Understanding the Reaction Mechanism of the Drug Metabolizing P450 Enzyme

Grant Winner

  • Reza Razeghifard, Ph.D. – Farquhar College of Arts and Sciences

Dean

  • Don Rosenblum, Ph.D. – Farquhar College of Arts and Sciences

Abstract

Award Winners

Mammalian P450s are unusual enzymes capable of binding and modifying many different drugs mainly through hydroxylation. The P450 protein uses a heme group as its reactive site. The hydroxylation reaction begins with oxygen binding to the heme iron. The oxygenated heme then changes its oxidation state forming some unstable intermediates. Understanding of the reaction mechanism is then depended on findings techniques to observe and identify the intermediates. Since heme can give a rich optical spectrum, attempts were made to apply optical spectroscopy for the detection of these intermediates. However, those measurements were not successful because they were not performed with reactive mammalian P450 enzymes are substrate/drug. One major achievement was made recently by finding butylated hydroxytoluene (BHT) and propofol as very reactive substrates for P4502B4 . P4502B4 is the model enzyme for human P450. Low-temperature spectroscopy of P4502B4 in the presence of BHT revealed that the purified P450 2B4 enzymes were in two different forms: inactive and active forms. It is the presence of the inactive forms of the P450 enzymes that makes resolving the optical spectra of the reaction intermediates so difficult. The goal of this project is first to improve the purification techniques needed for the isolation of P450 enzymes free from the inactive forms. Secondly, several reaction conditions will be tested to ensure that the purified P450 enzyme remains active during the time-course of optical spectroscopy measurements. If successful, all P450s will carry out a homogenous reaction with the same starting optical spectra, changing gradually while the intermediates are formed and reaching eventually to the same ending optical spectra. Detecting the intermediates will greatly advance our understanding the reaction mechanism of this very important enzyme.