HYBRID ENZYMES FOR STRUCTURE-FUNCTION ANALYSIS OF CYTOCHROME-P-450-2B11

Previous work has shown that P-450 2B11 is responsible for the unique ability of dogs to metabolize and eliminate certain highly-chlorinated biphenyls such as 2,2',4,4',5,5'-hexachlorobiphenyl (245-HCB), wherea s the related P-450 2B forms in rat and rabbit are unable to metaboliz e the compound to any significant degree. To determine the structural basis for this functional diversity, hybrid enzymes were generated. Su ccess with this approach required a careful choice of second enzyme an d common substrate with which to assess the functional integrity of th e hybrid proteins. The choices of P-450 2B5 from rabbit as the second enzyme and androstenedione as the substrate were based in part on the finding that P-450 2B11 and P-450 2B5 hydroxylate androstenedione with similar overall activities but distinct profiles. Enzymatic studies w ith eight hybrid enzymes provided evidence for two regions of P-450 2B 11 and 2B5, between residues 95-239 and 240-370, that appear to be inv olved in defining substrate specificity for androstenedione, and three regions of P-450 2B11, between residues 95-239, 240-370, and 371-494, that contain amino acids necessary for metabolism of 245-HCB. This de liberate approach to the creation of hybrid cytochromes P-450 has gene rated a series of enzymes that will be central to further structure-fu nction studies of the cytochromes P-450 2B.