Purification and mechanism of human aldehyde oxidase expressed in Escherichia coli.

Human aldehyde oxidase 1 (AOX1) has been subcloned into a vector suitable for expression in Escherichia coli, and the protein has been expressed. The resulting protein is active, with sulfur being incorporated in the molybdopterin cofactor. Expression levels are modest, but 1 liter of cells supplies enough protein for both biochemical and kinetic characterization. Partial purification is achieved by nickel affinity chromatography through the addition of six histidines to the amino-terminal end of the protein. Kinetic analysis, including kinetic isotope effects and comparison with xanthine oxidase, reveal similar mechanisms, with some subtle differences. This expression system will allow for the interrogation of human aldehyde oxidase structure/function relationships by site-directed mutagenesis and provide protein for characterizing the role of AOX1 in drug metabolism.

Cytochrome P450 2C9 type II binding studies on quinoline-4-carboxamide analogues.

CYP2C9 is a significant P450 protein responsible for drug metabolism. With the increased use of heterocyclic compounds in drug design, a rapid and efficient predrug screening of these potential type II binding compounds is essential to avoid adverse drug reactions. To understand binding modes, we use quinoline-4-carboxamide analogues to study the factors that determine the structure-activity relationships. The results of this study suggest that the more accessible pyridine with the nitrogen para to the linkage can coordinate directly with the ferric heme iron, but this is not seen for the meta or ortho isomers. The pi-cation interaction of the naphthalene moiety and Arg 108 residue may also assist in stabilizing substrate binding within the active-site cavity. The type II substrate binding affinity is determined by the combination of steric, electrostatic, and hydrophobicity factors; meanwhile, it is enhanced by the strength of lone pair electrons coordination with the heme iron.

Modeling and synthesis of novel tight-binding inhibitors of cytochrome P450 2C9.

Cytochrome P450 2C9 (2C9) is one of the three major drug metabolizing cytochrome P450 enzymes in human liver. Although the crystal structure of 2C9 has been solved, the important physicochemical properties of substrate-enzyme interactions remain difficult to be determined. This is due in part to the conformational flexibility of mammalian P450 enzymes. Therefore, probing the active-site with high-affinity substrates is important in further understanding substrate-enzyme interactions. Three-dimensional quantitative structure-activity relationships (3D-QSAR) and docking experiments have been shown to be useful tools in correlating biological activity with structure. In particular we have previously reported that the very tight-binding inhibitor benzbromarone can provide important information about the active-site of 2C9. In this study we report the binding affinities and potential substrate-enzyme interactions of 4H-chromen-4-one analogs, which are structurally similar to benzbromarone. The chromenone structures are synthetically accessible inhibitors and give inhibition constants as low as 4.2 nM, comparable with the very tightest-binding inhibitors of 2C9. Adding these compounds to our previous 2C9 libraries for CoMFA models reinforces the important electrostatic and hydrophobic features of substrate binding. These compounds have also been docked in the 2C9 crystal structure and the results indicate that Arg 108 plays significant roles in the binding of chromenone substrates.

ATLes: the strategic application of Web-based technology to address learning objectives and enhance classroom discussion in a veterinary pathology course.

A case-based program called ATLes (Adaptive Teaching and Learning Environments) was designed for use in a systemic pathology course and implemented over a four-year period. Second-year veterinary students working in small collaborative learning groups used the program prior to their weekly pathology laboratory. The goals of ATLes were to better address specific learning objectives in the course (notably the appreciation of pathophysiology), to solve previously identified problems associated with information overload and information sorting that commonly occur as part of discovery-based processes, and to enhance classroom discussion. The program was also designed to model and allow students to practice the problem-oriented approach to clinical cases, thereby enabling them to study pathology in a relevant clinical context. Features included opportunities for students to obtain additional information on the case by requesting specific laboratory tests and/or diagnostic procedures. However, students were also required to justify their diagnostic plans and to provide mechanistic analyses. The use of ATLes met most of these objectives. Student acceptance was high, and students favorably reviewed the online ''Content Links'' that made useful information more readily accessible and level appropriate. Students came to the lab better prepared to engage in an in-depth and high-quality discussion and were better able to connect clinical problems to underlying changes in tissue (lesions). However, many students indicated that the required time on task prior to lab might have been excessive relative to what they thought they learned. The classroom discussion, although improved, was not elevated to the expected level-most likely reflecting other missing elements of the learning environment, including the existing student culture and the students' current discussion skills. This article briefly discusses the lessons learned from ATLes and how similar case-based exercises might be combined with other approaches to enhance and enliven classroom discussions in the veterinary curriculum.