Cytochrome P450 (P450) 2D6 is an important human drug-metabolizing enzyme responsible for the oxidation of greater than 30 widely used therapeutic agents. Another significant feature of P450 2D6 is its apparent genetic polymorphism which results in the reduction or absence of the functional protein. Although the substrates of P450 2D6 are quite diverse they usually possess a basic nitrogen that is located 5-7 Angstrom units from the site of oxidation P450 2D6. The major working hypothesis to be addressed is that basic character found on substrates interacts via a salt bridge with a point negative charge on P450 2D6 that facilitates substrate binding and orientation in the active site. The identity of the point charge has been postulated to be contributed by the carboxylate of Asp301. To test this hypothesis further, Asp301 will be replaced by a positively-charged amino acid (e.g. Lys or Arg) with the goal of generating an enzyme that interacts in an equal or similar manner with carboxy-derivatives of known P4502D6 substrates as P4502D6 interacts with its substrates. Further studies will involve the study of individual P4502D6 reaction steps to determine which is rate- limiting. Finally, modified P4502D6 expressed in Escherichia coli represents an attractive target for protein crystallization attempts, in light of its demonstrated solubility compared with other mammalian P450s that are expressed under similar conditions.
