unapproved medicines, by allowing the patient, doctor, and drug company to arrange for access without federal oversight. Critics of the law argued that it would not meaningfully increase access to experimental medicines, because federal oversight was not the obstacle in the first place. And they were correct. U.S. law already permitted companies to provide terminally ill patients with early access to unapproved medicines. The problem was instead that companies did not take advantage of this option. This Article offers new insights into U.S. law on early access, as well as the new rightto- try law, by offering a comparative perspective using French law. We explore the historical, legal, and cultural differences between France and the United States that may explain differences in their early access systems and why the right-to-try law emerged in one country but not the other. The differing approaches reflect in part differing reactions to arguments grounded in personal autonomy and patients\u27 rights, when held up against utilitarian arguments for premarket approval and traditions of medical paternalism. Using the French experience, this Article also considers the possibility that the key to increasing use of expanded access in the United States might be financial: making it worthwhile for companies, by allowing them to profit from sales, and making the medicines and associated healthcare services free for patients through insurance coverage

The Evergreening Metaphor in Intellectual Property Scholarship

This article is a plea for changes in the scholarly dialogue about “evergreening” by drug companies. Allegations that drug companies engage in “evergreening” are pervasive in legal scholarship, economic scholarship, medical and health policy scholarship, and policy writing, and they have prompted significant policymaking proposals. This Article was motivated by concern that the metaphor has not been fully explained and that policymaking in response might therefore be premature. It canvasses and assesses the scholarly literature—more than 300 articles—discussing or mentioning “evergreening.” It catalogues the definitions, the examples, and the empirical studies. Scholars use the term when describing certain actions taken by the innovative companies that develop and introduce new medicines to market. But they are inconsistent in their descriptions of the circumstances to which the term applies. And though most claim the innovator has “extended” something in these circumstances, they do not agree on the particulars. The literature is similarly in disarray about what has been “evergreened”—an invention, a product, a price, a patent, or something else entirely. All of this makes it hard to know from the literature what exactly scholars are writing about. After sorting through the definitions and examples—and considering the legal framework and practical landscape in which drug innovators and their generic competitors operate—this Article offers an answer and, more importantly, identifies the implicit normative claim. In simple terms, the normative claim in the literature is something like this: “an innovator should not enjoy an exclusive market and supra-competitive pricing for innovations that stem in some fashion from a separate innovation for which it already enjoyed a 20-year patent term. Or at least, a drug innovator should not.” This Article does not defend, or reject, this normative claim. Instead, it makes a different claim: that policymaking should be based on descriptive scholarship that is careful and precise about the relevant law and facts, normative work that is clear and candid about its claim and thorough in its reasoning, and empirical studies that document the actual problem the normative proposals and policymaking proposals are meant to address. Significant policymaking would be premature today, because we have not yet produced this body of work. Constant use of the “evergreening” metaphor may be obscuring this failure. The Article concludes with recommendations for scholars continuing to work on these topics, focusing on ways that we can provide quality work to assist policymakers considering the normative claim

The Evergreening Metaphor in Intellectual Property Scholarship

This article is a plea for changes in the scholarly dialogue about evergreening by drug companies. Allegations that drug companies engage in evergreening are pervasive in legal scholarship, economic scholarship, medical and health policy scholarship, and policy writing, and they have prompted significant policymaking proposals. This Article was motivated by concern that the metaphor has not been fully explained and that policymaking in response might therefore be premature. It canvasses and assesses the scholarly literature-more than 300 articles discussing or mentioning evergreening. It catalogues the definitions, the examples, and the empirical studies. Scholars use the term when describing certain actions taken by the innovative companies that develop and introduce new medicines to market. But they are inconsistent in their descriptions of the circumstances to which the term applies. And though most claim the innovator has extended something in these circumstances, they do not agree on the particulars. The literature is similarly in disarray about what has been evergreened -an invention, a product, a price, a patent, or something else entirely. All of this makes it hard to know from the literature what exactly scholars are writing about. After sorting through the definitions and examples-and considering the legal framework and practical landscape in which drug innovators and their generic competitors operate - this Article offers an answer and, more importantly, identifies the implicit normative claim. In simple terms, the normative claim in the literature is something like this: an innovator should not enjoy an exclusive market and supracompetitive pricing for innovations that stem in some fashion from a separate innovation for which it already enjoyed a 20-year patent term. Or at least, a drug innovator should not. This Article does not defend, or reject, this normative claim. Instead, it makes a different claim: that policymaking should be based on descriptive scholarship that is careful and precise about the relevant law and facts, normative work that is clear and candid about its claim and thorough in its reasoning, and empirical studies that document the actual problem the normative proposals and policymaking proposals are meant to address. Significant policymaking would be premature today, because we have not yet produced this body of work. Constant use of the evergreening metaphor may be obscuring this failure. The Article concludes with recommendations for scholars continuing to work on these topics, focusing on ways that we can provide quality work to assist policymakers considering the normative clai

A Brief History of 180-Day Exclusivity Under the Hatch-Waxman Amendments to the Federal Food, Drug, and Cosmetic Act

This article summarizes the history of the 180-day exclusivity provision in the Hatch- Waxman Amendments to the Federal Food, Drug, and Cosmetic Act (FDCA). Part II presents the statutory language, as amended in the Medicare Prescription Drug, Improvement, and Modernization Act of 2003 (MMA), and summarizes the law that applies to new abbreviated new drug applications (ANDAs) (those filed after December 8, 2003, provided there was no paragraph IV certification to the listed drug prior to December 8), as well as the law that applies to all other ( old ) ANDAs. Part III describes the legislative history of the original 1984 provision and traces its judicial and administrative history through the present. Part IV describes the history of the 2003 amendments and describes the key changes made in 2003. While Congress addressed in 2003 a number of the interpretive issues that had arisen since 1984, the new law is intricate and undoubtedly will give rise to new interpretive questions in the months and years ahead

2004 Update - 180-Day Exclusivity under the Hatch-Waxman Amendments to the Federal Food, Drug, and Cosmetic Act

This article updates the author\u27s previously published article on the topic, provides some insight into recent events in this area of the law, and specifies a few minor items that were noted incorrectly in the earlier work