Structural studies of cytochrome P4501A1: Identification of substrate and cumene hydroperoxide binding regions in the active site of P4501A1 and characterization of the P4501A1 interaction with cytochrome P450 reductase

Cytochromes P450 are a superfamily of heme-thiolate proteins that function in a concert with another protein, cytochrome P450 reductase, as terminal oxidases of an enzymatic system catalyzing the metabolism of a variety of foreign compounds and endogenous substrates. In order to better understand P450s catalytic mechanism and substrate specificity, information about the structure of the active site is necessary. Given the lack of a crystal structure of mammalian P450, other methods have been used to elucidate the substrate recognition and binding site structure in the active center. In this project I utilized the photoaffinity labeling technique and site-directed mutagenesis approach to gain further structural insight into the active site of mammalian cytochrome P4501AI and examine the role of surface residues in the interaction of P4501A1 with the reductase. ^ Four crosslinked peptides were identified by photoaffinity labeling using diazido benzphetamine as a substrate analog. Alignment of the primary structure of cytochrome P4501A1 with that of bacterial cytochrome P450102 (the crystal structure of which is known) revealed that two of the isolated crosslinked peptides can be placed in the vicinity of heme (in the L helix region and β10-β11 sheet region of cytochrome P450102) and could be involved in substrate binding. The other two peptides were located on the surface of the protein with the label bound specifically to Lys residues that were proposed to be involved in reductase-P450 interaction. ^ Alternatively, it has been shown that some of the organic hydroperoxides can support P450 catalyzed reactions in the absence of NADPH, O2 and reductase. By means of photoaffinity labeling the cumene hydroperoxide binding region was identified. Using azidocumene as the photoaffinity label, the tripeptide T501-L502-K503 was shown to be the site where azidocumene covalently binds to P4501A1. The sequence alignment of cytochrome P4501A1 with cytochrome P450102 predicts that this region might correspond to β-sheet structure localized on the distal side of the heme ring near the I helix and the oxygen binding pocket. The role of Thr501 in the cumene hydroperoxide binding was confirmed by mutations of this residue and kinetic analysis of the effects of the mutations. ^ In addition, the role of two lysine residues, Lys271 and Lys279, in the interaction with reductase was examined by means of site-directed mutagenesis. The lysine residues were substituted with isoleucine and enzymatic activity of the wild type and the mutants were compared in reductase- and cumene hydroperoxide-supported systems. The lysine 279 residue has been shown to play a critical role in the P4501A1-reductase interaction. ^

Is family medicine meeting the HIV/AIDS challenge? A national survey of family physicians' beliefs, clinical competence, and experience regarding HIV disease

A national sample of family physicians was surveyed to (1) assess family physicians' beliefs about the human immunodeficiency virus (HIV) and individuals at risk for infection, their clinical competence regarding HIV-related issues, and their experiences with HIV disease; (2) present conclusions to the American Academy of Family Physicians (AAFP) to effect the development of an early clinical care protocol and a continuing medical education curriculum; and (3) collect base-line data for use in the evaluation of an early clinical care protocol and a continuing medical education curriculum, in the case that such programs are developed and disseminated. After considering retired or deceased respondents, of the 2,660 physicians surveyed, 1,678 (63.7%) responded. The resulting sample was representative of the active members of the AAFP. About 77% of the respondents were unable to accurately identify the universal precautions for blood and body fluids to prevent occupational transmission of HIV or hepatitis B virus (HBV). Residency trained and board certified physicians expressed fewer "external constraints," such as fear of losing patients, obviating them from providing treatment to individuals with HIV disease (p =.004 and p $<$.001, respectively). These physicians also manifested fewer "internal constraints" to the provision of HIV treatment, such as fear of becoming infected (p $<$.001 and p =.012, respectively). Residency trained physicians also expressed a greater comfort with discussing sexually-related topics with their patients than did non-residency trained physicians (p $<$.001). There were 67.1% of the physicians surveyed who reported never providing treatment to an individual with HIV disease. Residency trained and board certified physicians expressed a greater likelihood to provide treatment to HIV-infected patients (p $<$.001) than non-residency trained and non-board certified physicians.^ Among the various primary care specialties, family medicine is especially vulnerable to the current challenges of HIV/AIDS. These challenges are augmented by the epidemiologic pattern that characterizes AIDS. For the past several years, we have seen AIDS in this country assume a similar pattern to that seen in most other countries; HIV is becoming increasingly prevalent in the heterosexual population as well as in locations removed from metropolitan centers. This current phase of the epidemic generates greater pressures upon primary care physicians, particularly family physicians, to become better acquainted with the means to provide early care to HIV/AIDS patients and to prevent HIV/AIDS among their patients. Family medicine is especially appropriate for providing care to HIV patients because family medicine involves treatment to all age groups and conditions; other primary care specialties focus on limited patient populations or specific conditions. Family physicians should be armed with the expertise to confront HIV/AIDS. However, family physicians' clinical competence and experience with HIV is not known. The data collected in this survey describes their competencies, attitudes, and experiences. ^