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Cytochrome P450 3A4 (CYP3A4) is the most important drug-metabolizing enzyme in humans and has been associated with harmful drug interactions. The activity of CYP3A4 is known to be modulated by several compounds and by the electron transfer partner, cytochrome P450 reductase (CPR). The underlying mechanism of these effects, however, is poorly understood. We have used hydrogen-deuterium exchange mass spectrometry to investigate the impact of binding of CPR and of three different substrates (7-benzyloxy-4-trifluoromethyl-coumarin, testosterone, and progesterone) on the conformational dynamics of CYP3A4. Here, we report that interaction of CYP3A4 with substrates or with the oxidized or reduced forms of CPR leads to a global rigidification of the CYP3A4 structure. This was evident from the suppression of deuterium exchange in several regions of CYP3A4, including regions known to be involved in protein-protein interactions (helix C) and substrate binding and specificity (helices B and E, and loop K/β1). Furthermore, the bimodal isotopic distributions observed for some CYP3A4-derived peptides were drastically impacted upon binding to CPR and/or substrates, suggesting the existence of stable CYP3A4 conformational populations that are perturbed by ligand/CPR binding. The results have implications for understanding the mechanisms of ligand binding, allostery, and catalysis in CYP enzymes.

Physical activity has been recommended as a preventive measure against type 2 diabetes. To improve physical activity programs, it is important to understand the determinants of physical activity. To examine to which extent physical activity is determined by genetics, we sought to estimate the heritability of physical activity in 701 Old Order Amish (372 men and 329 women) of Lancaster, PA, linked within a single 13-generation pedigree. Subjects were aged 43.3 ± 14.0 yr (mean ± SD); BMI was 25.6 ± 3.2 (men) and 27.7 ± 5.4 kg/m² (women). Physical activity was measured with Actical accelerometers (firmware versions 8.2, 8.3, and 8.4; Mini Mitter, Bend, OR) worn on the hip for 7 consecutive days, capturing activity in 0.25 min epochs. Activity was expressed as mean daily total activity counts (a raw measure of body movement independent of body size) and, With the use of algorithms incorporating body size applied on a per-epoch basis, total energy expenditure and time in sedentary (including sleep) and moderate/vigorous activity (> 3 METs). Heritability was estimated by a variance component method. Mean daily total activity counts were higher in men than in women (465 ± 194 vs. 346 ± 183 x 10³, p < 0.0001) and diminished with age (p < 0.0001). The same pattern was observed for total energy expenditure and moderate/vigorous activity; conversely, sedentary activity was lower in men (797 ± 113 vs. 823 ± 131 min/d, p = 0.03). After adjustment for sex-specific age and age², occupation, season of measurement, and firmware version, the following heritabilities (h²) ± SE were determined: total activity counts, 0.31 ± 0.13 (p = 0.005); total energy expenditure, 0.36 ± 0.12 (p = 0.0003); time in sedentary activity, 0.41 ± 0.13 (p = 0.0006); time in moderate/vigorous activity, 0.29 ± 0.13 (p = 0.008). Physical activity level is a heritable trait in the Old Order Amish. Discovery of the underlying coding variants will inform strategies to increase physical activity in the general population. [ABSTRACT FROM AUTHOR]