Using Students' Concept-building Tendencies to Better Characterize Average-Performing Student Learning and Problem-Solving Approaches in General Chemistry.

We previously reported that students' concept-building approaches, identified a priori using a cognitive psychology laboratory task, extend to learning complex science, technology, engineering, and mathematics topics. This prior study examined student performance in both general and organic chemistry at a select research institution, after accounting for preparation. We found that abstraction learners (defined cognitively as learning the theory underlying related examples) performed higher on course exams than exemplar learners (defined cognitively as learning by memorizing examples). In the present paper, we further examined this initial finding by studying a general chemistry course using a different pedagogical approach (process-oriented guided-inquiry learning) at an institution focused on health science majors, and then extended our studies via think-aloud interviews to probe the effect concept-building approaches have on problem-solving behaviors of average exam performance students. From interviews with students in the average-achieving group, using problems at three transfer levels, we found that: 1) abstraction learners outperformed exemplar learners at all problem levels; 2) abstraction learners relied on understanding and exemplar learners dominantly relied on an algorithm without understanding at all problem levels; and 3) both concept-building-approach students had weaknesses in their metacognitive monitoring accuracy skills, specifically their postperformance confidence level in their solution accuracy.

Structural basis for cooperative binding of azoles to CYP2E1 as interpreted through guided molecular dynamics simulations.

CYP2E1 metabolizes a wide array of small, hydrophobic molecules, resulting in their detoxification or activation into carcinogens through Michaelis-Menten as well as cooperative mechanisms. Nevertheless, the molecular determinants for CYP2E1 specificity and metabolic efficiency toward these compounds are still unknown. Herein, we employed computational docking studies coupled to molecular dynamics simulations to provide a critical perspective for understanding a structural basis for cooperativity observed for an array of azoles from our previous binding and catalytic studies (Hartman et al., 2014). The resulting 28 CYP2E1 complexes in this study revealed a common passageway for azoles that included a hydrophobic steric barrier causing a pause in movement toward the active site. The entrance to the active site acted like a second sieve to restrict access to the inner chamber. Collectively, these interactions impacted the final orientation of azoles reaching the active site and hence could explain differences in their biochemical properties observed in our previous studies, such as the consequences of methylation at position 5 of the azole ring. The association of a second azole demonstrated significant differences in interactions stabilizing the bound complex than observed for the first binding event. Intermolecular interactions occurred between the two azoles as well as CYP2E1 residue side chains and backbone and involved both hydrophobic contacts and hydrogen bonds. The relative importance of these interactions depended on the structure of the respective azoles indicating the absence of specific defining criteria for binding unlike the well-characterized dominant role of hydrophobicity in active site binding. Consequently, the structure activity relationships described here and elsewhere are necessary to more accurately identify factors impacting the observation and significance of cooperativity in CYP2E1 binding and catalysis toward drugs, dietary compounds, and pollutants.

Structure of pyrazole derivatives impact their affinity, stoichiometry, and cooperative interactions for CYP2E1 complexes.

CYP2E1 plays a critical role in detoxification and carcinogenic activation of drugs, pollutants, and dietary compounds; however, these metabolic processes can involve poorly characterized cooperative interactions that compromise the ability to understand and predict CYP2E1 metabolism. Herein, we employed an array of ten azoles with an emphasis on pyrazoles to establish the selectivity of catalytic and cooperative CYP2E1 sites through binding and catalytic studies. Spectral binding studies for monocyclic azoles suggested two binding events, while bicyclic azoles suggested one. Pyrazole had moderate affinity toward the CYP2E1 catalytic site that improved when a methyl group was introduced at either position 3 or 4. The presence of methyl groups simultaneously at positions 3 and 5 blocked binding, and a phenyl group at position 3 did not improve binding affinity. In contrast, pyrazole fusion to a benzene or cyclohexane ring greatly increased affinity. The consequences of these binding events on CYP2E1 catalysis were studied through inhibition studies with 4-nitrophenol, a substrate known to bind both sites. Most pyrazoles shared a common mixed cooperative inhibition mechanism in which pyrazole binding rescued CYP2E1 from substrate inhibition. Overall, inhibitor affinities toward the CYP2E1 catalytic site were similar to those reported in binding studies, and the same trend was observed for binding at the cooperative site. Taken together, these studies identified key structural determinants in the affinity and stoichiometry of azole interactions with CYP2E1 and consequences on catalysis that further advance an understanding of the relationship between structure and function for this enzyme.

CYP2E1 substrate inhibition. Mechanistic interpretation through an effector site for monocyclic compounds.

In this study we offer a mechanistic interpretation of the previously known but unexplained substrate inhibition observed for CYP2E1. At low substrate concentrations, p-nitrophenol (pNP) was rapidly turned over (47 min(-1)) with relatively low K(m) (24 microM); nevertheless, at concentrations of >100 microM, the rate of pNP oxidation gradually decreased as a second molecule bound to CYP2E1 through an effector site (K(ss) = 260 microm), which inhibited activity at the catalytic site. 4-Methylpyrazole (4MP) was a potent inhibitor for both sites through a mixed inhibition mechanism. The K(i) for the catalytic site was 2.0 microM. Although we were unable to discriminate whether an EIS or ESI complex formed, the respective inhibition constants were far lower than K(ss). Bicyclic indazole (IND) inhibited catalysis through a single CYP2E1 site (K(i) = 0.12 microM). Similarly, 4MP and IND yielded type II binding spectra that reflected the association of either two 4MP or one IND molecule(s) to CYP2E1, respectively. Based on computational docking studies with a homology model for CYP2E1, the two sites for monocyclic molecules, pNP and 4MP, exist within a narrow channel connecting the active site to the surface of the enzyme. Because of the presence of the heme iron, one site supports catalysis, whereas the other more distal effector site binds molecules that can influence the binding orientation and egress of molecules for the catalytic site. Although IND did not bind these sites simultaneously, the presence of IND at the catalytic site blocked binding at the effector site.