EVIDENCE THAT ASPARTIC-ACID-301 IS A CRITICAL SUBSTRATE-CONTACT RESIDUE IN THE ACTIVE-SITE OF CYTOCHROME-P450 2D6

Model building studies have intimated a role for aspartic acid 301 in the substrate binding of cytochrome P450 2D6 (CYP2D6). We have tested this hypothesis by generating a range of CYP2D6 mutants substituting a variety of amino acids at this site. The mutant proteins, which inclu ded substitution with a negatively charged glutamic acid residue or ne utral asparagine, alanine, or glycine residues, were expressed in Sacc haromyces cerevisiae. In addition, a mutant where aspartic acid 301 wa s deleted was also tested. All the mutants expressed approximately equ ivalent amounts of recombinant apoprotein and, apart hom the alanine 3 01 and the aspartic acid 301 deletion mutants, gave carbon monoxide di fference spectra of similar magnitude to the wild type. In the cases o f the alanine and deletion mutants, the amount of holoprotein was sign ificantly reduced or absent relative to the amount of apoprotein, indi cating restricted heme incorporation. The glutamic acid mutant was sho wn to have similar catalytic properties to the wild type enzyme toward the substrates debrisoquine and metoprolol; however, some differences in regioselectivity and ligand binding were observed. The mutants con taining neutral amino acids at position 301 exhibited marked reduction s in catalytic activity. At low substrate concentrations little, if an y, activity toward debrisoquine and metoprolol was measured. However, at a higher substrate concentration (2 mM) some activity was observed (about 10-20% of wild type levels). Consistent with the above findings , the debrisoquine-induced spin changes in the mutant proteins were ma rkedly reduced These data collectively demonstrate that aspartic acid 301 plays an important role in determining the substrate specificity a nd activity of CYP2D6 and provide experimental evidence supporting the role of this amino acid in forming an electrostatic interaction betwe en the basic nitrogen atom in CYP2D6 substrates and the carboxylate gr oup of aspartic acid 301.